SAP30 promotes clear cell renal cell carcinoma proliferation and inhibits apoptosis through the MT1G/P53 axis

SAP30 promotes clear cell renal cell carcinoma proliferation and inhibits apoptosis through the MT1G/P53 axis | 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 SAP30 promotes clear cell renal cell carcinoma proliferation and inhibits apoptosis through the MT1G/P53 axis Wei Xue, Wei Guo, Shuwen Wang, Yu Dong, Zitong Yang, Zhinan Xia, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4164049/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 Sin3-associated polypeptide p30(SAP30) is an important component of the SIN/HDAC histone deacetylase complex that act as a scaffolding and facilitates target gene binding. SAP30 is highly expressed in a variety of tumors, however; its role in renal cell carcinoma is still unclear. In our study, we found that SAP30 was upregulated in tissues of renal clear cell carcinoma (ccRCC), and high SAP30 expression was associated with a poor prognosis. According to relevant studies, SAP30 may be associated with the growth, proliferation and apoptosis of renal cell carcinoma cells, and GO analysis of SAP30 downstream regulatory target genome showed that SAP30 repressed the expression of MT1G, a P53-binding protein. Mechanistically, SAP30 inhibits MT1G expression at the transcriptional level, reducing the ability of MT1G to deliver to zinc ions to P53, thus reducing P53 activity, and the downregulation of MT1G also attenuates the inhibition of MDM2, thereby reducing the stability of P53, which ultimately promotes the development of renal cell carcinoma. In summary, our study shows that SAP30 inhibits the P53 pathway by inhibiting MT1G, suggesting that SAP30 and MT1G may become markers of renal cell carcinoma prognosis and therapeutic targets. Transcription factor P53 SAP30 MT1G ccRCC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Renal cell carcinoma (RCC) is one of the most common malignancies in the urinary system and accounts for nearly 5% of all malignancies [ 1 ]. Of all the subtypes of RCC, clear cell renal cell carcinoma is the most common, accounting for more than 90% of cases [ 2 ]. Although surgery is still the primary treatment modality for ccRCC in the early stages, more than 30% of patients have developed distant metastases before diagnosis and postoperative survival is not particularly satisfactory [ 3 , 4 ]. In addition, its poor prognosis is closely linked to its insensitivity to radiotherapy/chemotherapy and the lack of effective biomarkers [ 5 ]. Thus, it is particularly urgent for us to find suitable biomarkers. Sin3-related polypeptide p30 (SAP30) is an essential part of the SIN/HDAC histone deacetylase complex, which is widely expressed in human tissues, especially haematopoietic tissues [ 6 ], and mainly functions as a transcriptional repressor [ 7 ]. SAP30 acts as a scaffold for SIN3, which acts as a repressor and corepressor of gene expression in the presence of binding proteins [ 8 , 9 ]. Its biological functions include DNA and histone methylation regulation, nucleosome remodelling, histone deacetylase scaffolding, and N-acetylglucosamine transferase activity [ 9 ]. SAP30 mainly performs transcriptional repression through two mechanisms. SAP30 binds to transcription factors such as YY1 and recruits HDAC1 [ 10 , 11 ]. SAP30 binds directly to DNA sequences, bending them structurally to achieve transcriptional inhibition [ 12 ]. The effect of knocking out the SAP30 gene in yeast was reported to be similar to that of knocking out SIN3 and Rpd3, which have various functions, such as affecting cell cycle regulation, apoptosis, mitochondrial metabolism, DNA replication and repair [ 6 , 13 – 15 ]. Therefore, it can be inferred that SAP30 knockdown may also affect cell cycle regulation and apoptosis [ 16 ]. However, SAP30 has rarely been studied in various tumors, especially the mechanism of action in the occurrence and development of ccRCC is still unclear. Here, we show that SAP30 inhibits the expression of metallothionein-1G (MT1G), a small cysteine-rich protein that plays an important role in metal homeostasis and protection against heavy metal toxicity, DNA damage, and oxidative stress, at the transcription level [ 17 ]. MT1G is expressed in a variety of tumors, and is involved in cell growth and proliferation as well as the EMT process [ 18 , 19 ]. It has been reported that MT1G increases the stability of P53 by inhibiting the P53 ubiquitinase MDM2 [ 20 ]. MT1G can also interact directly with P53 to provide zinc ions to P53, thereby increasing P53 transcriptional activity [ 20 ]. However, MT1G has been reported relatively little in kidney cancer. In this study, we demonstrated that SAP30 affects the proliferation and apoptosis of kidney cancer cells through the MT1G/P53 axis in vivo and in vitro. This molecular mechanism has the potential to be a new clinical therapeutic target with value for further research. 