ZBTB5 enhances the resistance of cervical cancer to paclitaxel by regulating BCL6

ZBTB5 enhances the resistance of cervical cancer to paclitaxel by regulating BCL6 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZBTB5 enhances the resistance of cervical cancer to paclitaxel by regulating BCL6 Yue He, Jing-Jing Zhang, Jian-Qing Xu, Yun-Fan Wang, Chen Ji, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2471463/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract PURPOSE: To preliminarily clarify the mechanism of paclitaxel (PTX) resistance in cervical squamous cell carcinoma with the involvement of ZBTB5. RESULTS: 1) Overexpression of ZBTB5 was cultured in PTX, and immunofluorescence showed that overexpression of ZBTB5 enhanced cervical squamous cell carcinoma resistance to PTX by functioning in the cell nucleus. 2) ZBTB5 knockdown inhibited cell cloningand proliferation and enhanced apoptosis of cervical squamous carcinoma cells in PTX and reduced resistance to PTX in SiHa cells compared with the non-knockdown group (p < 0.05). 3) A ChIP assay was performed after overexpression of ZBTB5, which suggested that the BCL6 gene is located 395 kb from the TSS region and may be regulated by ZBTB5, which may be the promoter of BCL6. This result was consistent with that of the IPA bioinformatics analysis. 4) Further in-gel enzymatic digestion and shotgun-mass spectrometry protein identification were performed after Co-IP, and 322 differential proteins were identified in the overexpression group. Bioinformatics analysis of different proteins was conducted and 13 proteins were selected for validation: U2AF2, RBM5, ILK, ENAH, JUP, RELA/P65, SQSTM1, YY1, STIM1, Integrin alpha V, EED, SUGT1, and NFKB1. Among them, U2AF2 was successfully detected in the input, suggesting a possible interaction of ZBTB5 with U2AF2. CONCLUSION: There is a protein–protein interaction between ZBTB5 and U2AF2, and ZBTB5 is involved in PTX resistance in cervical squamous cell carcinoma by regulating BCL6 . It was hypothesized that ZBTB5 may form a transcriptional complex with U2AF2 to regulate BCL6 to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma. ZBTB5 Paclitaxel Resistance Cervical Cancer Squamous Cell Carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cervical cancer is the fourth most common malignant tumor affecting women's health worldwide, 90% of which are cases of cervical squamous cell cancer [ 1 ]. According to data provided in 2018, there are 569,800 new cases of cervical cancer and 311,400 new deaths as a result of cervical cancer per year worldwide [ 2 ]. China has the highest number of new cases and deaths [ 3 ], with 131,500 new cases and 53,000 new deaths per year, accounting for 18.4% of female cancer deaths in China [ 4 ]. In underdeveloped countries, the 5-year survival rate of patients with cervical cancer is < 50%, and the survival rate for patients with stage III to IV cervical cancer is only 5–15%, making it a serious threat to women's lives [ 2 ]. Among the clinical treatment options for cervical cancer, paclitaxel (PTX) is the first-line chemotherapy agent, which is widely used in patients with intermediate and advanced cervical squamous cell cancer and plays an important role in chemotherapy for cervical squamous cell cancer. PTX resistance is the main cause of treatment failure in patients undergoing chemotherapy for cervical squamous cell cancer, and investigating strategies to overcome PTX resistance in cervical squamous cell cancer has become the most critical clinical subject for gynecologic oncologists. In this study, ZBTB5 was proposed, for the first time, as a transcription factor that regulates BCL6 to influence the PTX resistance of cervical squamous cell cancer, providing a new target for the precise treatment of PTX resistance in patients with cervical squamous cell cancer and giving it important clinical significance. 2. Materials And Methods 2.1. Materials The human cervical squamous carcinoma cell lines (SiHa) were obtained from the Laboratory of the School of Basic Medical Sciences, Capital Medical University. Materials also included Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, USA), fetal bovine serum (Evergreen, Hangzhou, China), PTX (Perilla frutescens, Beijing Union Pharmaceutical Factory, Lot number: 141101), ZBTB5 overexpression and ZBTB5 sgRNA lentiviruses (Shanghai Genechem, China), RIPA lysis buffer, NP-40 lysis buffer (Shanghai Dingguo Biotechnology), prestained protein marker (Thermo, USA), Apoptosis kit (eBioscience™ Annexin V USA), paraformaldehyde (Sigma, USA), ChIP kit (Pierce, USA), qPCR kit (Promega, USA), BCA protein assay kit (Beyotime, China), ANTI-FLAG® M2 Affinity Gel (Sigma, USA), 3X FLAG peptide (Sigma, USA), ECL-PLUS/kit (Thermo, USA), X-ray film developer powder (Shanghai Guanlong Photographic Materials Company, China), iodoacetamide (Sigma, USA), dithiothreitol (Sigma, USA), methanol, formic acid (Thermo, USA), and ultrafiltration centrifuge tubes (Sartorius, USA). 2.2. Instruments Instruments in the present study included a fluorescence microscope (Olympus), CO 2 incubator (Sanyo), inverted microscope (Shanghai Caikon Optical Instruments, China), centrifuges (Thermo Fisher Scientific, China; Eppendorf, USA), biosafety cabinet (Shanghai Zhen Zi Chuang Air Purification Equipment, China), SDS-PAGE protein electrophoresis instrument (Shanghai Tanon, China), protein transfer instrument (Shanghai Tanon, China), flow cytometer (Millipore, USA), metal bath (Hangzhou Miu Instruments, China), qPCR instrument (Bio-Rad, USA), high-speed frozen centrifuge, Easy-nLC chromatography system, Q Exactive Plus mass spectrometer (Thermo, USA), compact thermostatic mixer, rotating mixer (Dalong, China), and constant temperature incubator (Shanghai Jinghong Experiment Equipment, China). 2.3. Methods 2.3.1. Cell culture SiHa cells transfected with the ZBTB5 overexpression lentivirus, ZBTB5 sgRNA lentivirus, and control lentivirus were placed in the DMEM containing 10% fetal bovine serum and cultured in a 5% CO 2 incubator at 37°C. The DMEM was changed every 2–3 days, and cells in the logarithmic growth phase were taken for the experiment. 2.3.2. Immunofluorescence Infected cells in the logarithmic phase were formed into 1–2×10 4 single cell suspensions, inoculated in 96-well glass-bottom plates (i.e., special culture plates made for confocal laser scanning), placed in a CO 2 incubator, and incubated until the cells grew into a monolayer. Cell fixation: the plates were washed twice with PBS, fixed with 4% paraformaldehyde, and rinsed with PBS. Permeabilization: cells were perforated with 0.2% Triton, rinsed twice in PBS, and blocked. Antibody hybridization: 1) Primary antibody incubation: an antibody dilution (PBS containing 1% BSA, 2% sheep serum) was used to dilute the primary antibody at an appropriate ratio, and the primary antibody was then added dropwise to a wet box before being incubated at 37°C for 60 min or refrigerated at 4°C overnight then rinsed with PBS. 2) Secondary antibody incubation: an antibody dilution was used to dilute the fluorescent-labeled secondary antibody to an appropriate ratio, which was then added dropwise to a wet box, incubated at 37°C without light, and rinsed with PBS and distilled water. Re-staining: DAPI was incubated at 22–26℃for 10–15 min and rinsed with PBS. Fluorescence microscopy was used to observe the cells after staining. 2.3.3. Plate cloning experiment SiHa cells from the sgRNA and NC groups were retrieved, trypsin digested, and counted. The cell suspension was diluted and inoculated in six-well plates at a concentration of 500 cells/well. The cells were cultured in a cell incubator until they were visibly cloned. The DMEM was discarded, and the cells were fixed in 4% paraformaldehyde and stained with 1 mL of crystal violet for 10 min, then the staining solution was slowly washed away with running water. The clone formation rate was calculated by counting clones directly through visual observation or by counting the number of clones with > 50 cells under a microscope. The clone formation rate was calculated as follows: (number of clones / number of inoculated cells) × 100%. 2.3.4. MTT cell proliferation assay SiHa and Caski cells from the OE and NC groups were retrieved, trypsin digested, and counted. The cell density was adjusted to 2 × 10 3 cells/mL and the cells were inoculated into 96-well plates. The cells were then incubated in an incubator for 1, 2, 3, 4, and 5 days. After incubation, the DMEM was discarded and 5 mg/mL MTT was added to each well. DMSO was added to dissolve the methanogenic pellet, the OD value at 450 nm was measured by an enzyme-labeling instrument, and the relative cell viability was calculated from the OD value to plot the cell growth curve. 2.3.5. FACS single staining to detect apoptosis When the cells in each experimental group grew to approximately 70% coverage, apoptosis induced by drugs occurred, followed by trypsin digestion. A complete medium was used to resuspend dead cells into a cell suspension, which was then centrifuged, and the cell precipitate was washed with pre-cooled D-Hanks (pH = 7.2–7.4) at 4°C. The cell precipitate was washed once with 1× binding buffer, centrifuged at 1,300 rpm, and the cells were collected. Then, 1× binding buffer was used again to resuspend the cell precipitate. Subsequently, Annexin V-APC was added for staining and 1× binding buffer was replenished according to the number of cells. 2.3.6. IPA bioinformatics analysis Based on the list of genes provided, the genes in the annex were predicted to interact with the target gene ZBTB5 and the gene network was mapped. 