Knocking down of FANCI expression inhibits the biological behavior of glioma and mediates apoptosis by downregulating the Akt/Bcl2 pathway

Knocking down of FANCI expression inhibits the biological behavior of glioma and mediates apoptosis by downregulating the Akt/Bcl2 pathway | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Knocking down of FANCI expression inhibits the biological behavior of glioma and mediates apoptosis by downregulating the Akt/Bcl2 pathway Hanyun Li, Mingyao Wang, Yinrui Liu, Jilan Liu, Xianyun Qin, Jikui Sun, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3890361/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 Previous studies have shown that FANCI has cancer susceptibility, and high expression of FANCI promotes the progression of breast cancer, ovarian cancer, and other cancers. However, the potential mechanism of action of FANCI in glioma progression is unclear. Methods To explore the role of FANCI in glioma progression, we determined the expression of FANCI in glioma patients and its relationship with prognosis through database analysis and gene chip. And then further conducted in vitro functional experiments (overexpression and knockdown) and in vivo nude mouse xenograft model experiments. Results This study found that FANCI was significantly overexpressed in glioma, positively correlated with WHO grade, and closely related to patient prognosis. In vitro functional experiments showed that inhibiting the expression of FANCI could inhibit the proliferation, migration, and invasion of glioma and promote apoptosis. At the same time, the nude mouse xenograft model also confirmed that inhibition of FANCI could inhibit glioma in vivo. In addition, the low expression of FANCI inhibited the phosphorylation of Akt and the expression of Bcl-2 by western blotting. Conclusion FANCI promotes glioma growth and may mediate apoptosis by regulating Akt/Bcl-2. This study preliminarily explored the role of FANCI in glioma growth and provided data support for further application of FANCI in clinical practice. Glioma FANCI Malignant biological behavior Akt/Bcl2 Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gliomas, one of the lethal malignant neurological tumors, are the most frequent primary tumors in the brain. A survey shows that gliomas account for about 80% of primary malignant brain tumors in adults, with a 5-year survival rate of about 36% [ 1 ], and the onset is age-related, with a significant increase after the age of 40 [ 2 ]. We could divide the glioma into four types from the perspective of pathological histology: astrocytoma, oligodendroglioma, oligodendroglioma, and ependymoma [ 3 ]. The 2021 World Health Organization Classification of Central Nervous System Tumors divides gliomas into localized and diffuse according to the growth mode. The adult diffuse gliomas contain glioblastoma (GBM), isocitrate dehydrogenase (IDH) wild-type; astrocytoma, IDH mutation; Oligodendroglioma, IDH mutation, and 1p/19q combined deletion [ 4 ]. GBM is the most representative aggressive brain tumor in diffuse glioma, with a poor prognosis, and belongs to the WHO grade Ⅳ tumor. After years of medical technology development, the therapy methods of glioma have blossomed, from the classic surgical resection combined with radiotherapy and chemotherapy, and gradually advanced in all directions, such as electric field therapy, immunotherapy, targeted therapy, and gene therapy [ 5 – 6 ]. Unfortunately, however, relapse is inevitable as existing therapies do not have a long-lasting therapeutic effect [ 7 ]. Therefore, there is an urgent need to explore a molecular marker that could have a role in the development and development of glioma and drug-resistance therapy. In the case of standard treatment for glioma surgery combined with chemoradiotherapy, the goal is to induce DNA damage beyond the tumor's ability for repair. However, the literature shows that glioma cells have adapted several resistance mechanisms to maintain DNA integrity by combatting DNA damage due to chemoradiotherapy [ 8 ]. If tumor cells fail to repair DNA damage, it may destroy or block tumor cells, reducing the possibility of tumor cell mutations. Therefore, interfering with DNA repair mechanisms is a meaningful aspect of therapeutic research for gliomas, such as base excision repair, nucleotide excision repair, base mismatch repair, and single or double-strand breakage repair [ 9 ]. Interestingly, the classic DNA interstrand cross-linking repair pathway, also known as the FA (Fanconi anemia) pathway, is characterized by spontaneous DNA breaks and DNA interstrand cross-linking (ICL) phenomena [ 10 ]. Among more than 30 FA family-related proteins to date, FANCI (Fanconi anemia supplementation group I) has been identified as having a very influential position in ICL repair [ 11 ]. Moreover, studies have shown that FA has a research base in susceptibility to cancer [ 12 ] and, especially, has the potential to progress to brain tumors [ 13 ]. In addition, monoubiquitinated FANCI and FANCD2, the core complex of FA, and E3 ubiquitin ligase serve as prominent components of the FA pathway to maintain genome stability [ 14 – 15 ]. The FA family contains 22 genes with FANCA, FANCD2, FANCE, FANCI, FANCN, etc. Much of the literature suggests that FA family monoallelic mutations may be one of the causes of tumorigenesis, such as FANCA mutation carriers are significantly associated with the risk of breast cancer [ 16 ]. Patients with biallelic mutations in FANCD1 and FANCN are at increased risk of developing acute myeloid leukemia and embryonal tumors [ 17 ]. FANCI has mutations in hepatocellular carcinoma, colorectal cancer, breast cancer, and ovarian cancer. FANCI is a potential biomarker for the prognosis of hepatocellular carcinoma [ 18 ]. A lot of relevant papers have also shown that the high expression of FANCI in tumors is closely related to the survival rate and poor prognosis of cancer patients, such as FANCI may interact with extracellular matrix receptors to affect the poor prognosis of lung adenocarcinoma [ 19 ] and pathogenic FANCI mutations increase the risk of familial ovarian cancer [ 20 ]. However, the reports of the mechanism of FANCI in glioma development and progression are still very few. In this study, we first used TCGA and CGGA databases to analyze the expression of FANCI in different grades of glioma and its correlation with glioma patient’s prognosis. Then, the effects of FANCI on glioma cell growth in vitro and in vivo were explored at the cellular and animal level, respectively. Finally, we found that FANCI expression in glioma tissue was higher compared to non-tumor tissue, suggesting that FANCI upregulation may be closely related to glioma growth. After silencing the expression of FANCI, it inhibited the proliferation, migration, and invasion and promoted the apoptosis ability of cells. FANCI knockout may also promote tumor cell apoptosis through the Akt/Bcl2 pathway. To sum up, the findings suggest that FANCI may be a potential target for glioma therapy. Materials and methods Tissue specimens Tissue samples were retrieved from HuanHu Hospital in Tianjin, China, with a total of 5 cases of non-tumor brain tissue (traumatic brain injury) and 42 cases of glioma tissue. Include 9 cases of WHO Ⅰ grade (oligodendroglioma), 6 cases of WHO Ⅱ grade (astrocytoma), 8 cases of WHO Ⅲ grade (five of anaplastic oligodendroglioma and three of anaplastic astrocytoma), and 19 cases of WHO Ⅳ grade (GBM). The patients from whom the samples were derived did not receive any anti-cancer therapies in preoperative treatments, such as chemoradiotherapy. Samples be placed at -80°C for later use. Cell culture The human glioma cells (U251, LN229, U87, A172) and normal glial cells (NHA) came from the Laboratory of Medical Research Center, Affiliated Hospital of Jining Medical College. All cell lines were grown using DMEM medium (DMEM, Gibco) supplementing with 10% fetal bovine serum (FBS, Excell) and 1% penicillin-streptomycin (Gibco) in a 37°C, 5% CO2 cell culture incubator. Cell transfection Four different FANCI-specific small interfering RNA (siRNA) sequences (Shanghai, Genepharma) was used in this study to inhibit the expression of FANCI in glioma cells. Next, pcDNA3.1-FANCI overexpression plasmid (Shanghai, Genechem) targeting human FANCI were synthesized to promote the expression of FANCI. And the effect of transfection verified by real-time PCR and western blot. Sequence 2267: sense (5'-3'): GCACCAGUAUUGGCAUAAATT; antisense (5'-3'): UUUUUGCCAAUACUGGUGCTT Sequence 1292: sense (5'-3'): GCCCAAGUCUUUAGAAUTT; antisense (5'-3'): AUUCUAGAAAGACUUGGGCTT Sequence 2097: sense (5'-3'): GGCCUGGUAUAAGAAUACATT; antisense (5'-3'): UGUAUUCUAUACCAGGCCTT Sequence 573: sense (5'-3'): CAGGUGGGAUCAGCAAUAUTT; antisense (5'-3'): AUAUUGCUGAUCCCACCUGTT Negative control sequence: sense (5'-3'): UUCUCCGAACGUGUCACGUTT; antisense (5'-3'): ACGUGACACGUUCGGAGAATT Cells are seeded in 6-well plates one day before transfection according to the company's guidelines to ensure a cell density of 30%-40% at seeding. siRNA or plasmid transfected by Lipofectamine 3000 reagent (Invitrogen, USA), and subsequent cell function experiments after transfection for 48h. siRNA 1292 was selected as the subsequent lentiviral (Shanghai, Genechem) infection sequence based on the transfection results. After stable transfection 48h, select the target cells with 0.5 µg/mL puromycin for around 14 days. Quantitative Reverse-Transcription PCR Extract total cellular RNA using Trizol reagent (Invitrogen) and store at -80°C for later use after determining RNA concentration. According to the Fastking RT Kit (with gDNase) (Tiangen) Usage Guide, synthesize cDNA and store it at -20°C for later use. Using the SYBR Green Assay Mix Kit (Kang Wei Century) on the real-time PCR detection system (Thermo Fisher) perform Real-time PCR experiments. The total working solution system is 20ul, including 0.4ul of forward primer, 0.4ul of reverse primer, 9ul of SYBR reagent, 2ul of cDNA, and 8.6ul of deionized water. The 2 −∆∆Ct method determines the relative expression of the target gene. The relative expression of FANCI in each glioma cell was standardized with GAPDH as an internal reference. All experiments were performed in three replicates and were independent. Primer sequences (AG, Accurate Biology) are as follows: FANCI: F: 5’-CTCCTCCAAGGGAAGCAGAAAGA-3’ R: 5’-GGCACAGTGACAACATCCAATAGC-3’ CCK-8 Cell Proliferation assay