2 Methods and Materials 2.1 Cell lines, cell culture and tissue specimens Human renal cancer cell lines (786-O, CAKI-1 OS-RC-2, and CAKI-2) and human renal tubular epithelial cells (HK-2) were all purchased from the Cell Resources Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. All cell lines were cultivated in the recommended medium supplemented with 10% heat-inactivated foetal bovine serum (Gibco, Beijing, China) and 1% streptomycin/penicillin (Keygen, Nanjing, China) at 37°C and 5% CO2. RCC tissue specimens were obtained from 40 patients who were treated by radical nephrectomy or preserved kidney surgery at the Department of Urology, First Affiliated Hospital of Harbin Medical University between 2010 and 2019. All diagnoses were confirmed by histopathological examination. 2.2 Antibodies and reagents The following antibodies were used for Western blotting: SAP30 (#LS-C676447, Lsbio), MT1G (#323730, US Biological), P53 (#60283-2-Ig, Proteintech), Bax (#50599-2-Ig, Proteintech), Bcl2 (#12789-AP, Proteintech), CCNB1 (#28603-AP, Proteintech), CCND1 (#60186-I-Ig, Proteintech), and CCNL2(#LS-C749918, LSBio). And the anti-SAP30 antibody for IHC was purchased from Lsbio(#LS-C676447). The anti-SAP30 antibody for ChIP-PCR was purchased from Abcam (#ab231804). Sunitinib was purchased from MCE (#HY-10255A). 2.3 siRNA and plasmid transfection The siRNAs for SAP30 and MT1G were purchased from GenePharma (Suzhou, China), and their sequences are shown in Supplementary Table S1 . Transfection of the siRNA-SAP30 and siRNA-MT1G vectors was performed using jetPRIME (PolyPlus, Shanghai, China). The plasmids (pENTER-SAP30, pENTER-NC, pCMV3-SAP30 and pCMV3-NC) were purchased from Weizhen (Shandong, China) and SinoBiological (Beijing, China). Briefly, 1.2–1.3 x10 6 cells were loaded in a 6 cm culture dish to prepare for transfection and collect cells for subsequent experiments after 47–72 hours. 2.4 Quantitative real-time PCR (qRT‒PCR) Total RNA from cells and tissues was extracted using TRIzol reagent (Ambion,USA). Reverse transcription kits (TianGen, Beijing, China) and PCR kits (SYBR Green) (TianGen, Beijing, China) were used to perform RT ‒ qPCR according to the manufacturer’s instructions. The sequences of primers were provided in the Supplementary Table S1 . The results were analysed by the 2 -ΔΔCt method to quantify the fold change. 2.5 Western blotting analysis and immunohistochemical staining (IHC) Proteins from nude mouse kidney cancer cells and tissue and tumor cell lines were extracted with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Quantified proteins were separated by SDS ‒ PAGE and transferred to PVDF membrane. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with the corresponding antibodies. For the IHC, paraffin-embedded 4-µm-thick kidney cancer tissue sections were deparaffinized, subjected to antigen retrieval, blocked with 5% bovine serum albumin (BSA) for 2 hours at room temperature, and then incubated with antibodies against SAP30 (1:400) at 4°C. The next day peroxidase-conjugated polymer was added for 30 min. Finally, visualization was performed with DAB (Beyotime, Shanghai, China). 2.6 CCK-8 cell proliferation assay We used the CCK-8 assay (MCE, China) to analyse cell proliferation. Transfected cells were grown in 96-well plates (3000 cells/well) for 12 hours. Then 10% CCK8 solution was added at different time periods (0/24/48/72 h) according to the CCK8 kit manufacturer's protocol. The OD absorbance values were measured at 450 nm using a 96-well plate reader. 2.7 Colony formation assay Cell colony formation capacity was assessed by a plate colony formation assay. Transfected 786-O, CAKI-1 and OS-RC-2 cells (1 × 10 3 cells/well) were seeded in 6-well plates for 14 days to form colonies, which were subsequently fixed in paraformaldehyde and then stained with crystal violet. 2.8 Hoechst staining A apoptosis of transfected cells was induced with H2O2. After fixation with paraformaldehyde, the cells were washed with PBS 3 times. Finally, the cells were stained with Hoechst dye (#C1017, Beyotime, China) for 5 min and photographed under a microscope. 2.9 Flow cytometry First, the transfected cells were trypsinized with EDTA-free trypsin and washed with cold PBS according to the manufacturer's instructions of the ANNEXIN V-FITC/PI Apoptosis Kit (Solarbio, Beijing, China). Second, apoptosis was examined in the dark with Annexin V-FITC/propidium iodide (PI) staining. Finally, cell cycle profiles were analysed by PI staining and flow cytometry according to the manufacturer's instructions of the Cell Cycle Detection Kit (Beyotime, Shanghai, China). 