2.3.7. ChIP screening combined with target genes (1) Cell lysis and nuclease digestion Cells were counted and incubated overnight until they attached to the wall. Paraformaldehyde was then added to the cell medium, and the cells were incubated. The cross-linking reaction was observed under a optical microscope. First, Halt Cocktail was added, followed by Lysis Buffer 1 containing protease inhibitor then MNase Digestions Buffer and Micrococcal Nuclease. The solution was then well-mixed in a 37°C water bath for 15 min while inverting. MNase Stop Solution was added to terminate the reaction, followed by an ice bath. Lysis Buffer 2 containing protease / phosphatase inhibitor was used to resuspend the precipitate, followed by an ice bath. The precipitate was centrifuged and the supernatant was transferred to a new 1.5 mL pre-cooled centrifuge tube. (2) Western blotting for target protein expression First, 12% separation gel was configured and left to solidify after the addition of isopropyl alcohol, and the isopropyl alcohol was then aspirated. Second, the residual isopropyl alcohol was rinsed with water and the residue was aspirated with blotting paper. Then, the preconfigured 5% concentrated gel was added, and the comb was inserted. Next, it was left to stand until the concentrated gel solidified, and the comb was removed. This was followed by membrane transfer, blocking, and PBS washing, which was repeated three times. The primary antibody (diluted with PBS) and secondary antibody were incubated, and two luminescent agents were added to the kit to develop and fix the image and scan the results. (3) Chromatin immunoprecipitation The supernatant from the above step was stored at − 20°C and used as the input by adding to a centrifuge tube containing 1 × immunoprecipitation (IP) dilution buffer. For each IP, diluted lysate was added to the plug spin column, the primary antibody was added, and the IP was evenly mixed overnight at 4°C. Positive control IP: H3K27ac antibody; negative control IP: normal rabbit IgG; target-specific IP: antibody. ChIP grade protein A/G plus agarose were added to each IP, which were then incubated at 4°C in a shaker. The collected material was discarded after centrifugation, wash buffer 1 was added, and IP was centrifuged. Wash buffer 2 was then added and IP was centrifuged, and, finally, wash buffer 3 was added and IP was centrifuged. (4) Elution of chromatin immunoprecipitation The plug column was returned into the centrifuge tube and 1× IP elution buffer was added to wash the resin. This was then incubated at 65°C, the magnetic beads were resuspended, and the solution was centrifuged. Next, 5 M NaCl and 20 mg/mL proteinase K were added and mixed. Afterward, the input was thawed and the elution buffer was added to IP, and 5 M NaCl and 20 mg/mL proteinase K were added. Finally, the IP was placed into a 65°C metal bath. (5) DNA recovery DNA binding buffer was added to a DNA Clean-Up column and centrifuged. Then, remaining samples were added to the same DNA Clean-Up column and this was centrifuged again. The column was then placed in a collection tube, and DNA column wash buffer was added before centrifuging. Finally, DNA elution solution was added, and this was then centrifuged before performing positive qPCR and input and targeted sequencing. 2.3.8. Co-IP screening combined with mass spectrometry (1) Construction of a stable cell line with 3× FLAG-ZBTB5 expression Through PCR, the gene sequence encoding the 3× FLAG tag was fused to the 5'-end of the ZBTB5 gene (target) to be studied to generate the 3× FLAG-target fusion gene. The 3× FLAG-target fusion gene was inserted into the corresponding lentiviral expression vector to prepare the expression plasmid, p3×FLAG-target. The lentiviral empty vector and p3× FLAG-target plasmid were co-transfected with helper plasmids, respectively, to harvest and prepare lentiviral particles; namely, lenti-control and lenti-3× FLAG-target. These were then infected with the target cells to be studied, respectively, and the screening of stable cell lines was performed using puromycin, i.e., control cells ("NC") and 3× FLAG-target cells (“OE”). (2) Western blotting to identify the stable cell lines NC and OE stable cell lines were cultured in large numbers, and the cells above 80% confluence were collected. RIPA lysis buffer was used to lyse the cells and sonicate them on ice, and the supernatant was collected after centrifugation of the whole cell lysate for BCA protein quantification. Aliquots were loaded for western blotting of 3× FLAG-ZBTB5 to determine the protein expression levels. (3) Co-IP pre-experiment Since there was a 3× FLAG tag fused at the N-terminal end of the target gene, 3× FLAG-target proteins were enriched by the FLAG antibody-mediated Co-IP technique. The detailed Co-IP process was carried out according to the user manual for FLAG-beads (Sigma, A2220). (4) Co-IP formal experiment: Purification of 3× FLAG-target interaction complexes NC and OE stable cell lines were cultured in large numbers, respectively, and culture plates and cells above 80% confluence were collected. RIPA lysis buffer was used to lyse the cells and sonicate them on ice, and the supernatant total cell protein was collected by centrifugation for BCA protein quantification. Equal amounts of both groups of proteins were taken, and Co-IP was performed with FLAG-beads (Sigma) to purify the protein complexes. Co-IP samples were subjected to SDS-PAGE and Coomassie Brilliant Blue staining. (5) In-gel enzymatic digestion and shotgun-mass spectrometry protein identification According to each sample lane to be measured on gummed paper in Part V, the gummed tapes were cut off, and the proteins in each gummed tape sample were cleaved into peptides by enzymatic digestion with trypsin separately. Each peptide sample was identified by LC-MS. The raw files of the mass spectra of each sample were checked by PD/MASCOT software, and the library was checked to obtain the protein identification results. 2.3.9. WB validation of Co-IP screening proteins The above SiHa cells with ZBTB5 overexpression were centrifuged and the protein concentration was determined using a BCA protein quantification kit. A 10% separating gel and 4% stacking gel were prepared and sampled for electrophoresis. The stacking gel was set at 70 V and the separating gel at 100 V. Electrophoresis was terminated when bromophenol blue electrophoresis was applied to the bottom of the gel. The membrane was electrotransferred for 60 min and the proteins were transferred to the PVDF membrane. Then, the membrane was blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated with diluted primary antibodies (mouse anti-human Flag, rabbit anti-human U2AF2, rabbit anti-human RBM5, rabbit anti-human ILK, rabbit anti-human ENAH, mouse anti-human JUP, rabbit anti-human RELA/P65, mouse anti-human SQSTM1, rabbit anti-human YY1, rabbit anti-human STIM1, rabbit anti-human Integrin alpha V, rabbit anti-human EED, rabbit anti-human SUGT1, and rabbit anti-human NFKB1; 1:5,000–1:500) overnight at 4°C. Secondary human antibody (corresponding anti-mouse or anti-rabbit secondary antibody; 1:10,000) was added and incubated with the membrane together at room temperature for 1 h. After incubation, LAS-3000 was used to observe the transfer effect under exposure. The appearance of a chromogenic strip was considered to be a positive result, and its absence was considered negative. The clearest image of the band was selected for analysis. 2.4. Statistical methods SPSS 20.0 software was used for statistical analysis and plotting, and the measurement data are expressed as the mean ± standard deviation (x ± s). A t-test was used for the comparison of means between two groups, an ANOVA was used for the comparison of means between multiple groups, and a Chi-squared test was used for the comparison of rates between groups. Differences were considered statistically significant at p < 0.05. Analysis software included ProteomeDiscoverer 2.1 (Thermo, USA), Perseus 1.3 (Max Planck Institute of Biochemistry in Martinsried, Germany), MASCOT 2.5, and R version 3.0.3 (Matrix Science, USA). 3. Results 3.1. Involvement of ZBTB5 in the mechanism of PTX resistance in cervical squamous cell cancer 3.1.1. Use of cervical squamous carcinoma cell line (SiHa cells) as an in vitro model of ZBTB5 affecting PTX resistance Using the CRISPR/Cas9 gene-editing tool, six sgRNAs were designed against the ZBTB5 gene, and plasmids were constructed, packaged with lentivirus, and infected the human cervical cancer cell line to obtain SiHa cell lines with knockdown of ZBTB5. The ploidy data of each group of cells were analyzed by treating each of the six SiHa cell lines with 4 ng/mL PTX with drug solvent for 120 h (see Table 1). The optimal ZBTB5 knockdown target was selected, and knockdown of ZBTB5 significantly reduced the PTX resistance of SiHa cervical squamous carcinoma cells. Table 1 Assessment of optimal ZBTB5 knockdown targets using SiHa cell lines for PTX resistance Target Number of cell doubling (120 h) Sensitization folds Notes sgRNA-NC 7.93 1.00 sgRNA-NC-PTX 2.24 ZBTB5-sgRNA(05084) 7.49 1.37 ZBTB5-sgRNA(05084)-PTX 1.54 ZBTB5-sgRNA(05085) 7.52 1.57 ZBTB5-sgRNA(05085)-PTX 1.35 ZBTB5-sgRNA(05086) 7.5 1.16 ZBTB5-sgRNA(05086)-PTX 1.82 ZBTB5-sgRNA(05087) 7.04 1.64 Final target selection ZBTB5-sgRNA(05087)-PTX 1.21 ZBTB5-sgRNA(05088) 6.62 1.34 ZBTB5-sgRNA(05088)-PTX 1.4 ZBTB5-sgRNA(05089) 7.68 1.05 ZBTB5-sgRNA(05089)-PTX 2.07 3.1.2. Immunofluorescence shows that overexpression of ZBTB5 enhances PTX resistance in cervical squamous cell cancer by functioning in the nucleus When cultured in 4 ng/mL PTX for 14 days, normal SiHa cells had a rounded morphology and smaller pseudopods than SiHa cells with ZBTB5 overexpression, indicating that overexpression of ZBTB5 increased cell adhesion and invasion, thereby enhancing the resistance of cervical squamous SiHa cells to PTX and leading to PTX resistance. ZBTB5 was labeled with red light, and cytoplasmic red fluorescence intensity after overexpression of ZBTB5 decreased and accumulated near the punctate nucleus, indicating that the relative amount of ZBTB5 in the cytoplasm decreased, aggregated towards the nucleus, and increased PTX resistance by functioning in the nucleus (see Figure 1). 3.2. Involvement of ZBTB5 in cervical cancer cell function 3.2.1. Effect on the cell clone-forming ability To investigate the effect of ZBTB5 knockdown on the cloning of SiHa cervical squamous carcinoma cells, the effect of ZBTB5 overexpression on the clone-forming ability of cervical squamous carcinoma cells was observed using a plate clone formation assay, and the number of cell clones was counted after the above cells were cultured for 9 days, where the study cells were incubated with PTX on the fourth day. The results showed that in SiHa cells, the number of clones was 245 ± 11 in the sgCtrl group, 108 ± 8 in the sgCtrl PTX group, 227 ± 19 in the sgZBTB5 group, and 55 ± 7 in the sgZBTB5 PTX group. The difference between the sgCtrl PTX / sgCtrl ratio and the sgZBTB5 PTX / sgZBTB5 ratio was statistically significant with weaker cell cloning ability (p < 0.05) (see Figure 2). ZBTB5 knockdown inhibited cell cloning and increased sensitivity to PTX. 3.2.2. Effect on cell proliferation To investigate the effect of sgZBTB5 on the proliferation of SiHa cervical squamous carcinoma cells, two groups were incubated with PTX for 6 days and treated with MTT for 4 h. The absorbance of light at a wavelength of 490 nm over time for each group was compared in an enzyme-labeling instrument. Here, OD490 reflected the number of viable cells. As shown in Figure 3-1 and Figure 3-2, the cells in the sgZBTB5+ group were more sensitive to the drug compared to the sgCtrl+ group (p < 0.05). 3.2.3. ZBTB5 can negatively regulate the apoptosis ability of the cervical squamous carcinoma cell line SiHa under PTX (4 ng/mL) treatment conditions Compared with the control group, the apoptosis ability of SiHa cells with ZBTB5 knockdown was significantly enhanced after PTX treatment, which reduced resistance to PTX and reduced PTX resistance in SiHa cells (as shown in Figure 4). 3.3. IPA bioinformatics analysis: The ZBTB5 gene affects downstream gene expression by regulating BCL6 (see Figure 5) Through genome-wide IPA analysis, ZBTB5 may indirectly affect the expression of downstream genes through the BCL6 gene, which overexpresses to promote tumor proliferation or inhibit apoptosis, and the ZBTB5 gene is consistent with its function. 3.4. ZBTB5 knockdown can downregulate BCL6 gene expression levels (see Figure 6) The qPCR and WB results suggested that ZBTB5 knockdown downregulated BCL6 gene expression (mRNA) and that ZBTB5 was correlated with BCL6. 