About 2,000 cells per well were seeded in a 96-well plate, with five replicate wells a day, placed in a cell culture incubator for 1, 2, 3, 4, and 5 days. Each well-added 100µl working solution, which mixed 10µl CCK-8 reagent (Dojindo, Shanghai) and 90µl DMEM, was next put back incubated for 2 hours and then represented proliferative capacity of glioma cells as the absorbance of each well at 450 nm. EdU Cell Proliferation assay Place suitable coverslips into 24-well plates, then seed around 8,000 cells per well and incubate for 24–48 hours. Add into per well with 250µl the diluted Edu working solution (20µM) with DMEM, and continue the incubation for two hours. Remove the 24-well plate for 4% paraformaldehyde fixation, and use the EdU cell proliferation kit (Abbkine) to prepare an appropriate amount of staining solution according to the instructions. After staining, BSA is washed twice. Prepare an appropriate amount of 1× Hoechst reaction solution and perform DNA staining for 10 min. BSA wash 2 times and dry moderately. Observe and photograph under an inverted fluorescence microscope and manipulate the relative proliferation rate of cells using ImageJ software. Wound healing assay Mark the back of the 6-well plates in advance. Then, inoculate the cells in the 6-well plates at about 1×10^ 6 cells per well to ensure 100% confluency the next day. Scrape the cell monolayer vertically with a 200µl sterile pipette, wash with PBS, and discard the floating cells. The lower serum DMEM (2%FBS) was substituted for complete DMEM (10%FBS). Remove at 0 and 48h, observe under an inverted microscope, and take pictures. Cell migration area comparison was performed using ImageJ software. Cell migration and invasion assays For cell migration, aliquots of 2×10 ^5 cells with serum-free DMEM per well in the upper chamber of the Transwell insert chambers (pore size 8µm, Corning) without Matrigel, add 600ul of DMEM containing 10% FBS in the lower chamber. For cell invasion experiments, an upper chamber was precoated with Matrigel (Corning). After 48h, the chamber was removed and fixed with 4% paraformaldehyde 30min, stained 10min with crystal violet, and gently wiped the Transwell chamber inner surface with a cotton swab. After properly air-drying, observe it with a microscope and took photography. The number of cells passed through each field of view using ImageJ software processing. Flow Cytometry Briefly, take about 1×10 ^6 cells after cell transfection 48h, with 1800rpm, centrifugation for 5min. cells were resuspended with 400µl of 1× Binding Buffer from apoptosis kit (BD, Biosciences). Then, in the dark conditions add 5µl of propidium iodide (PI) and 5µl Annexin V, mix thoroughly, and incubate at room temperature for 10min. Finally, analyze the apoptosis rate of cells by flow cytometer (Beckman Coulter). Apoptosis rate calculation = (Q1-UR + Q1-LR)/Q1-LL%. Q1-UR represents the proportion of late-stage apoptotic cells in the upper right quadrant, Q1-LR represents the proportion of early-stage apoptotic cells in the lower right quadrant, and Q1-LL represents the proportion of viable cells in the lower left quadrant. Western blotting The extraction of total cell protein using RIPA lysate with protease inhibitors and protein quantification by BCA Protein Quantitation Kit (Beyotime). Prepare a 10% protein gel and polyvinylidene difluoride membranes (0.45µm PVDF, Millipore, USA). After the protein is transferred to the membrane, the membrane is closed with 5% skimmed milk powder for two hours, and the primary antibody is incubated overnight at 4°C. The membrane reacted with the corresponding secondary antibody binding reaction at room temperature for 1–2 h the next day, followed by band development using ECL Western Blot luminescence solution (Millipore). In the end, protein band gray value analysis using ImageJ software. The antibodies were as follows: anti-GAPDH (Affinity, 1:1000), anti-FANCI (Abcam, 1:5000), Akt (ABclonal, 1:1000), Phospho-Akt (Ser473) (Always, 1:1000), Bcl-2 (Cell Signaling, 1:1000), Bax (Cell Signaling, 1:1000), HRP-anti-rabbit/mouse (Affinity, 1:5000-1:8000). Subcutaneous tumor xenografts in nude mice BALB/c nude mice (4–5 weeks old) were purchased from Peng Yue Animal Breeding Center in Jinan, China. And these mice raised in the SPF-conditioned animal house of Jining Medical College. Stably transfected U251 cells with knockdown for FANCI or negative controls were injected into the right subcutaneous part of each mouse (8 mice in total). After two weeks, observed the growth of the tumor every five days, and sacrificed the mice at four weeks later. Tumors were weighed and treated with 4% paraformaldehyde, and we retained a part of tumor tissue for further histological analysis and RNA extraction. We promise that All experiments involving animals conducted following the protocols of the animal ethics committee approved by the institution. Fluorescent immunohistochemistry The tissue sections are first deparaffinized and fixed. The tissue is then repaired with sodium citrate repair solution at high temperatures, and remove the residual solution. Follow the instructions for the fluorescent immunohistochemistry kit (Absin). Block with 5% BSA for one hour at room temperature. Then, incubate with rabbit Ki-67 (Affinity, 1:200) primary antibody for 1 hour at room temperature, and rinse with PBS 3 times for 5 min each time. Dropwise the secondary antibodies, and at room temperature for 1 hour, rinse with PBS 3 times, 5 minutes each time. After DAPI staining, rinse with PBS. Finally, after adding an appropriate amount of autofluorescence quenching reagent to the tissue, the mounting was observed and photographed under a fluorescence microscope after mounting treatment. Statistical analyses Data statistical analysis using GraphPad software 8.0. Values from at least three independent experiments. We used the two-tailed unpaired Student’s t -tests to test the statistical differences between the two groups. The One-way ANOVA test for the between-group comparison. The Kaplan-Meier method and log-rank test plotted the survival curve with statistical differences. A p -value less than 0.05 indicates a statistically significant difference. Result FANCI is highly expressed in glioma tissues and is associated with poor prognosis in glioma patients The TCGA database utilizes the TIMER ( http://timer.xistrome.org ) analysis tool to show that FANCI is highly expressed in a variety of cancers, including gliomas (Fig. 1 A). The TCGA database used the GEPIA ( http://gepia.cancer-pku.cn/ ) analysis tool to show that FANCI was significantly overexpressed in GBM (Fig. 1 B), and low expression of FANCI was associated with a good prognosis (Fig. 1 C). The results of CGGA database showed that the high expression of FANCI in both primary gliomas and recurrent gliomas indicated poor prognosis (Fig. 1 D-E). Moreover, the higher the WHO grade, the higher the expression levels of FANCI (Fig. 1 F). To verify the above information, we further analyzed the relationship between FANCI and glioma using the human glioma tissue chip (HBraG160Su01, Shanghai Outdo Biotech Co., Ltd.), and the immunohistochemistry results again showed that FANCI positively correlated with glioma WHO grade (Fig. 1 G). The survival rate was worse in patients with high FANCI expression (Fig. 1 H-I). In addition, there were statistically significant differences in the expression of FANCI in recurrent or non-recurrent gliomas, as well as between WHO low-grade or high-grade (Fig. 1 J-K). The chi-square analysis results of the differences between the clinicopathological characteristics and the expression of FANCI in 152 of glioma microarray are shown in Table 1 . The results showed that the age ≥ 40 ( χ 2 = 6.848, P <0.009), grade Ⅲ-Ⅳ ( χ 2 = 16.882, P <0.001), high level of PD-L1 expression ( χ 2 = 4.746, P <0.029) were statistically significant with FANCI expression ( P < 0.05). Furthermore, Cox regression analysis showed that age ≥ 40, grade Ⅲ-Ⅳ, and Ki67 high expression were independent factors predicting the prognosis of glioma patients (Table 2 , P < 0.05). Table 1 Correlation between FANCI expression and clinicopathological characteristics (N = 152) variables FANCI expression total χ 2 p value low high Age (year) 6.848 0.009 ** < 40 48 13 61 ≥ 40 53 38 91 Sex 0.161 0.688 Female 37 17 54 Male 64 34 98 Grade 16.882 < 0.001 *** I-II 69 17 86 III-IV 32 34 66 ki67 0.160 0.689 Low 51 24 75 High 50 27 77 EGFR 0.337 0.561 Low 57 26 83 High 43 24 67 PDL-1 4.746 0.029 * Low 75 29 104 High 26 22 48 * p < 0.05; ** p < 0.01; *** P < 0.001 Table 2 Prognostic Value of FANCI Expression with Overall survival in Patients with Gliomas Variables Univariate analysis Multivariate analysis HR 95%CI p value HR 95%CI p value Sex (female/male) 1.684 0.930–3.049 0.085 1.448 0.766–2.737 0.255 Age (< 40/≥40) 3.971 1.942–8.119 < 0.001 * 3.582 1.688–7.598 0.001 * Grade (I-II/ III-IV) 14.267 7.136–28.523 < 0.001 * 15.753 7.061–35.145 < 0.001 * Ki67 (low/high) 0.500 0.288–0.866 0.013 0.389 0.213–0.710 0.002 * EGFR (low/high) 1.106 0.647–1.892 0.713 - - - PDL1 (low/high) 1.078 0.608–1.909 0.798 - - - FANCI (low/high) 3.499 3.023–6.053 < 0.001 * - - - * Statistically significant(p < 0.05) We also obtained the expression levels of FANCI in clinical glioma tissue, which included five normal peritumor tissues and 34 glioma tissues (10 cases of lower grade and 24 cases of higher grade), by the qRT-PCR analysis. Our results are consistent with the results of the public bioinformatics database analysis described above, where FANCI has higher mRNA levels in glioma high-grade tissues than in lower-grade tissues (Fig. 2 A). The above results preliminarily indicate that FANCI may be a critical gene for glioma development. FANCI regulates the biological behavior of glioma cells in vitro In this study, we first verified the expression of FANCI in glioma cell lines (NHA, LN229, U251, U87, A172). The results showed that FANCI was expressed higher in LN229 and U251 cells (Fig. 2 B-D), so in subsequent cell function experiments, we selected knockdown LN229 and U251 cells with high FANCI expression and overexpression A172 cells with low FANCI to verify our results. Considering that the knockdown transfection effect of the sequence si-1292 on LN229/U251 glioma cells was good, we chose to use the si-1292 sequence as the lentivirus template in the future (Fig. 2 E, G). In addition, the results showed that the low expression of FANCI in