2.10 Chromatin immunoprecipitation assay (ChIP) The chromatin immunoprecipitation assay with 786-O and CAKI-1 cells was performed according to the protocol for the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads). First, 1x10 7 cells were fixed with 1% formaldehyde and 1.25 M glycine was added to terminate the reaction. The cells were washed with cold PBS containing protease inhibitor and collected into a centrifuge for the next step. The last collected cells were sonicated in IP mixed medium and centrifuged at 14, 000 × g for 30 min at 4°C. Protein A/G agarose beads were mixed with the above supernatant and centrifuged, and the chromatin in the supernatant was immunoprecipitated overnight with antibodies against SAP30 or IgG. The protein A/G agarose beads were then washed with mixed wash buffer, LiCl/detergent and TE buffer. The beads were heated overnight at 65°C in a buffer mixture containing 1% SDS, 0.1 M NaHCO3 to crosslink the beads in reverse. The beads were then treated with 1 ml of RNaseA for 15 min at 37°C and digested with 2 ml of proteinase K solution for 1 hr at 37°C. Finally, DNA was purified by extraction with LiCl, phenol/chloroform and ethanol, and the specific MT1G promoter region was amplified with DNA template. Supplementary Table S1 lists the primer sequences designed to detect specific promoters. 2.11 Luciferase reporter assay The SAP30 overexpressing plasmid pcDNA3.3-SAP30 and the plasmid pGL4.18WT + binding site (BS1, BS2 and BS3) containing the promoter region of MT1G were constructed. Binding site sequences for promoter reporter plasmid construction are listed in Supplementary Table S1 . 293T Cells cultured in 6-well plates were transfected with overexpression plasmids or empty vectors. Luciferase activity was measured using a dual luciferase reporter system according to the manufacturer's instructions (E1910, Promega, USA). 2.12 Detection of EdU incorporation Cells (3 x 10 5 cells/well) were seeded on coverslips in 6-well plates for 24 hours. Cells were then cultured in culture medium containing EdU for 2 h after 48 h of transfection. The cultures were subsequently stained with Azide 488 and Hoechst 33342. EdU-positive cells were observed under a fluorescence microscope and photographed. The EdU binding rate was expressed as the ratio of EdU-positive cells (green cells) to total Hoechst-labelled cells (blue cells). 2.13 Immunofluorescence (IF) Cells (3 × 10 5 cells/well) were inoculated in 6-well plates with coverslips for 24 hours. First, after washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.05% Triton for 30 min, blocked with 5% BSA for 2 h, and incubated with antibodies against SAP30 overnight at 4°C. The next day, the type A fluorescent secondary antibody was incubated for 1 hour at room temperature. Finally, nuclear staining was performed with DAPI and subsequent imaging was performed with fluorescence microscopy. 2.14 Xenograft tumor growth 786-O cells (3 × 10 7 cells/ml) were injected into the posterior ventral sides of 6-week-old male nude mouse, purchased from Charles River Laboratories (Beijing, China). After 6 days, visible tumors were visible, their volume was measured, and transfection was performed according to the instructions of the EntransterTM-In Vivo kit (Engreen, Beijing, China) and repeated every 3 days. The transfection was repeated every 3 days. Forty-eight days later, the nude mice were sacrificed, the tumors were removed, and their weights and volumes were measured. The protocol complied with the regulations of the Animal Ethics Committee of Harbin Medical University. 2.15 Statistical analysis Statistical analysis was carried out by GraphPad software (version: 7.0). Data are expressed as the mean ± standard deviation (SD). A t test was used to assess statistical significance, and a p value < 0.05 was considered significant. 3 Result 3.1 SAP30 is highly expressed in ccRCC cells and tissues and correlated with an unfavourable prognosis Transcription factors play an important role in the regulation of cancer development [ 21 , 22 ]. Thus, we first used the intersection of renal cancer differentially expressed genes in The Cancer Genome Atlas (TCGA) dataset and transcription factor ENCODE database to find the transcriptional suppressor SAP30 (Fig. 1 A). SAP30 was found to be highly expressed in kidney renal clear cell carcinoma (KIRC) on TCGA (Fig. 1 B). We then examined the mRNA and protein expression levels of SAP30 in human ccRCC using samples from our own group. The PCR results showed that SAP30 was higher in tumor tissue than in normal tissue in 34 pairs of samples (Fig. 1 C) and was higher in other renal cancer cell lines compared to normal renal tubular cells (HK-2) (Fig. 1 D). In addition, the western blotting results showed that SAP30 expression was higher in kidney cancer cell lines than in HK-2 cells (Fig. 1 E). To further validate the differential expression of SAP30 in ccRCC, we performed immunohistochemical staining (IHC) in 40 pairs of tumor and adjacent noncancerous tissues, and SAP30 was significantly upregulated in tumor tissues versus adjacent nontumor tissues (Fig. 1 F, 1 G). Finally, we revealed that high SAP30 expression was associated with shorter OS times in ccRCC TCGA patients (Fig. 1 H). In summary, SAP30 is highly expressed in ccRCC and is associated with a poor prognosis. 