3.5. ChIP screening and validation of downstream genes with possible ZBTB5 action 3.5.1. ChIP detection of downstream genes with possible ZBTB5 action A ChIP assay was performed after overexpression of ZBTB5, where the ZBTB5 protein was screened, gene fragments 0~3 kb away from the transcription start site (TSS) were selected for gene sequencing, and the distribution of reads (sequenced fragments) 0~3 kb close to the TSS was determined. The peak refers to a gene fragment near the ZBTB5 protein promoter (395 kb away from the TSS). GO functional enrichment analysis was performed on peak-related genes, and these were classified according to their functions. Results of the GO functional enrichment analysis are shown in Figure 7-1, and KEGG pathway enrichment analysis was performed on peak-related genes in Figure 7-2, which showed that the BCL6 gene was in the signaling pathway of these genes. The above analyses revealed that the BCL6 gene is a downstream gene of ZBTB5 gene action, and the ZBTB5 gene affects cell proliferation and apoptosis by regulating the signaling pathway of the BCL6 gene, thus, affecting resistance to PTX-based chemotherapy in patients with cervical cancer, which is consistent with the results of IPA bioinformatics analysis (see Table 2). Table 2 BCL gene sequences that may be regulated downstream of ZBTB5 Seq Start End Width Annotation Distance t o TSS Gene chr3 187843762 187843935 174 Distal Intergenic − 98035 BCL6 transcription repressor chr3 187765104 187765282 179 Distal Intergenic − 19377 BCL6 transcription repressor chr3 187729721 187729865 145 3' UTR − 4098 BCL6 transcription repressor chr3 187725021 187725228 208 Promoter ( ≤ 1 kb) 395 BCL6 transcription repressor 3.5.2. Co-IP screening combined with mass spectrometry of target gene interaction complexes Co-IP was performed in the OE and NC groups, and further in-gel enzymatic digestion and shotgun-mass spectrometry protein identification was performed. Overall, 1,371 proteins were identified in the normal group and 1,336 in the overexpression group, and a total of 322 differential proteins were identified. Bioinformatics analysis was performed on the differential proteins to analyze 247 proteins with possible relevance and their mechanism of action, including their possible involvement in cell proliferation, metastasis, apoptosis, and invasion, as shown in Figure 8. 3.5.3. WB validation of proteins identified by Co-IP screening From the proteins identified by the above screening, 13 were selected for validation after bioinformatics analysis: U2AF2, RBM5, ILK, ENAH, JUP, RELA/P65, SQSTM1, YY1, STIM1, Integrin alpha V, EED, SUGT1, and NFKB1, among which U2AF2 was successfully detected in the input. The target bands were detected in NC and OE and, compared with NC, U2AF2 expression was significantly increased in the OE group, suggesting that the 3× Flag-ZBTB5 protein pulled down the U2AF2 protein and there may be an interaction between the two proteins (see Figure 9). 4. Discussion Cervical cancer is a disease that leads to serious health risks for women. As the main treatment for cervical cancer, PTX-based chemotherapy is pivotal in the treatment of advanced and recurrent cervical cancer. PTX-based chemotherapy regimens have been published in the National Comprehensive Cancer Network [ 5 ], International Federation of Gynecology and Obstetrics, and European Society of Medical Oncology guidelines as the standard of care for intermediate-to-advanced cervical cancer. However, in cases treated with PTX as a first-line chemotherapeutic agent, drug resistance rates in patients with intermediate-to-advanced squamous cervical cancer ranges from 20–44% [ 6 ]. Resistance to PTX in patients with cervical squamous cell carcinoma leads to disease progression and affects prognosis with a 20–30% reduction in five-year survival. Studies in the United States and Japan have reported [ 7 – 8 ] that among patients with locally advanced cervical squamous cell cancer, 20% were resistant to PTX-based neoadjuvant chemotherapy, resulting in treatment failure. PTX resistance is the main cause of treatment failure in patients with cervical squamous cell cancer. Therefore, it has become a critical clinical issue with utmost concern for gynecologic oncologists to explore strategies to overcome PTX resistance in cervical squamous cell cancer. The main mechanisms of chemoresistance include anti-apoptosis, pro-survival, increased intracellular drug excretion, cellular detoxification, enhanced DNA repair function, and dysregulation of microtubule dynamic balance. To date, the reported antitumor mechanisms of PTX mainly include (1) PTX binding to intracellular microtubules, enhancing microtubule stability, causing cell cycle arrest and making cells unable to divide, which eventually leads to the apoptosis of tumor cells [ 9 ]; (2) PTX inhibiting cell proliferation, migration, and infiltration by inhibiting the expression of androgen receptor (AR) and limiting AR signaling pathway viability to kill tumor cells [ 10 ]; (3) PTX promoting tumor cell apoptosis by inhibiting the expression of the anti-apoptotic BLC-2 proteins, promoting the phosphorylation of BCL-2, and inhibiting the Bax/Caspase 3 signaling pathway [ 11 ]. The BCL-6 proto-oncogene belongs to the anti-apoptotic family, and the main function of BCL-6 is transcriptional repression. The target genes regulated by BCL-6 are mainly associated with cell activation, differentiation, and proliferation. The function of BCL-6 is to repress transcription, thus, inhibits apoptosis. The human BCL-6 proto-oncogene is located in the 3q27 region of the chromosome, is 26 kb in length, and contains 10 exons and seven introns. Bcl-6 is widely distributed in cell activation, differentiation, cell cycle regulation, DNA damage repair, and other processes. Target genes of BCL-6 competitively repress STAT6, CD23, and IgE as well as repress CD69, CD44, Blimp-1, p27kip1, and CyclinD2. In the early stage, our research team successfully knocked out ZBTB5 gene in human cervical squamous cell carcinoma cell line SiHa by CRISPR/Cas9 gene editing technology [ 12 ]. ZBTB5 is a newly discovered BTB / POK protein that is mainly located in the nucleus and is expressed in all normal tissues of the body, especially in tumor tissues, where it is highly expressed and may be an oncogene. ZBTB5 possesses a POZ domain and two unique zinc-finger domains, which can be involved in gene regulation as a transcription factor. ZBTB5 represses the p21 gene during the cell cycle and promotes the proliferation and growth of tumor cells, but the regulatory mechanism of ZBTB5 for other genes is unclear [ 13 ]. According to TCGA database, ZBTB5 is highly expressed in a variety of tumor tissues, including bladder and ureteral, gall bladder, intestinal, head and neck, esophageal, and renal cancers ( http://starbase.sysu.edu.cn/panGeneDiffExp.php ). ZBTB5 is also significantly highly expressed in cervical cancer, especially in young Asian patients with cervical squamous cell cancer, and affects prognosis. Recent studies have reported that ZBTB5 is a proto-oncogene that stimulates cell proliferation. zBTB5 represses p21 / CDKN1A by competing with p53 and recruiting complexes, and the oncoprotein c-Myc directly activates the transcription of ZBTB5. In the absence of p53, acetylated ZBTB5 K597 interacts with p300 and activates NOXA transcription, thereby inducing apoptosis. In contrast, in the presence of p53, ZBTB5 interacts with p53 and blocks ZBTB5 K597 acetylation. Thus, the cell fate determination by c-Myc depends on ZBTB5, p53, and p300, as well as the acetylation of ZBTB5K597 [ 14 ]. In this study, it was clarified that ZBTB5 functions in the nucleus, and that ZBTB5 knockdown inhibits cell cloning, cell proliferation, significantly enhances apoptosis in cervical squamous carcinoma cells in PTX, and reduces PTX resistance in SiHa cells. The ChIP assay after ZBTB5 overexpression suggested that ZBTB5 is involved in PTX resistance in cervical squamous cell carcinoma by regulating BCL6 , for which ZBTB5 may be the promoter, which is consistent with the results of IPA bioinformatics analysis. After Co-IP, further screening and identification of ZBTB5-interacting proteins showed that there was protein-to-protein interaction between ZBTB5 and U2AF2, and it was hypothesized that ZBTB5 might form a transcriptional complex with U2AF2 to regulate BCL6 to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma. Whether there was a similar relationship between BCL-6 and ZBTB5 to that between c-Myc and ZBTB5 still requires to be confirmed by further basic studies. 5. Conclusion In summary, this study preliminarily speculated that ZBTB5 may form a transcriptional complex with U2AF2 to regulate BCL6 to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma, and this speculation needs to be further confirmed by subsequent clinical and lab-based experiments. Abbreviations PTX paclitaxel IP immunoprecipitation ZBTB5 Zinc finger and BTB domain-containing 5 Declarations Acknowledgments (1)We are grateful to all the reviewers and editors who provided comments that substantially improved the manuscript. (2)We are grateful to Shanghai GeneChem Co., Ltd for the support of experiment progress during the COVID-19 pandemic in China. Authors’ contributions Conceptualization: H.Y., W.Y.M.; Data curation: Z.J.J, H.Y.; Formal analysis: H.Y.; Funding acquisition: H.Y., W.Y.M.; Methodology: J.C., L.Y., W.M.; Project administration: W.Y.M.; Resources: H.S.B., X.J.Q, W.Y.F.; Supervision: W.Y.M.; Writing-original draft: H.Y.; Writing review & editing: W.Y., W.Y.M. The author(s) read and approved the fnal manuscript. Authors’ information Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article/supplementary materials. Futher inquiries can be directed to the corresponding authors. Ethics approval and consent to participate Not applicable. There is no studies involving human participants. Consent for publication Not applicable. Competing interests No potential confict of interest relevant to this article was reported Funding Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (grant No.ZYLX201705). National Natural Science Foundation of China (grand No.82102711). References Calys-Tagoe BNL, Aheto JMK, Mensah G, et al. Cervical cancer screening practices among women in Ghana: evidence from wave 2 of the WHO study on global AGEing and adult health [J]. BMC Womens Health, 2020, 20 (1): 49. de Martel C, Georges D, Bray F, et al. Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis [J]. Lancet Glob Health, 2020, 8 (2): e180-e190. Duan R, Qiao Y, Clifford G, et al. Cancer burden attributable to human papillomavirus infection by sex, cancer site, age, and geographical area in China [J]. Cancer Med, 2020, 9 (1): 374-384. Arbyn M, Weiderpass E, Bruni L, et al. Estimates of incidence and mortality of cervical cancer in 2018: A worldwide analysis [J]. Lancet Glob Health, 2020, 8 (2), e191-e203. [5] Koh WJ, Abu-Rustum NR, Bean S, et al. Cervical cancer, version 3.2019, NCCN clinical practice guidelines in oncology [J]. J Natl Compr Canc Netw, 2019, 17 (1) : 64-84. Kastritis E, Bamias A, Efstathiou E, et al. The outcome of advanced or recurrent non-squamous carcinoma of the uterine cervix after platinum based combination chemotherapy [J]. Gynecol Oncol, 2005, 99(2): 376-382. Mori T, Makino H, Okubo T, et al. Multi-institutional phase II study of neoadjuvant irinotecan and nedaplatin followed by radical hysterectomy and the adjuvant chemotherapy for locally advanced, bulky uterine cervical cancer: A Kansai Clinical Oncology Group study (KCOG-G1201) [J]. J Obstet Gynaecol Res, 2019, 45(3): 671-678. de la Torre M. Neoadjuvant chemotherapy in woman with early or locally advanced cervical cancer [J]. Rep Pract Oncol Radiother, 2018, 23(6): 528-532. Qi Z, Yin L, Xu Y. Pegylated liposomal paclitaxel induces ovarian cancer cell apoptosis via TNF induced ERK/AKT signaling pathway [J]. Mol Med Rep, 2018, 17(6): 7497-7504. Shin S, Woo S, Yim H. Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer [J]. Ther Adv Med Oncol, 2019, eCollection 2019. Shi X, Sun X. Regulation of paclitaxel activity by microtubule-associated proteins in cancer chemotherapy [J]. Cancer Chemother Pharmacol, 2017, 80(5): 909-917. Wei W, He Y, Wu YM. Identification of genes associated with SiHa cell sensitivity to paclitaxel by CRISPR-Cas9 knockout screening [J].Int J Clin Exp Pathol, 2018, 11(3): 1972-1978. Koh DI, Choi WI, Jeon BN, et al. A novel POK family transcription factor, ZBTB5, represses transcription of p21CIP1 gene [J]. J Biol Chem, 2009, 284(30): 19856-19866. Choi SH, Koh DI, Ahn H, et al. Cell fate decisions by c-Myc depend on ZBTB5 and p53[J]. Biochem Biophys Res Commun, 2020, 533(4): 1247-1254. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2471463","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":167389018,"identity":"637f4c42-2bc6-4d00-8272-fd91f7c6d8e9","order_by":0,"name":"Yue He","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"He","suffix":""},{"id":167389019,"identity":"0056a845-1c16-45ed-95c1-7b781ac38330","order_by":1,"name":"Jing-Jing Zhang","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing-Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":167389021,"identity":"3dab56b1-1895-4d17-b73d-442f7e159e68","order_by":2,"name":"Jian-Qing Xu","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jian-Qing","middleName":"","lastName":"Xu","suffix":""},{"id":167389023,"identity":"98994084-5d7f-4494-b7cd-057939ef9578","order_by":3,"name":"Yun-Fan Wang","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yun-Fan","middleName":"","lastName":"Wang","suffix":""},{"id":167389026,"identity":"9f55b3b6-e45a-4d64-bdaf-5be7bddc38bc","order_by":4,"name":"Chen Ji","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University. 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Beijing Maternal and Child Health Care Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yu-Mei","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2023-01-12 13:59:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2471463/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2471463/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31653734,"identity":"03ff8395-9098-4fe7-85ac-a47c37de3e26","added_by":"auto","created_at":"2023-01-16 21:56:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":477295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSiHa cell cultured in PTX or non-PTX\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/392ff91baa3818f72792dd7f.png"},{"id":31654658,"identity":"830488ed-3a90-4560-b869-4a479100a10e","added_by":"auto","created_at":"2023-01-16 22:20:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":454868,"visible":true,"origin":"","legend":"\u003cp\u003eCell cloning assay to detect the effect of ZBTB5 overexpression on SiHa cell clones\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/1816dc14a2a2ce386119f252.png"},{"id":31653737,"identity":"e02f07a5-0d41-413e-9c08-01a05cda14ec","added_by":"auto","created_at":"2023-01-16 21:56:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40300,"visible":true,"origin":"","legend":"\u003cp\u003eMTT assay to detect the effect of sgZTBTB5 on the proliferation of SiHa cells\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/25e979f1340ab5d6b430bdae.png"},{"id":31653735,"identity":"7a278f78-820d-407e-9c13-3ac743b62f63","added_by":"auto","created_at":"2023-01-16 21:56:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25316,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ZBTB5 knockdown on the apoptosis ability of the SiHa cell line after PTX treatment\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/1cb709a58b461434c938c02b.png"},{"id":31654774,"identity":"906e5980-12cf-4438-9eaf-cf2b891dabaf","added_by":"auto","created_at":"2023-01-16 22:28:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247410,"visible":true,"origin":"","legend":"\u003cp\u003eGenome-wide IPA analysis of ZBTB5 acting on downstream genes\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/35472ec4e231e5381dfa43bd.png"},{"id":31654339,"identity":"d825eabc-dd40-4ced-845e-753be91fc9e5","added_by":"auto","created_at":"2023-01-16 22:12:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13644,"visible":true,"origin":"","legend":"\u003cp\u003eBCL6 mRNA expression levels in the cervical squamous carcinoma cell line SiHa with ZBTB5 knockdown\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/a0f07ec41c3ae2ba89f7d9ce.png"},{"id":31653736,"identity":"839bed8e-f7bb-464f-a4e5-595b57591dc5","added_by":"auto","created_at":"2023-01-16 21:56:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":140044,"visible":true,"origin":"","legend":"\u003cp\u003eGO functional enrichment analysis of peak-related genes\u003c/p\u003e\n\u003cp\u003eKEGG pathway enrichment analysis of peak-related genes\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/7416311c4baa9f63f71af246.png"},{"id":31653982,"identity":"ca7f526d-f294-445f-82a1-33abb7b9c551","added_by":"auto","created_at":"2023-01-16 22:04:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":237599,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics analysis of target proteins screened by Co-IP combined with mass spectrometry\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/cd7f3c832afe8805e4c8c8ac.png"},{"id":31653742,"identity":"adbb28d8-5716-4aca-8b62-1734aa9904a8","added_by":"auto","created_at":"2023-01-16 21:56:06","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":168247,"visible":true,"origin":"","legend":"\u003cp\u003eWB validation of the proteins identified by Co-IP screening\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/426944266beb82a1c9fb1b38.png"},{"id":31654776,"identity":"9abb30e1-aee4-44b7-b5f2-5ea7057104c1","added_by":"auto","created_at":"2023-01-16 22:28:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2505053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2471463/v1/4afcd885-592f-4fa2-8a9d-6e585febfc9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"ZBTB5 enhances the resistance of cervical cancer to paclitaxel by regulating BCL6","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCervical cancer is the fourth most common malignant tumor affecting women's health worldwide, 90% of which are cases of cervical squamous cell cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to data provided in 2018, there are 569,800 new cases of cervical cancer and 311,400 new deaths as a result of cervical cancer per year worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. China has the highest number of new cases and deaths [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], with 131,500 new cases and 53,000 new deaths per year, accounting for 18.4% of female cancer deaths in China [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In underdeveloped countries, the 5-year survival rate of patients with cervical cancer is \u0026lt;\u0026thinsp;50%, and the survival rate for patients with stage III to IV cervical cancer is only 5\u0026ndash;15%, making it a serious threat to women's lives [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among the clinical treatment options for cervical cancer, paclitaxel (PTX) is the first-line chemotherapy agent, which is widely used in patients with intermediate and advanced cervical squamous cell cancer and plays an important role in chemotherapy for cervical squamous cell cancer. PTX resistance is the main cause of treatment failure in patients undergoing chemotherapy for cervical squamous cell cancer, and investigating strategies to overcome PTX resistance in cervical squamous cell cancer has become the most critical clinical subject for gynecologic oncologists. In this study, ZBTB5 was proposed, for the first time, as a transcription factor that regulates BCL6 to influence the PTX resistance of cervical squamous cell cancer, providing a new target for the precise treatment of PTX resistance in patients with cervical squamous cell cancer and giving it important clinical significance.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe human cervical squamous carcinoma cell lines (SiHa) were obtained from the Laboratory of the School of Basic Medical Sciences, Capital Medical University. Materials also included Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (HyClone, USA), fetal bovine serum (Evergreen, Hangzhou, China), PTX (Perilla frutescens, Beijing Union Pharmaceutical Factory, Lot number: 141101), ZBTB5 overexpression and ZBTB5 sgRNA lentiviruses (Shanghai Genechem, China), RIPA lysis buffer, NP-40 lysis buffer (Shanghai Dingguo Biotechnology), prestained protein marker (Thermo, USA), Apoptosis kit (eBioscience\u0026trade; Annexin V\u0026ensp;USA), paraformaldehyde (Sigma, USA), ChIP kit (Pierce, USA), qPCR kit (Promega, USA), BCA protein assay kit (Beyotime, China), ANTI-FLAG\u0026reg; M2 Affinity Gel (Sigma, USA), 3X FLAG peptide (Sigma, USA), ECL-PLUS/kit (Thermo, USA), X-ray film developer powder (Shanghai Guanlong Photographic Materials Company, China), iodoacetamide (Sigma, USA), dithiothreitol (Sigma, USA), methanol, formic acid (Thermo, USA), and ultrafiltration centrifuge tubes (Sartorius, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Instruments\u003c/h2\u003e \u003cp\u003eInstruments in the present study included a fluorescence microscope (Olympus), CO\u003csub\u003e2\u003c/sub\u003e incubator (Sanyo), inverted microscope (Shanghai Caikon Optical Instruments, China), centrifuges (Thermo Fisher Scientific, China; Eppendorf, USA), biosafety cabinet (Shanghai Zhen Zi Chuang Air Purification Equipment, China), SDS-PAGE protein electrophoresis instrument (Shanghai Tanon, China), protein transfer instrument (Shanghai Tanon, China), flow cytometer (Millipore, USA), metal bath (Hangzhou Miu Instruments, China), qPCR instrument (Bio-Rad, USA), high-speed frozen centrifuge, Easy-nLC chromatography system, Q Exactive Plus mass spectrometer (Thermo, USA), compact thermostatic mixer, rotating mixer (Dalong, China), and constant temperature incubator (Shanghai Jinghong Experiment Equipment, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Methods\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Cell culture\u003c/h2\u003e \u003cp\u003eSiHa cells transfected with the ZBTB5 overexpression lentivirus, ZBTB5 sgRNA lentivirus, and control lentivirus were placed in the DMEM containing 10% fetal bovine serum and cultured in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. The DMEM was changed every 2\u0026ndash;3 days, and cells in the logarithmic growth phase were taken for