lentivirus-infected cells (KD-FANCI) was well (Fig. 2 F-G). The results of the CCK-8 proliferation assay of LN229 and U251 cells showed that the low expression of FANCI inhibited the proliferation of cells compared with the control group (NC) (Fig. 2 H). In addition, we performed an Edu cell proliferation assay to verify the proliferation rate of glioma cells, and the results showed that inhibition of FANCI expression led to a decrease in Edu-positive cells (Fig. 2 I). Next, cell migration experiments (wound scratch healing experiment and transwell migration experiment) analyzed the effect of FANCI on glioma cell migration ability. The results of the wound healing experiment showed that low of FANCI expression significantly reduced the migration distance of LN229/U251 cells (Fig. 3 A). Further, we used the Transwell assay to verify the effect of FANCI on the migration ability of glioma cells, such as the results of the scratch assay were consistent, and the low of FANCI expression significantly inhibited the migration of glioma cells (Fig. 3 B). The transwell invasion assay (including matrigel) evaluated the effect of FANCI on the aggressiveness of glioma cells, and the results also showed that knocking down FANCI expression inhibited the aggressiveness of glioma cells (Fig. 3 C). That prompts that FANCI may be involved in the movement of glioma cells through the body. In addition, the results of apoptosis experiments suggest that decreased FANCI expression promotes the apoptosis ability of glioma cells (Fig. 3 D). At the same time, we also back-validated the above results by FANCI overexpression (Fig. 4 A-B), and our results showed that FANCI overexpression promoted the proliferation (Fig. 4 C-D), invasion ability (Fig. 4 E), migration (Fig. 4 F-G), and inhibition of apoptosis of glioma cells (Fig. 4 H). Therefore, data from cell function experiments prove that FANCI is involved in the biological processes of proliferation, migration, invasion, and apoptosis. FANCI may mediate apoptosis by regulating the Akt/Bcl2 pathway In addition, since the PI3K/Akt pathway is one of the classical signaling pathways in malignant glioma, and the literature indicates that Akt is also an upstream factor of the Bcl2-mediated apoptosis pathway [ 21 ], we speculate that FANCI may regulate the apoptosis process of glioma cells through the PI3K/Akt pathway. And, it is worth mentioning that the results of our Western blot experiment (Fig. 5 ) showed that knocking down of FANCI expression in LN229 cells and U251 cells inhibited the phosphorylation expression of Akt (P-Akt ser473) and also reduced the expression of Bcl-2. The results may suggest that FANCI influences apoptosis in glioma through the Akt/Bcl2 pathway possibly. Knocking down of FANCI inhibit the growth of nude Xenografts tumor in vivo To further verify whether FANCI affects the growth of gliomas in vivo, we established a nude mouse xenograft tumor model by subcutaneous injection in the lentiviral transfection U251 cell control group (CON) and knocking down group (KD-FANCI). The results showed that the size of xenograft tumors formed by nude mice of KD-FANCI group was significantly smaller than that of the control group (Fig. 6 A-B), and the weight of the tumors was also lower than that of the control group significantly (Fig. 6 C). The qRT-PCR technology and Western blotting technology verified the expression of FANCI in xenograft tumor tissues, and the results confirmed that the expression of FANCI in the KD-FANCI group was lower than that in the CON group (Fig. 6 D-E). At the same time, the results of immunopathological staining (Fig. 6 F) and immunofluorescence (Fig. 6 G) showed that the expression levels of Ki-67 in tumor tissues of the KD-FANCI group decreased. These in vivo data suggest that FANCI inhibits the progression of gliomas in vivo. Discussion In recent years, many studies have found that silencing FANCI expression can inhibit the occurrence and development of cancers, such as ovarian cancer and lung adenocarcinoma [ 12 , 22 ], and also enhance the chemotherapy sensitivity of ovarian cancer [ 22 ]. Glioma has become a worldwide problem because of its resistance to chemoradiotherapy and poor prognosis. Therefore, FANCI has important practical significance in the occurrence and development of glioma and the resistance mechanism of chemoradiotherapy. In this study, we will preliminarily explore the role of FANCI in the development of glioma. Not only was the FANCI expression elevated in glioma tissues and cells, but it also correlates with WHO grade positively. At the same time, the FANCI expression is close to the survival and disease-free survival in glioma patients. It is similar to the results of a meta-analysis that there is a possibility that the tumor stage may affect the potential prognostic value [ 23 ]. The higher FANCI expression may not significantly affect OS in GBM patients. It is worth noting that the results of COX regression analysis indicated that Ki-67 was an independent prognostic factor for OS in glioma, which is also consistent with the literature [ 24 ]. Unexpectedly, FANCI was not an independent prognostic factor for OS in glioma patients. It also may imply that glioma grade is one of the reasons interfering with the relationship between FANCI and patient OS. However, these results suggest that FANCI is involved in the malignant biological process of glioma and related to the prognosis of glioma patients. The experiments have shown that FANCI promotes the occurrence and development of glioma by promoting the proliferation, migration, invasion, and inhibition of apoptosis of glioma cells. Consistent with previous studies in lung cancer tissues, the knockdown of FANCI expression will inhibit tumor growth [ 25 ]. Taken together, FANCI may be a potential target for glioma therapy. One cause of cancer is endogenous DNA replication mutations or exogenous DNA damage. However, some specialists suggest that exogenous factors may account for the majority [ 26 ]. But in any case, genomic instability is one of the bases of cancer development, which can accelerate the acquisition of genetic diversity and the occurrence of inflammatory responses in organisms [ 27 ]. FA is a gene-disease known for chromosomal instability, and evidence is accumulating that a wide variety of cancers with different FA pathway defects. The FA pathway may be helpful to hyperresponsive to cross-linkers to obtain more effective therapy by interference with it [ 28 ]. Interestingly, the literature has shown that the FA pathway is closely related to the PI3K/Akt pathway, and FANCI is a vital regulator of Akt activation [ 29 ]. The PI3K-Akt signaling pathway is one of the classic activation pathways in cancer, which controls cell proliferation, cell metabolism, cell motility, apoptosis, and genomic instability [ 30 – 31 ]. And the PI3K/Akt is also involved in the mechanism of cell drug resistance [ 7 ]. Moreover, Akt kinase is a key effector molecule of this pathway and is implicated in DNA damage [ 32 ]. One of the mechanisms by which cells survive is that Akt inhibits pro-apoptotic proteins and apoptotic processes, such as the anti-apoptotic protein Bcl-2 [ 33 ]. There is a strong link between Akt activation and glioma prognosis, with a retrospective study in children showing elevated p-Akt (phosphorylation of Akt) expression in more than half of patients with high-grade glioma and a declining trend in overall survival [ 34 ]. According to the literature, phosphorylation of Ser 473 residues is necessary for the complete activation of Akt [ 33 ]. In conclusion, our results suggest that FANCI has a carcinogenic effect in glioma and may be one of the crucial molecules in the Akt/Bcl2-mediated anti-apoptotic pathway, which is worthy of further exploration of the potential of FANCI as a combination therapy regimen for PI3K/Akt signaling pathway in the treatment of glioma. However, the mechanism of action of FANCI in the AKT/Bcl2 pathway needs to be further verified and explored. Declarations Funding Statement We thank Tianjin Huanhu Hospital for providing clinical samples very much. This study was supported by the Natural Science Foundation of Shandong Province [ZR2022MH140], China Postdoctoral Science Foundation [2022M711324], Shandong First Medical University youth Science Foundation [202201-089], Project of Scientific Developmental Program of Shandong Provincial Administration of Traditional Chinese Medicine [Q-2022136], Project of the Key Research and Development Program of Jining Science and Technology [2023YXNS045], and Key Project of Shandong Provincial Traditional Chinese Medicine Science and Technology Project [Z-2022082]. Available of data The raw data are listed in the article, please contact the corresponding author for further needs. Author Contributions The idea of this study was proposed by SJK. LHY, LJL and QXY was responsible for the experimental operation and LHY writing this article, WMY and LYR conducted the animal experiment part. SJK, JF and LY revised the final draft. All authors have read and agree to publish the final manuscript. Disclosure The authors declare no proprietary or commercial interest in any product mentioned or concept discussed in this article. References Schaff LR, Mellinghoff IK (2023) Glioblastoma and Other Primary Brain Malignancies in Adults: A Review. JAMA 329: 574-587. doi: 10.1001/jama.2023.0023. Ostrom QT, Cioffi G, Waite K, Kruchko C, Barnholtz-Sloan JS (2021) CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018. Neuro Oncol 23: iii1-iii105. doi: 10.1093/neuonc/noab200. Lim M, Xia Y, Bettegowda C, Weller M (2018) Current state of immunotherapy for glioblastoma. Nat Rev Clin Oncol 15: 422-442. doi: 10.1038/s41571-018-0003-5. 