3.2 SAP30 knockdown inhibits ccRCC cell proliferation and promotes cell apoptosis To verify the biological functional significance of SAP30 in ccRCC, we knocked down its expression using its specific siRNAs in three different ccRCC cell lines (786-O, CAKI-1, OS-RC-2) (Fig. 2 A, 2 B). The CCK8 assay showed that knockdown of SAP30 had an inhibitory effect on tumor cells in three cell lines (Fig. 2 C). Consistent with this, the inhibition of proliferation was also reflected by the decreased colony formation under SAP30 silencing in ccRCC cells (Fig. 2 D, 2 E). We suspected that SAP30 may affect cell viability through apoptosis, and consistent with this speculation, Hoechst staining revealed that SAP30 knockdown promotes cell apoptosis (Fig. 2 F, G). Moreover, flow cytometry apoptosis detection and a cell cycle assay indicated that knockdown of SAP30 enhanced cell apoptosis (Fig. 2 H, 2 I) and blocked the G1 to G2 phase transition (Fig. 2 J, 2 K) in three cell lines. Given the effect of SAP30 on the abovementioned related phenotypes, we also assessed changes in apoptosis and cell cycle related proteins after SAP30 knockdown. The expression of cell cycle-related proteins, such as CCNB1, CCND1 and CCNL2, was significantly downregulated upon SAP30 silencing in three cell lines (Fig. 2 L). In addition, SAP30 knockdown also increased the expression of the apoptotic protein Bax and decreased the expression of the antiapoptotic protein Bcl-2 (Fig. 2 M). Importantly, we found that P53 protein expression increased with SAP30 knockdown (Fig. 2 M), indicating that SAP30 may be a key affecting cell proliferation and apoptosis [ 23 ]. Thus, these data suggest that SAP30 may promote cell proliferation, inhibit apoptosis and affect cell cycle arrest by inhibiting the P53 signalling pathway in ccRCC. 3.3 Overexpression of SAP30 promotes ccRCC cell proliferation and inhibits cell apoptosis In contrast, the SAP30-OE constructs were transfected into 786-O and CAKI-1 cells to ectopically overexpress SAP30 (Fig. 3 A, 3 B). CCK-8 and EdU assays showed that overexpression of SAP30 promoted the proliferation of renal cancer cells (Fig. 3 C- 3 E), and flow cytometry verified that overexpression of SAP30 also inhibited apoptosis (Fig. 3 F, 3 G). In addition, overexpression of SAP30 reduced the proportion of cells in the G1-phase of the cell cycle (Fig. 3 H, 3 I). These results indicated that overexpression of SAP30 could promote the proliferation and inhibit the apoptosis of tumor cells 3.4 SAP30 affects the proliferation and apoptosis of tumor cells through the MT1G/P53 pathway in ccRCC To dentify the targets of SAP30 causing the above phenotype, we obtained 251 targets of interest by intersecting the possible targets of SAP30 in the ENCODE database with the low expression differential base of kidney cancer in the TCGA database (Fig. 4 A). Then, GO term analysis of the 251 resulting target genes was performed, and growth-related metallothionein caught our attention (Fig. 4 B). Because studies have shown that MT1G can interact with P53 to promote the expression of P53 in hepatocellular carcinoma [ 20 ], we hypothesized that MT1G/P53 is required for the mechanism by which SAP30 affects proliferation and apoptosis in ccRCC. Subsequently, Western blot analysis showed increased protein expression of MT1G and P53 after knockdown of SAP30 in 786-O and CAKI-1 cell lines (Fig. 4 C). We next knocked down MT1G and SAP30 simultaneously, and found that knocking down MT1G reversed the effect of SAP30 silencing on the P53 protein (Fig. 4 D). These results suggested that SAP30 regulates the expression of P53 protein through MT1G. Indeed, CCK-8 and colony formation assays showed that knockdown of MT1G restored tumor cell viability, partially reversing the inhibitory effect of SAP30 silencing on tumor cell growth (Fig. 4 E- 4 G). Then, we used flow cytometry and found that knockdown of MT1G could also partially restore the number of apoptosis induced by SAP30 silencing (Fig. 4 H, 4 I). The above findings suggest that SAP30 affects the proliferation and apoptotic phenotype of kidney cancer cells by inhibiting the P53 protein mainly through affecting MT1G. 3.5 SAP30, as a transcription repressor, binds to the promoter of MT1G to inhibit its transcription Given that SAP30 is a transcriptional repressor, we used immunofluorescence microscopy to assess SAP30 protein localization and found that it was mainly localized in the nucleus (Fig. 5 A). Subsequently, we