the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Immunofluorescence\u003c/h2\u003e \u003cp\u003eInfected cells in the logarithmic phase were formed into 1\u0026ndash;2\u0026times;10\u003csup\u003e4\u003c/sup\u003e single cell suspensions, inoculated in 96-well glass-bottom plates (i.e., special culture plates made for confocal laser scanning), placed in a CO\u003csub\u003e2\u003c/sub\u003e incubator, and incubated until the cells grew into a monolayer. Cell fixation: the plates were washed twice with PBS, fixed with 4% paraformaldehyde, and rinsed with PBS. Permeabilization: cells were perforated with 0.2% Triton, rinsed twice in PBS, and blocked. Antibody hybridization: 1) Primary antibody incubation: an antibody dilution (PBS containing 1% BSA, 2% sheep serum) was used to dilute the primary antibody at an appropriate ratio, and the primary antibody was then added dropwise to a wet box before being incubated at 37\u0026deg;C for 60 min or refrigerated at 4\u0026deg;C overnight then rinsed with PBS. 2) Secondary antibody incubation: an antibody dilution was used to dilute the fluorescent-labeled secondary antibody to an appropriate ratio, which was then added dropwise to a wet box, incubated at 37\u0026deg;C without light, and rinsed with PBS and distilled water. Re-staining: DAPI was incubated at 22\u0026ndash;26℃for 10\u0026ndash;15 min and rinsed with PBS. Fluorescence microscopy was used to observe the cells after staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Plate cloning experiment\u003c/h2\u003e \u003cp\u003eSiHa cells from the sgRNA and NC groups were retrieved, trypsin digested, and counted. The cell suspension was diluted and inoculated in six-well plates at a concentration of 500 cells/well. The cells were cultured in a cell incubator until they were visibly cloned. The DMEM was discarded, and the cells were fixed in 4% paraformaldehyde and stained with 1 mL of crystal violet for 10 min, then the staining solution was slowly washed away with running water. The clone formation rate was calculated by counting clones directly through visual observation or by counting the number of clones with \u0026gt;\u0026thinsp;50 cells under a microscope. The clone formation rate was calculated as follows: (number of clones / number of inoculated cells) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. MTT cell proliferation assay\u003c/h2\u003e \u003cp\u003eSiHa and Caski cells from the OE and NC groups were retrieved, trypsin digested, and counted. The cell density was adjusted to 2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/mL and the cells were inoculated into 96-well plates. The cells were then incubated in an incubator for 1, 2, 3, 4, and 5 days. After incubation, the DMEM was discarded and 5 mg/mL MTT was added to each well. DMSO was added to dissolve the methanogenic pellet, the OD value at 450 nm was measured by an enzyme-labeling instrument, and the relative cell viability was calculated from the OD value to plot the cell growth curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5. FACS single staining to detect apoptosis\u003c/h2\u003e \u003cp\u003eWhen the cells in each experimental group grew to approximately 70% coverage, apoptosis induced by drugs occurred, followed by trypsin digestion. A complete medium was used to resuspend dead cells into a cell suspension, which was then centrifuged, and the cell precipitate was washed with pre-cooled D-Hanks (pH\u0026thinsp;=\u0026thinsp;7.2\u0026ndash;7.4) at 4\u0026deg;C. The cell precipitate was washed once with 1\u0026times; binding buffer, centrifuged at 1,300 rpm, and the cells were collected. Then, 1\u0026times; binding buffer was used again to resuspend the cell precipitate. Subsequently, Annexin V-APC was added for staining and 1\u0026times; binding buffer was replenished according to the number of cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6. IPA bioinformatics analysis\u003c/h2\u003e \u003cp\u003eBased on the list of genes provided, the genes in the annex were predicted to interact with the target gene ZBTB5 and the gene network was mapped.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.7. ChIP screening combined with target genes\u003c/h2\u003e\u003cp\u003e \u003cb\u003e(1) Cell lysis and nuclease digestion\u003c/b\u003e \u003c/p\u003e\u003cp\u003eCells were counted and incubated overnight until they attached to the wall. Paraformaldehyde was then added to the cell medium, and the cells were incubated. The cross-linking reaction was observed under a optical microscope. First, Halt Cocktail was added, followed by Lysis Buffer 1 containing protease inhibitor then MNase Digestions Buffer and Micrococcal Nuclease. The solution was then well-mixed in a 37\u0026deg;C water bath for 15 min while inverting. MNase Stop Solution was added to terminate the reaction, followed by an ice bath. Lysis Buffer 2 containing protease / phosphatase inhibitor was used to resuspend the precipitate, followed by an ice bath. The precipitate was centrifuged and the supernatant was transferred to a new 1.5 mL pre-cooled centrifuge tube.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(2) Western blotting for target protein expression\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFirst, 12% separation gel was configured and left to solidify after the addition of isopropyl alcohol, and the isopropyl alcohol was then aspirated. Second, the residual isopropyl alcohol was rinsed with water and the residue was aspirated with blotting paper. Then, the preconfigured 5% concentrated gel was added, and the comb was inserted. Next, it was left to stand until the concentrated gel solidified, and the comb was removed. This was followed by membrane transfer, blocking, and PBS washing, which was repeated three times. The primary antibody (diluted with PBS) and secondary antibody were incubated, and two luminescent agents were added to the kit to develop and fix the image and scan the results.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(3) Chromatin immunoprecipitation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe supernatant from the above step was stored at \u0026minus;\u0026thinsp;20\u0026deg;C and used as the input by adding to a centrifuge tube containing 1 \u0026times; immunoprecipitation (IP) dilution buffer. For each IP, diluted lysate was added to the plug spin column, the primary antibody was added, and the IP was evenly mixed overnight at 4\u0026deg;C. Positive control IP: H3K27ac antibody; negative control IP: normal rabbit IgG; target-specific IP: antibody.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eChIP grade protein A/G plus agarose were added to each IP, which were then incubated at 4\u0026deg;C in a shaker. The collected material was discarded after centrifugation, wash buffer 1 was added, and IP was centrifuged. Wash buffer 2 was then added and IP was centrifuged, and, finally, wash buffer 3 was added and IP was centrifuged.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003e(4) Elution of chromatin immunoprecipitation\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe plug column was returned into the centrifuge tube and 1\u0026times; IP elution buffer was added to wash the resin. This was then incubated at 65\u0026deg;C, the magnetic beads were resuspended, and the solution was centrifuged. Next, 5 M NaCl and 20 mg/mL proteinase K were added and mixed. Afterward, the input was thawed and the elution buffer was added to IP, and 5 M NaCl and 20 mg/mL proteinase K were added. Finally, the IP was placed into a 65\u0026deg;C metal bath.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(5) DNA recovery\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDNA binding buffer was added to a DNA Clean-Up column and centrifuged. Then, remaining samples were added to the same DNA Clean-Up column and this was centrifuged again. The column was then placed in a collection tube, and DNA column wash buffer was added before centrifuging. Finally, DNA elution solution was added, and this was then centrifuged before performing positive qPCR and input and targeted sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.8. Co-IP screening combined with mass spectrometry\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(1) Construction of a stable cell line with 3\u0026times; FLAG-ZBTB5 expression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThrough PCR, the gene sequence encoding the 3\u0026times; FLAG tag was fused to the 5'-end of the ZBTB5 gene (target) to be studied to generate the 3\u0026times; FLAG-target fusion gene. The 3\u0026times; FLAG-target fusion gene was inserted into the corresponding lentiviral expression vector to prepare the expression plasmid, p3\u0026times;FLAG-target. The lentiviral empty vector and p3\u0026times; FLAG-target plasmid were co-transfected with helper plasmids, respectively, to harvest and prepare lentiviral particles; namely, lenti-control and lenti-3\u0026times; FLAG-target. These were then infected with the target cells to be studied, respectively, and the screening of stable cell lines was performed using puromycin, i.e., control cells (\"NC\") and 3\u0026times; FLAG-target cells (\u0026ldquo;OE\u0026rdquo;).\u003c/p\u003e \u003cp\u003e \u003cb\u003e(2) Western blotting to identify the stable cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNC and OE stable cell lines were cultured in large numbers, and the cells above 80% confluence were collected. RIPA lysis buffer was used to lyse the cells and sonicate them on ice, and the supernatant was collected after centrifugation of the whole cell lysate for BCA protein quantification. Aliquots were loaded for western blotting of 3\u0026times; FLAG-ZBTB5 to determine the protein expression levels.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(3) Co-IP pre-experiment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince there was a 3\u0026times; FLAG tag fused at the N-terminal end of the target gene, 3\u0026times; FLAG-target proteins were enriched by the FLAG antibody-mediated Co-IP technique. The detailed Co-IP process was carried out according to the user manual for FLAG-beads (Sigma, A2220).\u003c/p\u003e \u003cp\u003e \u003cb\u003e(4) Co-IP formal experiment: Purification of 3\u0026times; FLAG-target interaction complexes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNC and OE stable cell lines were cultured in large numbers, respectively, and culture plates and cells above 80% confluence were collected. RIPA lysis buffer was used to lyse the cells and sonicate them on ice, and the supernatant total cell protein was collected by centrifugation for BCA protein quantification. Equal amounts of both groups of proteins were taken, and Co-IP was performed with FLAG-beads (Sigma) to purify the protein complexes. Co-IP samples were subjected to SDS-PAGE and Coomassie Brilliant Blue staining.