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Fierheller CT, Guitton-Sert L, Alenezi WM, Revil T, Oros KK, Gao Y, Bedard K, Arcand SL, Serruya C, Behl S, Meunier L, Fleury H, Fewings E, Subramanian DN, Nadaf J, Bruce JP, Bell R, Provencher D, Foulkes WD, El Haffaf Z, Mes-Masson AM, Majewski J, Pugh TJ, Tischkowitz M, James PA, Campbell IG, Greenwood CMT, Ragoussis J, Masson JY, Tonin PN (2021) A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene. Genome Med 13: 186. doi:10.1186/s13073-021-00998-5. Zhong ZH, Wang YM, Guo H, Sagare A, Fernández JA, Bell RD, Barrett TM, Griffin JH, Freeman RS, Zlokovic BV (2010) Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region. J Neurosci 30: 15521-15534. doi:10.1523/JNEUROSCI.4437-10.2010. Li YQ, Zhang YN, Yang Q, Zhou XT, Guo YY, Ding F, Liu ZH, Luo A (2022) Silencing of FANCI Promotes DNA Damage and Sensitizes Ovarian Cancer Cells to Carboplatin. Curr Cancer Drug Targets 22: 591-602. doi:10.2174/1568009622666220331091709. Wu G, Song XH, Liu J, Li SZ, Gao WQ, Qiu MX, Yang CJ, Ma YM, Chen YH. (2020) Expression of CD44 and the survival in glioma: a meta-analysis. Biosci Rep 40: BSR20200520. doi: 10.1042/BSR20200520. Tini P, Yavoroska M, Mazzei MA, Miracco C, Pirtoli L, Tomaciello M, Marampon F, Minniti G (2023) Low expression of Ki-67/MIB-1 labeling index in IDH wild type glioblastoma predicts prolonged survival independently by MGMT methylation status. J Neurooncol 163: 339-344. doi: 10.1007/s11060-023-04342-2. Zhang JG, Wang JD, Wu JC, Huang JY, Lin ZX, Lin X (2022) UBE2T regulates FANCI monoubiquitination to promote NSCLC progression by activating EMT. Oncol Rep 48: 139. doi:10.3892/or.2022.8350. Nalepa G, Clapp DW (2018) Fanconi anaemia and cancer: an intricate relationship. Nat Rev Cancer 18: 168-185. doi:10.1038/nrc.2017.116. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674. doi:10.1016/j.cell.2011.02.013. Meijer GA (2007) The 13th Fanconi anemia gene identified: FANCI--importance of the 'Fanconi anemia pathway' for cellular oncology. Cell Oncol 29: 181-182. doi: 10.1155/2007/871608. Zhang XS, Lu XY, Akhter S, Georgescu MM, Legerski RJ (2016) FANCI is a negative regulator of Akt activation. Cell Cycle 15: 1134-1143. doi: 10.1080/15384101.2016.1158375. Stebbing J, Lit LC, Zhang H, Darrington RS, Melaiu O, Rudraraju B, Giamas G (2014) The regulatory roles of phosphatases in cancer. Oncogene 33: 939-953. doi:10.1038/onc.2013.80. Cao LJ, Xie HT, Chu ZX, Ma Y, Wang MM, Shi Z (2020) Tubeimoside‑1 induces apoptosis in human glioma U251 cells by suppressing PI3K/Akt‑mediated signaling pathways. Mol Med Rep 22: 1527-1535. doi: 10.3892/mmr.2020.11224. Dummler B, Hemmings BA (2007) Physiological roles of PKB/Akt isoforms in development and disease. Biochem Soc Trans 35: 231-235. doi:10.1042/bst0350231. Chautard E, Ouédraogo ZG, Biau J, Verrelle P (2014) Role of Akt in human malignant glioma: from oncogenesis to tumor aggressiveness. J Neurooncol 117: 205-215. doi:10.1007/s11060-014-1382-9. Antonelli M, Massimino M, Morra I, Garrè ML, Gardiman MP, Buttarelli FR, Arcell A, Giangaspero F (2012) Expression of pERK and pAKT in pediatric high grade astrocytomas: correlation with YKL40 and prognostic significance. Neuropathology 32: 133-138. doi:10.1111/j.1440-1789.2011.01252.x. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-3890361","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268918389,"identity":"7aed37d4-1ba6-40bc-9b09-8e9ec2d2b107","order_by":0,"name":"Hanyun Li","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Hanyun","middleName":"","lastName":"Li","suffix":""},{"id":268918390,"identity":"c16baadd-2cbb-446d-9765-5f43b833ec68","order_by":1,"name":"Mingyao Wang","email":"","orcid":"","institution":"Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyao","middleName":"","lastName":"Wang","suffix":""},{"id":268918391,"identity":"c3adb9ed-1c4c-432e-971f-19c670b33144","order_by":2,"name":"Yinrui Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Shandong First Medical University \u0026 Shandong Provincial Qianfoshan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yinrui","middleName":"","lastName":"Liu","suffix":""},{"id":268918392,"identity":"b4f6ff18-0868-4566-aefd-ba37541b316e","order_by":3,"name":"Jilan Liu","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jilan","middleName":"","lastName":"Liu","suffix":""},{"id":268918393,"identity":"a6ae2fe5-7d3b-4cd2-a67f-3e7981dd2130","order_by":4,"name":"Xianyun Qin","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianyun","middleName":"","lastName":"Qin","suffix":""},{"id":268918394,"identity":"ae53ee39-f021-4837-a680-25b77d2556eb","order_by":5,"name":"Jikui Sun","email":"","orcid":"","institution":"The First Affiliated Hospital of Shandong First Medical University \u0026 Shandong Provincial Qianfoshan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jikui","middleName":"","lastName":"Sun","suffix":""},{"id":268918395,"identity":"6925b644-5a2c-42b6-a867-eb835adf2a86","order_by":6,"name":"Feng Jin","email":"","orcid":"","institution":"University of Health and Rehabilitation Sciences (Qingdao Central Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Jin","suffix":""},{"id":268918396,"identity":"826cdd8e-9305-4719-9322-0005176348f3","order_by":7,"name":"Yan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACAwY2EGXBwM/MfPgBKVokGCTb2dIMSNNicJ5HQYIoLebsx9KkC2okEjcf5gHqr7GJJqjFsiftmPSMYxKJ2w7zHnjAcCwtt4Ggww6kt0nzsIG08CUYMDYcJkLL+edALf+ADmvmMZAgTssNoMN42yQSNzATr+VZsjVvn4TxjMPAQE4gyi/n0wxv83yzke3vP3z4wYcaG8JaYMARrDKBWOUgYE+K4lEwCkbBKBhhAACBpTxd6xu2hgAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-23 07:59:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3890361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3890361/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50176004,"identity":"ae345ddc-8c39-4acb-9d05-61d98ff6846d","added_by":"auto","created_at":"2024-01-25 16:29:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1243786,"visible":true,"origin":"","legend":"\u003cp\u003eFANCI is highly expressed in gliomas and is associated with poor prognosis in patients. \u003cstrong\u003eA.\u003c/strong\u003e FANCI expression in a variety of different cancers analyzed by TIMER. \u003cstrong\u003eB.\u003c/strong\u003e Expression of FANCI in GBM in the TCGA database. \u003cstrong\u003eC.\u003c/strong\u003e The relationship between FANCI expression and clinical prognosis of glioma in TCGA database. \u003cstrong\u003eD-E.\u003c/strong\u003e High of FANCI expression in both primary and recurrent gliomas is associated with poor prognosis. \u003cstrong\u003eF.\u003c/strong\u003e The CGGA database showed that FANCI was positively correlated with the WHO grade of glioma. \u003cstrong\u003eG.\u003c/strong\u003e The results of immunohistochemistry assay showed that FANCI expression was positively correlated with WHO grade of glioma. \u003cstrong\u003eH-K.\u003c/strong\u003e The GBM microarray indicates that high levels of FANCI expression indicate a poor prognosis, and the expression is differential in recurrent tumors and WHO grade.\u003csup\u003e *\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/6260444c33345237675e347c.png"},{"id":50176006,"identity":"dba40c8e-22a5-4064-bd9a-c886e8ad11b3","added_by":"auto","created_at":"2024-01-25 16:29:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383708,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FANCI in glioma specimens and cells. The low level of FANCI expression inhibits the proliferation of glioma. \u003cstrong\u003eA.\u003c/strong\u003e FANCI is more expressed in low/high grade gliomas than in non-tumor tissues. \u003cstrong\u003eB-D.\u003c/strong\u003e FANCI expression in different glioma cell lines. \u003cstrong\u003eE.\u003c/strong\u003e siRNA interference in U251/LN229 cells. \u003cstrong\u003eF.\u003c/strong\u003e lentiviral transfection in U251/LN229 cells. \u003cstrong\u003eG.\u003c/strong\u003e Western blotting was used to verify siRNA interference and lentiviral transfection (KD-FANCI). \u003cstrong\u003eH-I. \u003c/strong\u003eCCK-8 assay and EdU proliferation assay were used to analyze the effect of FANCI knockdown on the proliferation ability of glioma cells, respectively. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/d22fb42025d6b7546cbaead6.png"},{"id":50175338,"identity":"0c28dd78-7c78-44ee-98cc-4d7fb99698bd","added_by":"auto","created_at":"2024-01-25 16:21:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1148379,"visible":true,"origin":"","legend":"\u003cp\u003eLow levels of FANCI expression inhibits the migration and invasion of glioma cells and promotes apoptosis. \u003cstrong\u003eA-B.\u003c/strong\u003e The scratch assay and the Transwell assay were used to detect the migration ability of LN229 cells and U251 cells after low expression of FANCI. \u003cstrong\u003eC.\u003c/strong\u003e Transwell (with Matrigel) invasion assay was used to analyze the motility of LN229 cells and U251 cells after low expression of FANCI. \u003cstrong\u003eD.\u003c/strong\u003e Apoptosis assay analysis of the effect of FANCI on the apoptosis of LN229 cells and U251 cells. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/6e49ab7013a20b0ff5632766.png"},{"id":50175339,"identity":"330f73a1-eb5c-4eb5-ba3b-c03ffde2e23a","added_by":"auto","created_at":"2024-01-25 16:21:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1063030,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of FANCI overexpression on the biological behavior of A172 cells. \u003cstrong\u003eA-B.\u003c/strong\u003e qRT-PCR and western blotting were used to verify the transfection effect of FANCI overexpression plasmid in A172 cells (OE-FANCI). \u003cstrong\u003eC-D.\u003c/strong\u003e CCK-8 assay and EdU proliferation assay verified the effect of FANCI overexpression on the proliferation ability of A172 cells. \u003cstrong\u003eE.\u003c/strong\u003e Transwell (Matrigel-included) invasion assay to analyze the effect of FANCI overexpression on the motility of A172 cells. \u003cstrong\u003eF-G.\u003c/strong\u003e Transwell assay and cell scratch assay to analyze the effect of overexpression of FANCI on the migration ability of A172 cells. \u003cstrong\u003eH.\u003c/strong\u003e Apoptosis assay was to detect the changes in the apoptosis ability of A172 cells after overexpression of FANCI. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01,\u003csup\u003e ***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/ee86cd6535b53e48b3ac789a.png"},{"id":50175336,"identity":"673c7c64-add1-4adf-bbca-62babfeff87c","added_by":"auto","created_at":"2024-01-25 16:21:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145019,"visible":true,"origin":"","legend":"\u003cp\u003eFANCI low expression inhibited Akt phosphorylation (Ser 473) and Bcl2 expression. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/652a5faec76bfbbef2ebd813.png"},{"id":50176005,"identity":"c50e8eca-2933-44cf-861f-f0f33b11ebfb","added_by":"auto","created_at":"2024-01-25 16:29:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1405541,"visible":true,"origin":"","legend":"\u003cp\u003eLow level of FANCI expression inhibits tumorigenesis in nude mice. \u003cstrong\u003eA-C.