analysed the expression level of MT1G of ccRCC based on TCGA database, and interestingly, MT1G was significantly downregulated in ccRCC (Fig. 5 B). Moreover, analysis of the TCGA database did not confirm a significant correlation between DNA methylation of MT1G and low expression of MT1G in renal cancer (Fig. 5 C). Because SAP30 is part of the histone deacetylation complex and MT1G has been reported to be induced by histone deacetylase inhibitor (HDACi) treatment [ 19 ], low expression of MT1G in renal cancer may be due to the regulation of deacetylation. As we speculated, either knockdown of SAP30 or the use of a histone deacetylase inhibitor (HDACi) has the potential to increase MT1G protein and mRNA expression (Fig. 5 D, 5 E). To further validate the correlation between SAP30 and MT1G/P53 in clinical specimens, we assessed their expression in 40 tomour samples via IHC (Fig. 5 F), which implied that SAP30 expression was significantly negatively correlated with MT1G/P53 expression (Fig. 5 G, 5 H). These results suggest that SAP30 can suppress MT1G expression at the transcriptional level. Moreover, a luciferase reporter assay showed that SAP30 suppressed MT1G gene transcription (Fig. 5 I). Next, to determine the ability of SAP30 to bind to the MT1G promoter region, chromatin immunoprecipitation (ChIP) assays were performed and showed that SAP30 knockdown reduced binding to the MT1G promoter (Fig. 5 J). The positions of SAP30-bingding sites were marked with colored boxes (Fig. 5 K). In summary, these data indicated that SAP30 binds to the MT1G promoter region and negatively regulates its transcription, thereby affecting downstream gene expression. 3.6 SAP30 knockdown sensitized ccRCC cells to sunitinib. To further verify the sensitivity of SAP30 to some small molecule inhibitors, we used the Cancer Therapeutics Response Portal (CTRP) database and found that SAP30 was associated with many targeted drugs, such as sorafenib, axitinib and sunitinib (Fig. 6 A). Sunitinib is a first-line targeted drug for patients with advanced renal cancer with dual antiangiogenic and proapoptotic effects [ 2 , 24 ]. Given the above findings of the antiapoptosis role of SAP30 in ccRCC cells, we hypothesized that SAP30 might modulate the sensibility of ccRCC cells to sunitinib. Next, we treated the 786-O, CAKI-1 cell lines with a concentration gradient of sunitinib, and the half inhibitory concentrations (IC50) were found to be 10.62 uM and 12.64 uM, respectively (Fig. 6 B). Consistent with our hypothesis, we treated renal cell carcinoma cell lines with 10uM sunitinib, and SAP30 protein expression increased with increasing treatment time (Fig. 6 C). Compared to cells treated with sunitinib alone, 786-O and CAKI-1 cells expressing SAP30 siRNA were more sensitive to sunitinib treatment and showed stronger inhibition of cellular activity (Fig. 6 D- 6 F). Similarly, flow cytometry apoptosis assays also showed that knockdown of SAP30 could increase the sensitivity of tumor cells to sunitinib (Fig. 6 G, 6 H). To further identify the potential molecular mechanisms of SAP30 in its regulation of sensitivity to sunitinib in ccRCC cells, Western blot analysis revealed that the combination of si-SAP30 and sunitinib increased the expression of proapoptotic protein Bax and decreased the expression of antiapoptotic proteins Bcl-2 (Fig. 6 I). Therefore, SAP30 knockout could make ccRCC cells more sensitive to sunitinib, at least in part, by inducing more apoptosis. 3.7 Knocking down SAP30 suppressed tumor growth in a ccRCC xenograft mouse model. The effect of SAP30 on ccRCC development was further proven in vivo based on a xenograft mouse model. 786-O cells were subcutaneously injected into the flanks of nude mice (Fig. 7 A, 7 B). When the tumors could be clearly observed, the sizes of the formed tumors were measured once every three days. Mice were then sacrificed and tumor weight and volume were measured. Compared with the si-ctrl group, the volume and weight of tumor were significantly reduced in the SAP30 knockdown group, and this reduction was reversed with knockdown of MT1G (Fig. 7 C- 7 E). Notably, the regulation of MT1G and P53 expression by SAP30 was confirmed again in vivo. Consistent with the effect in vitro, RT‒PCR and Western blot analysis confirmed that the expression of MT1G and P53 was significantly increased after knockdown of SAP30 (Fig. 7 F, 7 G). The above in vivo experiments once again demonstrated that inhibiting the expression of SAP30 in ccRCC can suppress the progression of ccRCC by upregulating MT1G and P53. 