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(5) In-gel enzymatic digestion and shotgun-mass spectrometry protein identification\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAccording to each sample lane to be measured on gummed paper in Part V, the gummed tapes were cut off, and the proteins in each gummed tape sample were cleaved into peptides by enzymatic digestion with trypsin separately. Each peptide sample was identified by LC-MS. The raw files of the mass spectra of each sample were checked by PD/MASCOT software, and the library was checked to obtain the protein identification results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.3.9. WB validation of Co-IP screening proteins\u003c/h2\u003e \u003cp\u003eThe above SiHa cells with ZBTB5 overexpression were centrifuged and the protein concentration was determined using a BCA protein quantification kit. A 10% separating gel and 4% stacking gel were prepared and sampled for electrophoresis. The stacking gel was set at 70 V and the separating gel at 100 V. Electrophoresis was terminated when bromophenol blue electrophoresis was applied to the bottom of the gel. The membrane was electrotransferred for 60 min and the proteins were transferred to the PVDF membrane. Then, the membrane was blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated with diluted primary antibodies (mouse anti-human Flag, rabbit anti-human U2AF2, rabbit anti-human RBM5, rabbit anti-human ILK, rabbit anti-human ENAH, mouse anti-human JUP, rabbit anti-human RELA/P65, mouse anti-human SQSTM1, rabbit anti-human YY1, rabbit anti-human STIM1, rabbit anti-human Integrin alpha V, rabbit anti-human EED, rabbit anti-human SUGT1, and rabbit anti-human NFKB1; 1:5,000\u0026ndash;1:500) overnight at 4\u0026deg;C. Secondary human antibody (corresponding anti-mouse or anti-rabbit secondary antibody; 1:10,000) was added and incubated with the membrane together at room temperature for 1 h. After incubation, LAS-3000 was used to observe the transfer effect under exposure. The appearance of a chromogenic strip was considered to be a positive result, and its absence was considered negative. The clearest image of the band was selected for analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical methods\u003c/h2\u003e \u003cp\u003eSPSS 20.0 software was used for statistical analysis and plotting, and the measurement data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s). A t-test was used for the comparison of means between two groups, an ANOVA was used for the comparison of means between multiple groups, and a Chi-squared test was used for the comparison of rates between groups. Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eAnalysis software included ProteomeDiscoverer 2.1 (Thermo, USA), Perseus 1.3 (Max Planck Institute of Biochemistry in Martinsried, Germany), MASCOT 2.5, and R version 3.0.3 (Matrix Science, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Involvement of ZBTB5 in the mechanism of PTX resistance in cervical squamous cell cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1. Use of cervical squamous carcinoma cell line (SiHa cells) as an in vitro model of ZBTB5 affecting PTX resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the CRISPR/Cas9 gene-editing tool, six sgRNAs were designed against the ZBTB5 gene, and plasmids were constructed, packaged with lentivirus, and infected the human cervical cancer cell line to obtain SiHa cell lines with knockdown of ZBTB5. The ploidy data of each group of cells were analyzed by treating each of the six SiHa cell lines with 4 ng/mL PTX with drug solvent for 120 h (see Table 1). The optimal ZBTB5 knockdown target was selected, and knockdown of \u003cem\u003eZBTB5\u003c/em\u003e significantly reduced the PTX resistance of SiHa cervical squamous carcinoma cells.\u003c/p\u003e\n\u003cdiv style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"width: 94%;border: none;border-collapse:collapse;margin-left:6.75pt;margin-right:6.75pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width:100.0%;border:none;border-bottom: solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:4.5pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:center;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eTable 1 Assessment of optimal \u003cem\u003eZBTB5\u003c/em\u003e knockdown targets using SiHa cell lines for PTX resistance\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:33.0pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003eTarget\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:33.0pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003eNumber of cell doubling (120 h)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:17.42%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:33.0pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003eSensitization folds\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.82%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:33.0pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003eNotes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003esgRNA-NC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;border-bottom: solid windowtext 1.0pt;padding: 0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;border-bottom: solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003esgRNA-NC-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e2.24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05084)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:20.6pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05084)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:20.6pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.54\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05085)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.52\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.57\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:24.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05085)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:24.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:21.15pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05086)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:21.15pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;border-bottom: solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:21.15pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:21.15pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:11.2pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05086)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:11.2pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.82\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05087)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;border-bottom: solid black 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;border-bottom: solid black 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cspan style=\"font-size:13px;color:black;\"\u003eFinal target selection\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:24.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05087)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;background:#FFD966;padding:0mm 5.4pt 0mm 5.4pt;height:24.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05088)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e6.62\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.34\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;border-bottom:solid black 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:23.55pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:25.6pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05088)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:25.6pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:22.35pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05089)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:22.35pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e7.68\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:17.42%;border:none;border-bottom: solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:22.35pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e1.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:16.82%;border:none;border-bottom: solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:22.35pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:36.8%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:24.5pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003eZBTB5-sgRNA(05089)-PTX\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.92%;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:24.5pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:150%;'\u003e\u003cspan style=\"font-size:13px;line-height:150%;color:black;\"\u003e2.07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:center;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2. Immunofluorescence shows that overexpression of ZBTB5 enhances PTX resistance in cervical squamous cell cancer by functioning in the nucleus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen cultured in 4 ng/mL PTX for 14 days, normal SiHa cells had a rounded morphology and smaller pseudopods than SiHa cells with ZBTB5 overexpression, indicating that overexpression of ZBTB5 increased cell adhesion and invasion, thereby enhancing the resistance of cervical squamous SiHa cells to PTX and leading to PTX resistance. ZBTB5 was labeled with red light, and cytoplasmic red fluorescence intensity after overexpression of ZBTB5 decreased and accumulated near the punctate nucleus, indicating that the relative amount of ZBTB5 in the cytoplasm decreased, aggregated towards the nucleus, and increased PTX resistance by functioning in the nucleus (see Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Involvement of ZBTB5 in cervical cancer cell function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1. Effect on the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecell clone-forming ability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of \u003cem\u003eZBTB5\u003c/em\u003e knockdown on the cloning of SiHa cervical squamous carcinoma cells, the effect of ZBTB5 overexpression on the clone-forming ability of cervical squamous carcinoma cells was observed using a plate clone formation assay, and the number of cell clones was counted after the above cells were cultured for 9 days, where the study cells were incubated with PTX on the fourth day. The results showed that in SiHa cells, the number of clones was 245 \u0026plusmn; 11 in the sgCtrl group, 108 \u0026plusmn; 8 in the sgCtrl PTX group, 227 \u0026plusmn; 19 in the sgZBTB5 group, and 55 \u0026plusmn; 7 in the sgZBTB5 PTX group. The difference between the sgCtrl PTX / sgCtrl ratio and the sgZBTB5 PTX / sgZBTB5 ratio was statistically significant with weaker cell cloning ability (p \u0026lt; 0.05) (see Figure 2). ZBTB5 knockdown inhibited cell cloning and increased sensitivity to PTX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2. Effect on cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of sgZBTB5 on the proliferation of SiHa cervical squamous carcinoma cells, two groups were incubated with PTX for 6 days and treated with MTT for 4 h. The absorbance of light at a wavelength of 490 nm over time for each group was compared in an enzyme-labeling instrument. Here, OD490 reflected the number of viable cells. As shown in Figure 3-1 and Figure 3-2, the cells in the sgZBTB5+ group were more sensitive to the drug compared to the sgCtrl+ group (p \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.3. ZBTB5 can negatively regulate the apoptosis ability of the cervical squamous carcinoma cell line SiHa