\u003c/strong\u003e Image of five nude mice in the CON/KD-FANCI group with size and weight of tumor differences. \u003cstrong\u003eD-E.\u003c/strong\u003e qRT-PCR and western blotting to verify the expression of FANCI in nude mouse tumor tissues. \u003cstrong\u003eF-G.\u003c/strong\u003e HE staining and immunofluorescence chemistry to detect the expression of Ki-67 in nude mouse tumor tissues. scale bar = 100 µm.\u003csup\u003e *\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/bfe83c58ec3e6d861016fac9.png"},{"id":52846778,"identity":"a6e11728-2a92-4ee0-b934-72b4e0843ac8","added_by":"auto","created_at":"2024-03-17 18:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4361103,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3890361/v1/48a30e48-b67c-45e3-af93-6619a65a522b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Knocking down of FANCI expression inhibits the biological behavior of glioma and mediates apoptosis by downregulating the Akt/Bcl2 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGliomas, one of the lethal malignant neurological tumors, are the most frequent primary tumors in the brain. A survey shows that gliomas account for about 80% of primary malignant brain tumors in adults, with a 5-year survival rate of about 36% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and the onset is age-related, with a significant increase after the age of 40 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. We could divide the glioma into four types from the perspective of pathological histology: astrocytoma, oligodendroglioma, oligodendroglioma, and ependymoma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The 2021 World Health Organization Classification of Central Nervous System Tumors divides gliomas into localized and diffuse according to the growth mode. The adult diffuse gliomas contain glioblastoma (GBM), isocitrate dehydrogenase (IDH) wild-type; astrocytoma, IDH mutation; Oligodendroglioma, IDH mutation, and 1p/19q combined deletion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. GBM is the most representative aggressive brain tumor in diffuse glioma, with a poor prognosis, and belongs to the WHO grade Ⅳ tumor. After years of medical technology development, the therapy methods of glioma have blossomed, from the classic surgical resection combined with radiotherapy and chemotherapy, and gradually advanced in all directions, such as electric field therapy, immunotherapy, targeted therapy, and gene therapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Unfortunately, however, relapse is inevitable as existing therapies do not have a long-lasting therapeutic effect [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, there is an urgent need to explore a molecular marker that could have a role in the development and development of glioma and drug-resistance therapy.\u003c/p\u003e \u003cp\u003eIn the case of standard treatment for glioma surgery combined with chemoradiotherapy, the goal is to induce DNA damage beyond the tumor's ability for repair. However, the literature shows that glioma cells have adapted several resistance mechanisms to maintain DNA integrity by combatting DNA damage due to chemoradiotherapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. If tumor cells fail to repair DNA damage, it may destroy or block tumor cells, reducing the possibility of tumor cell mutations. Therefore, interfering with DNA repair mechanisms is a meaningful aspect of therapeutic research for gliomas, such as base excision repair, nucleotide excision repair, base mismatch repair, and single or double-strand breakage repair [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Interestingly, the classic DNA interstrand cross-linking repair pathway, also known as the FA (Fanconi anemia) pathway, is characterized by spontaneous DNA breaks and DNA interstrand cross-linking (ICL) phenomena [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among more than 30 FA family-related proteins to date, FANCI (Fanconi anemia supplementation group I) has been identified as having a very influential position in ICL repair [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, studies have shown that FA has a research base in susceptibility to cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and, especially, has the potential to progress to brain tumors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, monoubiquitinated FANCI and FANCD2, the core complex of FA, and E3 ubiquitin ligase serve as prominent components of the FA pathway to maintain genome stability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The FA family contains 22 genes with FANCA, FANCD2, FANCE, FANCI, FANCN, etc. Much of the literature suggests that FA family monoallelic mutations may be one of the causes of tumorigenesis, such as FANCA mutation carriers are significantly associated with the risk of breast cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Patients with biallelic mutations in FANCD1 and FANCN are at increased risk of developing acute myeloid leukemia and embryonal tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. FANCI has mutations in hepatocellular carcinoma, colorectal cancer, breast cancer, and ovarian cancer. FANCI is a potential biomarker for the prognosis of hepatocellular carcinoma [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A lot of relevant papers have also shown that the high expression of FANCI in tumors is closely related to the survival rate and poor prognosis of cancer patients, such as FANCI may interact with extracellular matrix receptors to affect the poor prognosis of lung adenocarcinoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and pathogenic FANCI mutations increase the risk of familial ovarian cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the reports of the mechanism of FANCI in glioma development and progression are still very few.\u003c/p\u003e \u003cp\u003eIn this study, we first used TCGA and CGGA databases to analyze the expression of FANCI in different grades of glioma and its correlation with glioma patient\u0026rsquo;s prognosis. Then, the effects of FANCI on glioma cell growth in vitro and in vivo were explored at the cellular and animal level, respectively. Finally, we found that FANCI expression in glioma tissue was higher compared to non-tumor tissue, suggesting that FANCI upregulation may be closely related to glioma growth. After silencing the expression of FANCI, it inhibited the proliferation, migration, and invasion and promoted the apoptosis ability of cells. FANCI knockout may also promote tumor cell apoptosis through the Akt/Bcl2 pathway. To sum up, the findings suggest that FANCI may be a potential target for glioma therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eTissue specimens\u003c/h2\u003e\n \u003cp\u003eTissue samples were retrieved from HuanHu Hospital in Tianjin, China, with a total of 5 cases of non-tumor brain tissue (traumatic brain injury) and 42 cases of glioma tissue. Include 9 cases of WHO Ⅰ grade (oligodendroglioma), 6 cases of WHO Ⅱ grade (astrocytoma), 8 cases of WHO Ⅲ grade (five of anaplastic oligodendroglioma and three of anaplastic astrocytoma), and 19 cases of WHO Ⅳ grade (GBM). The patients from whom the samples were derived did not receive any anti-cancer therapies in preoperative treatments, such as chemoradiotherapy. Samples be placed at -80\u0026deg;C for later use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eThe human glioma cells (U251, LN229, U87, A172) and normal glial cells (NHA) came from the Laboratory of Medical Research Center, Affiliated Hospital of Jining Medical College. All cell lines were grown using DMEM medium (DMEM, Gibco) supplementing with 10% fetal bovine serum (FBS, Excell) and 1% penicillin-streptomycin (Gibco) in a 37\u0026deg;C, 5% CO2 cell culture incubator.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCell transfection\u003c/h2\u003e\n \u003cp\u003eFour different FANCI-specific small interfering RNA (siRNA) sequences (Shanghai, Genepharma) was used in this study to inhibit the expression of FANCI in glioma cells. Next, pcDNA3.1-FANCI overexpression plasmid (Shanghai, Genechem) targeting human FANCI were synthesized to promote the expression of FANCI. And the effect of transfection verified by real-time PCR and western blot.\u003c/p\u003e\n \u003cp\u003eSequence 2267: sense (5\u0026apos;-3\u0026apos;): GCACCAGUAUUGGCAUAAATT;\u003c/p\u003e\n \u003cp\u003eantisense (5\u0026apos;-3\u0026apos;): UUUUUGCCAAUACUGGUGCTT\u003c/p\u003e\n \u003cp\u003eSequence 1292: sense (5\u0026apos;-3\u0026apos;): GCCCAAGUCUUUAGAAUTT;\u003c/p\u003e\n \u003cp\u003eantisense (5\u0026apos;-3\u0026apos;): AUUCUAGAAAGACUUGGGCTT\u003c/p\u003e\n \u003cp\u003eSequence 2097: sense (5\u0026apos;-3\u0026apos;): GGCCUGGUAUAAGAAUACATT;\u003c/p\u003e\n \u003cp\u003eantisense (5\u0026apos;-3\u0026apos;): UGUAUUCUAUACCAGGCCTT\u003c/p\u003e\n \u003cp\u003eSequence 573: sense (5\u0026apos;-3\u0026apos;): CAGGUGGGAUCAGCAAUAUTT;\u003c/p\u003e\n \u003cp\u003eantisense (5\u0026apos;-3\u0026apos;): AUAUUGCUGAUCCCACCUGTT\u003c/p\u003e\n \u003cp\u003eNegative control sequence: sense (5\u0026apos;-3\u0026apos;): UUCUCCGAACGUGUCACGUTT;\u003c/p\u003e\n \u003cp\u003eantisense (5\u0026apos;-3\u0026apos;): ACGUGACACGUUCGGAGAATT\u003c/p\u003e\n \u003cp\u003eCells are seeded in 6-well plates one day before transfection according to the company\u0026apos;s guidelines to ensure a cell density of 30%-40% at seeding. siRNA or plasmid transfected by Lipofectamine 3000 reagent (Invitrogen, USA), and subsequent cell function experiments after transfection for 48h. siRNA 1292 was selected as the subsequent lentiviral (Shanghai, Genechem) infection sequence based on the transfection results. After stable transfection 48h, select the target cells with 0.5 \u0026micro;g/mL puromycin for around 14 days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative Reverse-Transcription PCR\u003c/h2\u003e\n \u003cp\u003eExtract total cellular RNA using Trizol reagent (Invitrogen) and store at -80\u0026deg;C for later use after determining RNA concentration. According to the Fastking RT Kit (with gDNase) (Tiangen) Usage Guide, synthesize cDNA and store it at -20\u0026deg;C for later use. Using the SYBR Green Assay Mix Kit (Kang Wei Century) on the real-time PCR detection system (Thermo Fisher) perform Real-time PCR experiments. The total working solution system is 20ul, including 0.4ul of forward primer, 0.4ul of reverse primer, 9ul of SYBR reagent, 2ul of cDNA, and 8.6ul of deionized water. The 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method determines the relative expression of the target gene. The relative expression of FANCI in each glioma cell was standardized with GAPDH as an internal reference. All experiments were performed in three replicates and were independent.