4. Discussion The high mortality rate of renal cell carcinoma is mainly due to the difficulty of early diagnosis, recurrence after surgery, high metastasis rate and insensitivity to radiotherapy, so it is significant to explore effective molecular biological treatment targets [ 25 ]. Genetic regulation relies heavily on transcription factors [ 26 ], which play important roles in a variety of diseases, including cancer [ 27 , 28 ]. The cancer dependency map project (DepMap) found that transcription factors play a key role in tumor development and maintenance, suggesting that they may be potential therapeutic targets for cancer [ 29 ]. Previous studies have identified SAP30 as a biomarker for many tumors, such as hepatocellular carcinoma, renal carcinoma, and basal cell carcinoma [ 30 – 32 ], but its biological function and mechanism of action are poorly understood. In our study, we found that SAP30 was highly expressed in ccRCC tissues through the TCGA database, and using our research group samples, we also found that SAP30 was highly expressed in ccRCC tissues, both mRNA and protein levels. In addition, high SAP30 expression was found to be associated with a poor prognosis. From detailed cell experiments to in vivo mouse models, we demonstrated that SAP30 plays an important role in promoting the proliferation of kidney cancer cells and inhibiting their apoptosis. Additionally, we also found that SAP30 can inhibit the P53 pathway. Overall, our data suggest that SAP30 facilitates the development of ccRCC and could be a potential therapeutic target for ccRCC. To further investigate the mechanism by which SAP30 promotes renal cancer cell growth and inhibits apoptosis, we used the ENCODE database and the TCGA database to identify targets for the downstream action of SAP30. We conducted a GO analysis of the target gene set we found. It was determined that MT1G may be a bridge between SAP30 and its effects on renal cancer cell proliferation and apoptosis. Dual-luciferase reporter assays and ChIP-PCR verified SAP30 as a transcriptional inhibitor of MT1G. The contribution of MT1G to P53 stability and activity has been validated in HCC [ 20 ]; we found that SAP30 can affect the expression of P53 through MT1G. Our findings show that the SAP30/MT1G/P53 signalling axis exerts an important effect on SAP30-induced ccRCC proliferation and apoptosis inhibition. However, our data analysis did not reveal which molecules of the HDAC family are recruited by SAP30 to exert transcriptional repression of MT1G. Moreover, our analysis also cannot exclude other signalling pathways of SAP30 in ccRCC that are parallel to the SAP30/MT1G/P53 axis. This requires us to perform into subsequent experiments. Small molecule inhibitors are one of the important means for the treatment of advanced renal cancer [ 33 ]. To determine the sensitivity of SAP30 to small molecule inhibitors, we used the CTRP database and found that knockdown of SAP30 may increase the sensitivity of ccRCC cells to sunitinib which is a first-line targeted drug for patients with advanced kidney cancer. Subsequent phenotypic experiments also verified our hypothesis, and western blotting partially explained that this sensitization phenomenon may be achieved by inducing more apoptosis in tumor cells. MT1G is one of eight functional (sub)isoforms of MT1 in the metallothionin family and is a small molecule protein rich in cysteine [ 17 ]. MT1G has been studied in liver cancer, prostate cancer, oesophageal cancer, pancreatic cancer, and it has been found to be closely related to ferroptosis, tumor drug resistance, cell proliferation and apoptosis, and tumor cell stem stemness [ 34 – 37 ]. However, there is particularly little evidence that MT1G is associated with kidney cancer. In our study we found that MT1G is expressed at low levels in ccRCC that and knockdown of MT1G can restore apoptosis and proliferation inhibition induced by knockdown of SAP30 in both cell lines and mouse models. Studies have shown that MT1G is necessary for SAP30-mediated regulation of tumor cell growth and apoptosis, which together regulate the development of ccRCC. Because SAP30 is part of the histone deacetylase complex, we demonstrated that HDACi can also inhibit the expression of MT1G at the mRNA and protein levels in kidney cancer cell lines. This also indirectly suggests that the low expression of MT1G in kidney cancer is at least partly regulated by histone deacetylation. It has been reported that low expression of MT1G in kidney cancer has no significant correlation with methylation [ 38 ], which indirectly supports our conclusions. It would be interesting to see if the low expression of MT1G in kidney cancer is mediated by other factors. It is well known that the TP53 gene encoding the p53 protein has an anticancer effect; in the process of cancer suppression, P53 binds to DNA response elements to induce multiple biological processes, such as cell cycle arrest, apoptosis, DNA repair, autophagy and ferroptosis [ 39 – 41 ]. Because P53 is a zinc ion-dependent transcription factor [ 42 ] and the P53 ubiquitinase MDM2 can affect its stability [ 43 ], when we knocked down SAP30, the P53 protein level was upregulated, and this upregulation was partially restored by knocking down MT1G. However, we cannot completely rule out SAP30 adjusting P53 in other ways. Collectively, our results suggested that P53 functions in accordance with SAP30 and MT1G to regulate ccRCC development. Altogether, our data highlight the critical role of SAP30 knockdown in inhibiting cell proliferation and enhancing apoptosis in inhibiting ccRCC progression. Our results also illustrated that the SAP30/MT1G/P53 axis affects the proliferation and apoptosis of ccRCC cells in vitro and in vivo, so this axis may become a future therapeutic target for ccRCC. 