under PTX (4 ng/mL) treatment conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the control group, the apoptosis ability of SiHa cells with ZBTB5 knockdown was significantly enhanced after PTX treatment, which reduced resistance to PTX and reduced PTX resistance in SiHa cells (as shown in Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. IPA bioinformatics analysis: The ZBTB5 gene affects downstream gene expression by regulating BCL6 (see Figure 5)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough genome-wide IPA analysis, ZBTB5 may indirectly affect the expression of downstream genes through the BCL6 gene, which overexpresses to promote tumor proliferation or inhibit apoptosis, and the ZBTB5 gene is consistent with its function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. ZBTB5 knockdown can downregulate BCL6 gene expression levels (see Figure 6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe qPCR and WB results suggested that ZBTB5 knockdown downregulated BCL6 gene expression (mRNA) and that ZBTB5 was correlated with BCL6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. ChIP screening and validation of downstream genes with possible ZBTB5 action\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1. ChIP detection of downstream genes with possible ZBTB5 action\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;A ChIP assay was performed after overexpression of ZBTB5, where the ZBTB5 protein was screened, gene fragments 0~3 kb away from the transcription start site (TSS) were selected for gene sequencing, and the distribution of reads (sequenced fragments) 0~3 kb close to the TSS was determined. The peak refers to a gene fragment near the ZBTB5 protein promoter (395 kb away from the TSS). GO functional enrichment analysis was performed on peak-related genes, and these were classified according to their functions. Results of the GO functional enrichment analysis are shown in Figure 7-1, and KEGG pathway enrichment analysis was performed on peak-related genes in Figure 7-2, which showed that the BCL6 gene was in the signaling pathway of these genes. The above analyses revealed that the BCL6 gene is a downstream gene of ZBTB5 gene action, and the ZBTB5 gene affects cell proliferation and apoptosis by regulating the signaling pathway of the BCL6 gene, thus, affecting resistance to PTX-based chemotherapy in patients with cervical cancer, which is consistent with the results of IPA bioinformatics analysis (see Table 2).\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width:426.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;height:25.35pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eTable 2 BCL gene sequences that may be regulated downstream of ZBTB5\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eSeq\u003c/span\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eStart\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eEnd\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:52.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eWidth\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:68.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eAnnotation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:57.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eDistance\u0026nbsp;\u003c/span\u003e\u003cspan style=\"color:black;\"\u003et\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eo TSS\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eGene\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003echr3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187843762\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187843935\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:52.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e174\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:68.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003eDistal Intergenic\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:57.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:baseline;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e\u0026minus;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e98035\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003eBCL6 transcription repressor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003echr3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187765104\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187765282\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:52.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e179\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:68.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003eDistal Intergenic\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:57.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e\u0026minus;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e19377\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003eBCL6 transcription repressor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003echr3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187729721\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family:SimSun;color:black;\"\u003e187729865\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:52.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e145\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:68.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e3\u0026apos; UTR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:57.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e\u0026minus;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003e4098\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:black;\"\u003eBCL6 transcription repressor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:37.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003echr3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:71.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family: SimSun;color:red;\"\u003e187725021\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;vertical-align:middle;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;font-family: SimSun;color:red;\"\u003e187725228\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:52.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e208\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:68.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003ePromoter\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e\u0026nbsp;(\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e\u0026le;\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003ekb)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:57.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003e395\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:78.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0mm 5.4pt 0mm 5.4pt;\"\u003e\n \u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:14px;color:red;\"\u003eBCL6 transcription repressor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0mm;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2. Co-IP screening combined with mass spectrometry of target gene interaction complexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-IP was performed in the OE and NC groups, and further in-gel enzymatic digestion and shotgun-mass spectrometry protein identification was performed. Overall, 1,371 proteins were identified in the normal group and 1,336 in the overexpression group, and a total of 322 differential proteins were identified. Bioinformatics analysis was performed on the differential proteins to analyze 247 proteins with possible relevance and their mechanism of action, including their possible involvement in cell proliferation, metastasis, apoptosis, and invasion, as shown in Figure 8. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.3. WB validation of proteins identified by Co-IP screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the proteins identified by the above screening, 13 were selected for validation after bioinformatics analysis: U2AF2, RBM5, ILK, ENAH, JUP, RELA/P65, SQSTM1, YY1, STIM1, Integrin alpha V, EED, SUGT1, and NFKB1, among which U2AF2 was successfully detected in the input. The target bands were detected in NC and OE and, compared with NC, U2AF2 expression was significantly increased in the OE group, suggesting that the 3\u0026times; Flag-ZBTB5 protein pulled down the U2AF2 protein and there may be an interaction between the two proteins (see Figure 9).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCervical cancer is a disease that leads to serious health risks for women. As the main treatment for cervical cancer, PTX-based chemotherapy is pivotal in the treatment of advanced and recurrent cervical cancer. PTX-based chemotherapy regimens have been published in the National Comprehensive Cancer Network [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], International Federation of Gynecology and Obstetrics, and European Society of Medical Oncology guidelines as the standard of care for intermediate-to-advanced cervical cancer. However, in cases treated with PTX as a first-line chemotherapeutic agent, drug resistance rates in patients with intermediate-to-advanced squamous cervical cancer ranges from 20\u0026ndash;44% [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Resistance to PTX in patients with cervical squamous cell carcinoma leads to disease progression and affects prognosis with a 20\u0026ndash;30% reduction in five-year survival. Studies in the United States and Japan have reported [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] that among patients with locally advanced cervical squamous cell cancer, 20% were resistant to PTX-based neoadjuvant chemotherapy, resulting in treatment failure. PTX resistance is the main cause of treatment failure in patients with cervical squamous cell cancer. Therefore, it has become a critical clinical issue with utmost concern for gynecologic oncologists to explore strategies to overcome PTX resistance in cervical squamous cell cancer.\u003c/p\u003e \u003cp\u003eThe main mechanisms of chemoresistance include anti-apoptosis, pro-survival, increased intracellular drug excretion, cellular detoxification, enhanced DNA repair function, and dysregulation of microtubule dynamic balance. To date, the reported antitumor mechanisms of PTX mainly include (1) PTX binding to intracellular microtubules, enhancing microtubule stability, causing cell cycle arrest and making cells unable to divide, which eventually leads to the apoptosis of tumor cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; (2) PTX inhibiting cell proliferation, migration, and infiltration by inhibiting the expression of androgen receptor (AR) and limiting AR signaling pathway viability to kill tumor cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]; (3) PTX promoting tumor cell apoptosis by inhibiting the expression of the anti-apoptotic BLC-2 proteins, promoting the phosphorylation of BCL-2, and inhibiting the Bax/Caspase 3 signaling pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe BCL-6 proto-oncogene belongs to the anti-apoptotic family, and the main function of BCL-6 is transcriptional repression. The target genes regulated by BCL-6 are mainly associated with cell activation, differentiation, and proliferation. The function of BCL-6 is to repress transcription, thus, inhibits apoptosis. The human BCL-6 proto-oncogene is located in the 3q27 region of the chromosome, is 26 kb in length, and contains 10 exons and seven introns. Bcl-6 is widely distributed in cell activation, differentiation, cell cycle regulation, DNA damage repair, and other processes. Target genes of BCL-6 competitively repress STAT6, CD23, and IgE as well as repress CD69, CD44, Blimp-1, p27kip1, and CyclinD2.