\u003c/p\u003e\n \u003cp\u003ePrimer sequences (AG, Accurate Biology) are as follows:\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003cp\u003eFANCI: F: 5\u0026rsquo;-CTCCTCCAAGGGAAGCAGAAAGA-3\u0026rsquo;\u003c/p\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003cp\u003eR: 5\u0026rsquo;-GGCACAGTGACAACATCCAATAGC-3\u0026rsquo;\u003c/p\u003e\n \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e\n \u003ch2\u003eCCK-8 Cell Proliferation assay\u003c/h2\u003e\n \u003cp\u003eAbout 2,000 cells per well were seeded in a 96-well plate, with five replicate wells a day, placed in a cell culture incubator for 1, 2, 3, 4, and 5 days. Each well-added 100\u0026micro;l working solution, which mixed 10\u0026micro;l CCK-8 reagent (Dojindo, Shanghai) and 90\u0026micro;l DMEM, was next put back incubated for 2 hours and then represented proliferative capacity of glioma cells as the absorbance of each well at 450 nm.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eEdU Cell Proliferation assay\u003c/h2\u003e\n \u003cp\u003ePlace suitable coverslips into 24-well plates, then seed around 8,000 cells per well and incubate for 24\u0026ndash;48 hours. Add into per well with 250\u0026micro;l the diluted Edu working solution (20\u0026micro;M) with DMEM, and continue the incubation for two hours. Remove the 24-well plate for 4% paraformaldehyde fixation, and use the EdU cell proliferation kit (Abbkine) to prepare an appropriate amount of staining solution according to the instructions. After staining, BSA is washed twice. Prepare an appropriate amount of 1\u0026times; Hoechst reaction solution and perform DNA staining for 10 min. BSA wash 2 times and dry moderately. Observe and photograph under an inverted fluorescence microscope and manipulate the relative proliferation rate of cells using ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eWound healing assay\u003c/h2\u003e\n \u003cp\u003eMark the back of the 6-well plates in advance. Then, inoculate the cells in the 6-well plates at about 1\u0026times;10^\u003csup\u003e6\u003c/sup\u003e cells per well to ensure 100% confluency the next day. Scrape the cell monolayer vertically with a 200\u0026micro;l sterile pipette, wash with PBS, and discard the floating cells. The lower serum DMEM (2%FBS) was substituted for complete DMEM (10%FBS). Remove at 0 and 48h, observe under an inverted microscope, and take pictures. Cell migration area comparison was performed using ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eCell migration and invasion assays\u003c/h2\u003e\n \u003cp\u003eFor cell migration, aliquots of 2\u0026times;10\u003csup\u003e^5\u003c/sup\u003e cells with serum-free DMEM per well in the upper chamber of the Transwell insert chambers (pore size 8\u0026micro;m, Corning) without Matrigel, add 600ul of DMEM containing 10% FBS in the lower chamber. For cell invasion experiments, an upper chamber was precoated with Matrigel (Corning). After 48h, the chamber was removed and fixed with 4% paraformaldehyde 30min, stained 10min with crystal violet, and gently wiped the Transwell chamber inner surface with a cotton swab. After properly air-drying, observe it with a microscope and took photography. The number of cells passed through each field of view using ImageJ software processing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eFlow Cytometry\u003c/h2\u003e\n \u003cp\u003eBriefly, take about 1\u0026times;10\u003csup\u003e^6\u003c/sup\u003e cells after cell transfection 48h, with 1800rpm, centrifugation for 5min. cells were resuspended with 400\u0026micro;l of 1\u0026times; Binding Buffer from apoptosis kit (BD, Biosciences). Then, in the dark conditions add 5\u0026micro;l of propidium iodide (PI) and 5\u0026micro;l Annexin V, mix thoroughly, and incubate at room temperature for 10min. Finally, analyze the apoptosis rate of cells by flow cytometer (Beckman Coulter). Apoptosis rate calculation = (Q1-UR\u0026thinsp;+\u0026thinsp;Q1-LR)/Q1-LL%. Q1-UR represents the proportion of late-stage apoptotic cells in the upper right quadrant, Q1-LR represents the proportion of early-stage apoptotic cells in the lower right quadrant, and Q1-LL represents the proportion of viable cells in the lower left quadrant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting\u003c/h2\u003e\n \u003cp\u003eThe extraction of total cell protein using RIPA lysate with protease inhibitors and protein quantification by BCA Protein Quantitation Kit (Beyotime). Prepare a 10% protein gel and polyvinylidene difluoride membranes (0.45\u0026micro;m PVDF, Millipore, USA). After the protein is transferred to the membrane, the membrane is closed with 5% skimmed milk powder for two hours, and the primary antibody is incubated overnight at 4\u0026deg;C. The membrane reacted with the corresponding secondary antibody binding reaction at room temperature for 1\u0026ndash;2 h the next day, followed by band development using ECL Western Blot luminescence solution (Millipore). In the end, protein band gray value analysis using ImageJ software. The antibodies were as follows: anti-GAPDH (Affinity, 1:1000), anti-FANCI (Abcam, 1:5000), Akt (ABclonal, 1:1000), Phospho-Akt (Ser473) (Always, 1:1000), Bcl-2 (Cell Signaling, 1:1000), Bax (Cell Signaling, 1:1000), HRP-anti-rabbit/mouse (Affinity, 1:5000-1:8000).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eSubcutaneous tumor xenografts in nude mice\u003c/h2\u003e\n \u003cp\u003eBALB/c nude mice (4\u0026ndash;5 weeks old) were purchased from Peng Yue Animal Breeding Center in Jinan, China. And these mice raised in the SPF-conditioned animal house of Jining Medical College. Stably transfected U251 cells with knockdown for FANCI or negative controls were injected into the right subcutaneous part of each mouse (8 mice in total). After two weeks, observed the growth of the tumor every five days, and sacrificed the mice at four weeks later. Tumors were weighed and treated with 4% paraformaldehyde, and we retained a part of tumor tissue for further histological analysis and RNA extraction. We promise that All experiments involving animals conducted following the protocols of the animal ethics committee approved by the institution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eFluorescent immunohistochemistry\u003c/h2\u003e\n \u003cp\u003eThe tissue sections are first deparaffinized and fixed. The tissue is then repaired with sodium citrate repair solution at high temperatures, and remove the residual solution. Follow the instructions for the fluorescent immunohistochemistry kit (Absin). Block with 5% BSA for one hour at room temperature. Then, incubate with rabbit Ki-67 (Affinity, 1:200) primary antibody for 1 hour at room temperature, and rinse with PBS 3 times for 5 min each time. Dropwise the secondary antibodies, and at room temperature for 1 hour, rinse with PBS 3 times, 5 minutes each time. After DAPI staining, rinse with PBS. Finally, after adding an appropriate amount of autofluorescence quenching reagent to the tissue, the mounting was observed and photographed under a fluorescence microscope after mounting treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analyses\u003c/h2\u003e\n \u003cp\u003eData statistical analysis using GraphPad software 8.0. Values from at least three independent experiments. We used the two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests to test the statistical differences between the two groups. The One-way ANOVA test for the between-group comparison. The Kaplan-Meier method and log-rank test plotted the survival curve with statistical differences. A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 indicates a statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eFANCI is highly expressed in glioma tissues and is associated with poor prognosis in glioma patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TCGA database utilizes the TIMER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.xistrome.org\u003c/span\u003e\u003c/span\u003e) analysis tool to show that FANCI is highly expressed in a variety of cancers, including gliomas (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The TCGA database used the GEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003c/span\u003e) analysis tool to show that FANCI was significantly overexpressed in GBM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), and low expression of FANCI was associated with a good prognosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results of CGGA database showed that the high expression of FANCI in both primary gliomas and recurrent gliomas indicated poor prognosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Moreover, the higher the WHO grade, the higher the expression levels of FANCI (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eTo verify the above information, we further analyzed the relationship between FANCI and glioma using the human glioma tissue chip (HBraG160Su01, Shanghai Outdo Biotech Co., Ltd.), and the immunohistochemistry results again showed that FANCI positively correlated with glioma WHO grade (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG). The survival rate was worse in patients with high FANCI expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). In addition, there were statistically significant differences in the expression of FANCI in recurrent or non-recurrent gliomas, as well as between WHO low-grade or high-grade (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eJ-K). The chi-square analysis results of the differences between the clinicopathological characteristics and the expression of FANCI in 152 of glioma microarray are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The results showed that the age\u0026thinsp;\u0026ge;\u0026thinsp;40 (\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;6.848, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.009), grade Ⅲ-Ⅳ (\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;16.882, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), high level of PD-L1 expression (\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.746, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.029) were