5. Conclusion In summary, our current study shows that the transcriptional repressor SAP30 promotes the proliferation and inhibits the apoptosis of ccRCC cells through the MT1G/P53 pathway. SAP30 binds to the MT1G promoter region and represses MT1G expression at the transcriptional level. Subsequently, it reduces the MT1G interaction with P53 and decreases P53 protein levels by promoting MDM2-mediated P53 degradation. Ultimately, it promotes tumor cell proliferation and inhibits tumor cell apoptosis. Declarations Ethics approval and consent to participate This study was conducted at the Department of Urology, First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China, which was authorized by the Institutional Review Board of Harbin Medical University, and the subjects provided informed consent for this study. Acknowledgements We would like to thank the TCGA database and ENCODE database, as well as the Department of Urology, the First Affiliated Hospital of Harbin Medical University. Consent for publication Not applicable. Availability of data and materials The original documents will be provided later. Author contributions The original draft was prepared and written by Wei Xue. Wei Guo and Shuwen Wang were responsible for the data processing. Yu Dong and Zitong Yang helped to review and revise all the diagrams, and Zhinan Xia helped to review and revise the article. Cheng Zhang designed the experiment and conducted the management project. 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Published 2021 Jul 15. 10.3389/fonc.2021.623313 Sun X, Niu X, Chen R et al (2016) Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology 64(2):488–500. 10.1002/hep.28574 Zhu L, Yang F, Wang L et al (2021) Identification the ferroptosis-related gene signature in patients with esophageal adenocarcinoma. Cancer Cell Int 21(1):124 Published 2021 Feb 18. 10.1186/s12935-021-01821-2 Li K, Zhang Z, Mei Y et al (2021) Metallothionein-1G suppresses pancreatic cancer cell stemness by limiting activin A secretion via NF-κB inhibition. Theranostics 11(7):3196–3212 Published 2021 Jan 1. 10.7150/thno.51976 Maleckaite R, Zalimas A, Bakavicius A, Jankevicius F, Jarmalaite S, Daniunaite K (2019) DNA methylation of metallothionein genes is associated with the clinical features of renal cell carcinoma. Oncol Rep 41(6):3535–3544. 10.3892/or.2019.7109 Hafner A, Bulyk ML, Jambhekar A, Lahav G (2019) The multiple mechanisms that regulate p53 activity and cell fate. Nat Rev Mol Cell Biol 20(4):199–210. 10.1038/s41580-019-0110-x Levine AJ (2019) The many faces of p53: something for everyone. J Mol Cell Biol 11(7):524–530. 10.1093/jmcb/mjz026 Janic A, Valente LJ, Wakefield MJ et al (2018) DNA repair processes are critical mediators of p53-dependent tumor suppression. Nat Med 24(7):947–953. 10.1038/s41591-018-0043-5 Méplan C, Richard MJ, Hainaut P (2000) Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 19(46):5227–5236. 10.1038/sj.onc.1203907 Pant V, Aryal NK, Xiong S, Chau GP, Fowlkes NW, Lozano G (2022) Alterations of the Mdm2 C-Terminus Differentially Impact Its Function In Vivo. Cancer Res 82(7):1313–1320. 10.1158/0008-5472.CAN-21-2381 Additional Declarations No competing interests reported. Supplementary Files SupplementarytableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies 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-4164049","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283890014,"identity":"4583a4dc-f2e1-4c27-9cc8-305cd09d35af","order_by":0,"name":"Wei Xue","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xue","suffix":""},{"id":283890015,"identity":"c81696e7-fc2d-4940-a65d-ce6b10a0c8a2","order_by":1,"name":"Wei Guo","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Guo","suffix":""},{"id":283890016,"identity":"3282bea6-7290-45cd-a3f1-b1673c49ee54","order_by":2,"name":"Shuwen Wang","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuwen","middleName":"","lastName":"Wang","suffix":""},{"id":283890017,"identity":"3d434a9b-8b6a-4428-b7f3-018016798478","order_by":3,"name":"Yu Dong","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Dong","suffix":""},{"id":283890018,"identity":"8104cd59-fe09-42fb-9150-c851a6f2b191","order_by":4,"name":"Zitong