\u003c/p\u003e \u003cp\u003eIn the early stage, our research team successfully knocked out ZBTB5 gene in human cervical squamous cell carcinoma cell line SiHa by CRISPR/Cas9 gene editing technology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. ZBTB5 is a newly discovered BTB / POK protein that is mainly located in the nucleus and is expressed in all normal tissues of the body, especially in tumor tissues, where it is highly expressed and may be an oncogene. ZBTB5 possesses a POZ domain and two unique zinc-finger domains, which can be involved in gene regulation as a transcription factor. ZBTB5 represses the p21 gene during the cell cycle and promotes the proliferation and growth of tumor cells, but the regulatory mechanism of ZBTB5 for other genes is unclear [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. According to TCGA database, ZBTB5 is highly expressed in a variety of tumor tissues, including bladder and ureteral, gall bladder, intestinal, head and neck, esophageal, and renal cancers (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://starbase.sysu.edu.cn/panGeneDiffExp.php\u003c/span\u003e\u003cspan address=\"http://starbase.sysu.edu.cn/panGeneDiffExp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). ZBTB5 is also significantly highly expressed in cervical cancer, especially in young Asian patients with cervical squamous cell cancer, and affects prognosis. Recent studies have reported that ZBTB5 is a proto-oncogene that stimulates cell proliferation. zBTB5 represses p21 / CDKN1A by competing with p53 and recruiting complexes, and the oncoprotein c-Myc directly activates the transcription of ZBTB5. In the absence of p53, acetylated ZBTB5 K597 interacts with p300 and activates NOXA transcription, thereby inducing apoptosis. In contrast, in the presence of p53, ZBTB5 interacts with p53 and blocks ZBTB5 K597 acetylation. Thus, the cell fate determination by c-Myc depends on ZBTB5, p53, and p300, as well as the acetylation of ZBTB5K597 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, it was clarified that ZBTB5 functions in the nucleus, and that ZBTB5 knockdown inhibits cell cloning, cell proliferation, significantly enhances apoptosis in cervical squamous carcinoma cells in PTX, and reduces PTX resistance in SiHa cells. The ChIP assay after ZBTB5 overexpression suggested that ZBTB5 is involved in PTX resistance in cervical squamous cell carcinoma by regulating \u003cem\u003eBCL6\u003c/em\u003e, for which ZBTB5 may be the promoter, which is consistent with the results of IPA bioinformatics analysis. After Co-IP, further screening and identification of ZBTB5-interacting proteins showed that there was protein-to-protein interaction between ZBTB5 and U2AF2, and it was hypothesized that ZBTB5 might form a transcriptional complex with U2AF2 to regulate \u003cem\u003eBCL6\u003c/em\u003e to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma. Whether there was a similar relationship between BCL-6 and ZBTB5 to that between c-Myc and ZBTB5 still requires to be confirmed by further basic studies.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, this study preliminarily speculated that ZBTB5 may form a transcriptional complex with U2AF2 to regulate \u003cem\u003eBCL6\u003c/em\u003e to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma, and this speculation needs to be further confirmed by subsequent clinical and lab-based experiments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epaclitaxel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eZBTB5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eZinc finger and BTB domain-containing 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1)We are grateful to all the reviewers and editors who provided comments that substantially improved the manuscript. \u003c/p\u003e\n\u003cp\u003e(2)We are grateful to Shanghai GeneChem Co., Ltd for the support of experiment progress during the COVID-19 pandemic in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: H.Y., W.Y.M.; Data curation: Z.J.J, H.Y.; Formal analysis: H.Y.; Funding acquisition: H.Y., W.Y.M.; Methodology: J.C., L.Y., W.M.; Project administration: W.Y.M.; Resources: H.S.B., X.J.Q, W.Y.F.; Supervision: W.Y.M.; Writing-original draft: H.Y.; Writing review \u0026amp; editing: W.Y., W.Y.M. \u003c/p\u003e\n\u003cp\u003eThe author(s) read and approved the fnal manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article/supplementary materials. Futher inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. There is no studies involving human participants.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eNo potential confict of interest relevant to this article was reported\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eBeijing Hospitals Authority Clinical Medicine Development of Special Funding Support (grant No.ZYLX201705). \u003c/li\u003e\n\u003cli\u003eNational Natural Science Foundation of China (grand No.82102711).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCalys-Tagoe BNL, Aheto JMK, Mensah G, et al. Cervical cancer screening practices among women in Ghana: evidence from wave 2 of the WHO study on global AGEing and adult health [J]. BMC Womens Health, 2020, 20 (1): 49. \u003c/li\u003e\n\u003cli\u003ede Martel C, Georges D, Bray F, et al. Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis [J]. Lancet Glob Health, 2020, 8 (2): e180-e190.\u003c/li\u003e\n\u003cli\u003eDuan R, Qiao Y, Clifford G, et al. Cancer burden attributable to human papillomavirus infection by sex, cancer site, age, and geographical area in China [J]. Cancer Med, 2020, 9 (1): 374-384. \u003c/li\u003e\n\u003cli\u003eArbyn M, Weiderpass E, Bruni L, et al. Estimates of incidence and mortality of cervical cancer in 2018: A worldwide analysis [J]. Lancet Glob Health, 2020, 8 (2), e191-e203.\u003c/li\u003e\n\u003cli\u003e[5] Koh WJ, Abu-Rustum NR, Bean S, et al. Cervical cancer, version 3.2019, NCCN clinical practice guidelines in oncology [J]. J Natl Compr Canc Netw, 2019, 17 (1) : 64-84.\u003c/li\u003e\n\u003cli\u003eKastritis E, Bamias A, Efstathiou E, et al. The outcome of advanced or recurrent non-squamous carcinoma of the uterine cervix after platinum based combination chemotherapy [J]. Gynecol Oncol, 2005, 99(2): 376-382.\u003c/li\u003e\n\u003cli\u003eMori T, Makino H, Okubo T, et al. Multi-institutional phase II study of neoadjuvant irinotecan and nedaplatin followed by radical hysterectomy and the adjuvant chemotherapy for locally advanced, bulky uterine cervical cancer: A Kansai Clinical Oncology Group study (KCOG-G1201) [J]. J Obstet Gynaecol Res, 2019, 45(3): 671-678.\u003c/li\u003e\n\u003cli\u003ede la Torre M. Neoadjuvant chemotherapy in woman with early or locally advanced cervical cancer [J]. Rep Pract Oncol Radiother, 2018, 23(6): 528-532. \u003c/li\u003e\n\u003cli\u003eQi Z, Yin L, Xu Y. Pegylated liposomal paclitaxel induces ovarian cancer cell apoptosis via TNF induced ERK/AKT signaling pathway [J]. Mol Med Rep, 2018, 17(6): 7497-7504. \u003c/li\u003e\n\u003cli\u003eShin S, Woo S, Yim H. Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer [J]. Ther Adv Med Oncol, 2019, eCollection 2019.\u003c/li\u003e\n\u003cli\u003eShi X, Sun X. Regulation of paclitaxel activity by microtubule-associated proteins in cancer chemotherapy [J]. Cancer Chemother Pharmacol, 2017, 80(5): 909-917. \u003c/li\u003e\n\u003cli\u003eWei W, He Y, Wu YM. Identification of genes associated with SiHa cell sensitivity to paclitaxel by CRISPR-Cas9 knockout screening [J].Int J Clin Exp Pathol, 2018, 11(3): 1972-1978.\u003c/li\u003e\n\u003cli\u003eKoh DI, Choi WI, Jeon BN, et al. A novel POK family transcription factor, ZBTB5, represses transcription of p21CIP1 gene [J]. J Biol Chem, 2009, 284(30): 19856-19866. \u003c/li\u003e\n\u003cli\u003eChoi SH, Koh DI, Ahn H, et al. Cell fate decisions by c-Myc depend on\u0026ensp;ZBTB5\u0026ensp;and p53[J]. Biochem Biophys Res Commun, 2020, 533(4): 1247-1254. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ZBTB5, Paclitaxel Resistance, Cervical Cancer, Squamous Cell Carcinoma ","lastPublishedDoi":"10.21203/rs.3.rs-2471463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2471463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePURPOSE: To preliminarily clarify the mechanism of paclitaxel (PTX) resistance in cervical squamous cell carcinoma with the involvement of ZBTB5.\u003c/p\u003e\n\u003cp\u003eRESULTS: 1) Overexpression of ZBTB5 was cultured in PTX, and immunofluorescence showed that overexpression of ZBTB5 enhanced cervical squamous cell carcinoma resistance to PTX by functioning in the cell nucleus. 2) \u003cem\u003eZBTB5\u003c/em\u003eknockdown inhibited cell cloningand proliferation and enhanced apoptosis of cervical squamous carcinoma cells in PTX and reduced resistance to PTX in SiHa cells compared with the non-knockdown group (p \u0026lt; 0.05). 3) A ChIP assay was performed after overexpression of ZBTB5, which suggested that the BCL6 gene is located 395 kb from the TSS region and may be regulated by ZBTB5, which may be the promoter of BCL6. This result was consistent with that of the IPA bioinformatics analysis. 4) Further in-gel enzymatic digestion and shotgun-mass spectrometry protein identification were performed after Co-IP, and 322 differential proteins were identified in the overexpression group. Bioinformatics analysis of different proteins was conducted and 13 proteins were selected for validation: U2AF2, RBM5, ILK, ENAH, JUP, RELA/P65, SQSTM1, YY1, STIM1, Integrin alpha V, EED, SUGT1, and NFKB1. Among them, U2AF2 was successfully detected in the input, suggesting a possible interaction of ZBTB5 with U2AF2.\u003c/p\u003e\n\u003cp\u003eCONCLUSION: There is a protein–protein interaction between ZBTB5 and U2AF2, and ZBTB5 is involved in PTX resistance in cervical squamous cell carcinoma by regulating \u003cem\u003eBCL6\u003c/em\u003e. It was hypothesized that ZBTB5 may form a transcriptional complex with U2AF2 to regulate \u003cem\u003eBCL6\u003c/em\u003e to affect tumor cell proliferation and participate in PTX resistance in cervical squamous cell carcinoma.\u003c/p\u003e","manuscriptTitle":"ZBTB5 enhances the resistance of cervical cancer to paclitaxel by regulating BCL6","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-16 21:56:00","doi":"10.21203/rs.3.rs-2471463/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2006392e-b2de-474e-a421-ce5be5d20578","owner":[],"postedDate":"January 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-24T07:29:30+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-16 21:56:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2471463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2471463","identity":"rs-2471463","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}