statistically significant with FANCI expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, Cox regression analysis showed that age\u0026thinsp;\u0026ge;\u0026thinsp;40, grade Ⅲ-Ⅳ, and Ki67 high expression were independent factors predicting the prognosis of glioma patients (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCorrelation between FANCI expression and clinicopathological characteristics (N\u0026thinsp;=\u0026thinsp;152)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003evariables\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFANCI expression\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003etotal\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003elow\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ehigh\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge (year)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.848\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.009\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026ge;\u0026thinsp;40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSex\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.688\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGrade\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.882\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eI-II\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIII-IV\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eki67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.160\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.689\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEGFR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.337\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.561\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.746\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.029\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLow\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e104\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e\u003csup\u003e*\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003csup\u003e***\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePrognostic Value of FANCI Expression with Overall survival in Patients with Gliomas\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVariables\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eUnivariate analysis\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMultivariate analysis\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95%CI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95%CI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSex (female/male)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.684\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.930\u0026ndash;3.049\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.085\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.448\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.766\u0026ndash;2.737\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.255\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge (\u0026lt;\u0026thinsp;40/\u0026ge;40)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.971\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.942\u0026ndash;8.119\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.582\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.688\u0026ndash;7.598\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGrade (I-II/ III-IV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.267\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.136\u0026ndash;28.523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.753\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.061\u0026ndash;35.145\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKi67 (low/high)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.288\u0026ndash;0.866\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.213\u0026ndash;0.710\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEGFR (low/high)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.106\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.647\u0026ndash;1.892\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.713\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePDL1 (low/high)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.078\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.608\u0026ndash;1.909\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.798\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFANCI (low/high)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.499\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.023\u0026ndash;6.053\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e* Statistically significant(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe also obtained the expression levels of FANCI in clinical glioma tissue, which included five normal peritumor tissues and 34 glioma tissues (10 cases of lower grade and 24 cases of higher grade), by the qRT-PCR analysis. Our results are consistent with the results of the public bioinformatics database analysis described above, where FANCI has higher mRNA levels in glioma high-grade tissues than in lower-grade tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The above results preliminarily indicate that FANCI may be a critical gene for glioma development.\u003c/p\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eFANCI regulates the biological behavior of glioma cells in vitro\u003c/h2\u003e\n\u003cp\u003eIn this study, we first verified the expression of FANCI in glioma cell lines (NHA, LN229, U251, U87, A172). The results showed that FANCI was expressed higher in LN229 and U251 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-D), so in subsequent cell function experiments, we selected knockdown LN229 and U251 cells with high FANCI expression and overexpression A172 cells with low FANCI to verify our results. Considering that the knockdown transfection effect of the sequence si-1292 on LN229/U251 glioma cells was good, we chose to use the si-1292 sequence as the lentivirus template in the future (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE, G). In addition, the results showed that the low expression of FANCI in lentivirus-infected cells (KD-FANCI) was well (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF-G).\u003c/p\u003e\n\u003cp\u003eThe results of the CCK-8 proliferation assay of LN229 and U251 cells showed that the low expression of FANCI inhibited the proliferation of cells compared with the control group (NC) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). In addition, we performed an Edu cell proliferation assay to verify the proliferation rate of glioma cells, and the results showed that inhibition of FANCI expression led to a decrease in Edu-positive cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI). Next, cell migration experiments (wound scratch healing experiment and transwell migration experiment) analyzed the effect of FANCI on glioma cell migration ability. The results of the wound healing experiment showed that low of FANCI expression significantly reduced the migration distance of LN229/U251 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Further, we used the Transwell assay to verify the effect of FANCI on the migration ability of glioma cells, such as the results of the scratch assay were consistent, and the low of FANCI expression significantly inhibited the migration of glioma cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eThe transwell invasion assay (including matrigel) evaluated the effect of FANCI on the aggressiveness of glioma cells, and the results also showed that knocking down FANCI expression inhibited the aggressiveness of glioma cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). That prompts that FANCI may be involved in the movement of glioma cells through the body. In addition, the results of apoptosis experiments suggest that decreased FANCI expression promotes the apoptosis ability of glioma cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eAt the same time, we also back-validated the above results by FANCI overexpression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), and our results showed that FANCI overexpression promoted the proliferation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D), invasion ability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE), migration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF-G), and inhibition of apoptosis of glioma cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). Therefore, data from cell function experiments prove that FANCI is involved in the biological processes of proliferation, migration, invasion, and apoptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eFANCI may mediate apoptosis by regulating the Akt/Bcl2 pathway\u003c/h2\u003e\n\u003cp\u003eIn addition, since the PI3K/Akt pathway is one of the classical signaling pathways in malignant glioma, and the literature indicates that Akt is also an upstream factor of the Bcl2-mediated apoptosis pathway [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], we speculate that FANCI may regulate the apoptosis process of glioma cells through the PI3K/Akt pathway. And, it is worth mentioning that the results of our Western blot experiment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) showed that knocking down of FANCI expression in LN229 cells and U251 cells inhibited the phosphorylation expression of Akt (P-Akt ser473) and also reduced the expression of Bcl-2. The results may suggest that FANCI influences apoptosis in glioma through the Akt/Bcl2 pathway possibly.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eKnocking down of FANCI inhibit the growth of nude Xenografts tumor in vivo\u003c/h2\u003e\n\u003cp\u003eTo further verify whether FANCI affects the growth of gliomas in vivo, we established a nude mouse xenograft tumor model by subcutaneous injection in the lentiviral transfection U251 cell control group (CON) and knocking down group (KD-FANCI). The results showed that the size of xenograft tumors formed by nude mice of KD-FANCI group was significantly smaller than that of the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B), and the weight of the tumors was also lower than that of the control group significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). The qRT-PCR technology and Western blotting technology verified the expression of FANCI in xenograft tumor tissues, and the results confirmed that the expression of FANCI in the KD-FANCI group was lower than that in the CON group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). At the same time, the results of immunopathological staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF) and immunofluorescence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG) showed that the expression levels of Ki-67 in tumor tissues of the KD-FANCI group decreased. These in vivo data suggest that FANCI inhibits the progression of gliomas in vivo.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, many studies have found that silencing FANCI expression can inhibit the occurrence and development of cancers, such as ovarian cancer and lung adenocarcinoma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and also enhance the chemotherapy sensitivity of ovarian cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Glioma has become a worldwide problem because of its resistance to chemoradiotherapy and poor prognosis. Therefore, FANCI has important practical significance in the occurrence and development of glioma and the resistance mechanism of chemoradiotherapy. In this study, we will preliminarily explore the role of FANCI in the development of glioma.\u003c/p\u003e \u003cp\u003eNot only was the FANCI expression elevated in glioma tissues and cells, but it also correlates with WHO grade positively. At the same time, the FANCI expression is close to the survival and disease-free survival in glioma patients. It is similar to the results of a meta-analysis that there is a possibility that the tumor stage may affect the potential prognostic value [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The higher FANCI expression may not significantly affect OS in GBM patients. It is worth noting that the results of COX regression analysis indicated that Ki-67 was an independent prognostic factor for OS in glioma, which is also consistent with the literature [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Unexpectedly, FANCI was not an independent prognostic factor for OS in glioma patients. It also may imply that glioma grade is one of the reasons interfering with the relationship between FANCI and patient OS. However, these results suggest that FANCI is involved in the malignant biological process of glioma and related to the prognosis of glioma patients. The experiments have shown that FANCI promotes the occurrence and development of glioma by promoting the proliferation, migration, invasion, and inhibition of apoptosis of glioma cells. Consistent with previous studies in lung cancer tissues, the knockdown of FANCI expression will inhibit tumor growth [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Taken together, FANCI may be a potential target for glioma therapy.\u003c/p\u003e \u003cp\u003eOne cause of cancer is endogenous DNA replication mutations or exogenous DNA damage. However, some specialists suggest that exogenous factors may account for the majority [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. But in any case, genomic instability is one of the bases of cancer development, which can accelerate the acquisition of genetic diversity and the occurrence of inflammatory responses in organisms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. FA is a gene-disease known for chromosomal instability, and evidence is accumulating that a wide variety of cancers with different FA pathway defects. The FA pathway may be helpful to hyperresponsive to cross-linkers to obtain more effective therapy by interference with it [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Interestingly, the literature has shown that the FA pathway is closely related to the PI3K/Akt pathway, and FANCI is a vital regulator of Akt activation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe PI3K-Akt signaling pathway is one of the classic activation pathways in cancer, which controls cell proliferation, cell metabolism, cell motility, apoptosis, and genomic instability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. And the PI3K/Akt is also involved in the mechanism of cell drug resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, Akt kinase is a key effector molecule of this pathway and is implicated in DNA damage [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. One of the mechanisms by which cells survive is that Akt inhibits pro-apoptotic proteins and apoptotic processes, such as the anti-apoptotic protein Bcl-2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. There is a strong link between Akt activation and glioma prognosis, with a retrospective study in children showing elevated p-Akt (phosphorylation of Akt) expression in more than half of patients with high-grade glioma and a declining trend in overall survival [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. According to the literature, phosphorylation of Ser 473 residues is necessary for the complete activation of Akt [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our results suggest that FANCI has a carcinogenic effect in glioma and may be one of the crucial molecules in the Akt/Bcl2-mediated anti-apoptotic pathway, which is worthy of further exploration of the potential of FANCI as a combination therapy regimen for PI3K/Akt signaling pathway in the treatment of glioma. However, the mechanism of action of FANCI in the AKT/Bcl2 pathway needs to be further verified and explored.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Tianjin Huanhu Hospital for providing clinical samples very much. This study was supported by the Natural Science Foundation of Shandong Province [ZR2022MH140], China Postdoctoral Science Foundation [2022M711324], Shandong First Medical University youth Science Foundation [202201-089], Project of Scientific Developmental Program of Shandong Provincial Administration of Traditional Chinese Medicine [Q-2022136], Project of the Key Research and Development Program of Jining Science and Technology [2023YXNS045], and Key Project of Shandong Provincial Traditional Chinese Medicine Science and Technology Project [Z-2022082].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailable of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data are listed in the article, please contact the corresponding author for further needs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe idea of this study was proposed by SJK. LHY, LJL and QXY was responsible for the experimental operation and LHY writing this article, WMY and LYR conducted the animal experiment part. SJK, JF and LY revised the final draft. All authors have read and agree to publish the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no proprietary or commercial interest in any product mentioned or concept discussed in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSchaff LR, Mellinghoff IK (2023) Glioblastoma and Other Primary Brain Malignancies in Adults: A Review. JAMA 329: 574-587. doi: 10.1001/jama.2023.0023.\u003c/li\u003e\n \u003cli\u003eOstrom QT, Cioffi G, Waite K, Kruchko C, Barnholtz-Sloan JS (2021) CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018. 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Mol Med Rep 22: 1527-1535. doi: 10.3892/mmr.2020.11224.\u003c/li\u003e\n \u003cli\u003eDummler B, Hemmings BA (2007) Physiological roles of PKB/Akt isoforms in development and disease. Biochem Soc Trans 35: 231-235. doi:10.1042/bst0350231.\u003c/li\u003e\n \u003cli\u003eChautard E, Ou\u0026eacute;draogo ZG, Biau J, Verrelle P (2014) Role of Akt in human malignant glioma: from oncogenesis to tumor aggressiveness. J Neurooncol 117: 205-215. doi:10.1007/s11060-014-1382-9.\u003c/li\u003e\n \u003cli\u003eAntonelli M, Massimino M, Morra I, Garr\u0026egrave; ML, Gardiman MP, Buttarelli FR, Arcell A, Giangaspero F (2012) Expression of pERK and pAKT in pediatric high grade astrocytomas: correlation with YKL40 and prognostic significance. Neuropathology 32: 133-138. doi:10.1111/j.1440-1789.2011.01252.x.\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":"Glioma, FANCI, Malignant biological behavior, Akt/Bcl2, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-3890361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3890361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that FANCI has cancer susceptibility, and high expression of FANCI promotes the progression of breast cancer, ovarian cancer, and other cancers. However, the potential mechanism of action of FANCI in glioma progression is unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo explore the role of FANCI in glioma progression, we determined the expression of FANCI in glioma patients and its relationship with prognosis through database analysis and gene chip. And then further conducted in vitro functional experiments (overexpression and knockdown) and in vivo nude mouse xenograft model experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis study found that FANCI was significantly overexpressed in glioma, positively correlated with WHO grade, and closely related to patient prognosis. In vitro functional experiments showed that inhibiting the expression of FANCI could inhibit the proliferation, migration, and invasion of glioma and promote apoptosis. At the same time, the nude mouse xenograft model also confirmed that inhibition of FANCI could inhibit glioma in vivo. In addition, the low expression of FANCI inhibited the phosphorylation of Akt and the expression of Bcl-2 by western blotting.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFANCI promotes glioma growth and may mediate apoptosis by regulating Akt/Bcl-2. This study preliminarily explored the role of FANCI in glioma growth and provided data support for further application of FANCI in clinical practice.\u003c/p\u003e","manuscriptTitle":"Knocking down of FANCI expression inhibits the biological behavior of glioma and mediates apoptosis by downregulating the Akt/Bcl2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 16:21:09","doi":"10.21203/rs.3.rs-3890361/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":"631fa152-8cdc-4019-894d-6b882167e245","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T18:02:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-25 16:21:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3890361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3890361","identity":"rs-3890361","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}