Yang","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zitong","middleName":"","lastName":"Yang","suffix":""},{"id":283890019,"identity":"9970c7b2-e60a-472f-9100-5e7dd3da784b","order_by":5,"name":"Zhinan Xia","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhinan","middleName":"","lastName":"Xia","suffix":""},{"id":283890020,"identity":"02404e77-94d1-436c-8b4d-0fdb01c520db","order_by":6,"name":"Cheng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDCCAxAqAYjZf/+pkJCTJ0ILYwNUC4MEzxkLY8MGkrTwtlUkwuzFCfiONz9/8HGPXR6/dPsFA8l5EgmMDcwPH93Ao0XyzDHDxhnPkosl55wpSDDcJpHHzsBmbJyDR4vBjRzGZp4DBxI33MhJOJC4TaKYsYGHTZooLftv5CQ2HJwjkdhwgFgtGyTSDzM2NhChBeSXmTMOJCfOuJHDxsxwTMLYsJmAX4Ah9uDDhwN2if0z0p8xM9TUycmzNz98jE8LEuAxgNDMxCkHAfYHxKsdBaNgFIyCEQUASUlTs4lNi98AAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-25 14:32:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4164049/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4164049/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53759609,"identity":"8a27827c-b5f2-4222-aefa-2dda53e01fe3","added_by":"auto","created_at":"2024-03-29 19:35:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":999536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAP30 is highly expressed in ccRCC and is highly correlated with patient prognosis\u003c/strong\u003e. (A) The Venn diagrams show the crossed genes shared by upregulated differential genes in the KIRC TCGA (|Log2FC|＞2; *p value \u0026lt; 0.05) and ENCODE databases. (B) Data on SAP30 expression were obtained and plotted from the TCGA (|Log2FC| Cut-off: 1; *p value \u0026lt; 0.01). (C) SAP30 mRNA expression in 34 pairs of ccRCC samples. Mean ± SD is shown. Statistical analysis was conducted using a t test (*: p\u0026lt;0.05). (D) The mRNA expression of SAP30 in 5 types of cell lines. (E) The protein expression of SAP30 in 5 types of cell lines. (F-G) Immunohistochemical verification of SAP30 protein levels in 40 pairs of cancer and adjacent nontumor tissues. (H) Overall survival (OS) Kaplan–Meier curve for SAP30 based on TCGA.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/eddceced5af207bd164f7026.png"},{"id":53759608,"identity":"67766347-0376-4a9c-9bb6-e1b893ddaef4","added_by":"auto","created_at":"2024-03-29 19:35:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1180180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SAP30 inhibits the proliferation, but promotes the apoptosis of tumor cells.\u003c/strong\u003e(A) The protein expression of SAP30 in si-Ctrl, si-SAP30-1 and si-SAP30-2 groups. (B) The mRNA expression of SAP30 in si-Ctrl, si-SAP30-1 and si-SAP30-2 groups. (C-E) CCK-8 and colony formation assay to analyze the effect of si-SAP30 on cell proliferation in 786-O, CAKI-1 and OS-RC-2 cells. (F-G) The cell apoptosis number detected by the Hoechst in si-Ctrl and si-SAP30 groups, which marked arrow were apoptotic cells. (H-K) Flow cytometry was used to detect the cell apoptosis and cycle in si-Ctrl and si-SAP30 groups in 786-O, CAKI-1 and OS-RC-2 cell lines, respectively. (L) The expression level of cycle-related proteins in the SAP30 knockdown 786-O, CAKI-1 and OS-RC-2 cells. (M) The expression level of apoptosis related proteins in the SAP30 knockdown 786-O, CAKI-1 and OS-RC-2 cells. *: P＜0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/1b8bd026ae3ab8bea270e7f0.png"},{"id":53759610,"identity":"50f1fbfc-1a06-408c-882d-c41128b23351","added_by":"auto","created_at":"2024-03-29 19:35:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1164520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of SAP30 promotes the proliferation but inhibits the apoptosis of tumor cells. \u003c/strong\u003e(A-B) The protein and mRNA expression of SAP30 in the Ctrl and SAP30-OE groups. (C-E) CCK-8 and EdU (green: EdU positive) assays were used to analyse the effect of SAP30-OE on cell proliferation in 786-O and CAKI-1 cells. (F-I) Flow cytometry was used to detect cell apoptosis and cycle progressionin the Ctrl and SAP30-OE groups in 786-O and CAKI-1 cell lines, respectively. *: P＜ 0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/dad7e3b9c8239a0209850697.png"},{"id":53759613,"identity":"2e693811-2b39-417c-aa2c-a7d9d4b7299c","added_by":"auto","created_at":"2024-03-29 19:35:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":921783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SAP30 promotes the expression of MT1G and P53 proteins, while knockdown of MT1G restores the alterations in P53 protein and proliferation and apoptosis-related phenotypes due to knockdown of SAP30.\u003c/strong\u003e(A) The Venn diagrams show the crossed genes shared by downregulated differential genes in the KIRC TCGA (|Log2FC|＞2; *p value \u0026lt; 0.05) and ENCODE databases. (B) The top 30 enriched GO terms for 251 genes. (C) The protein expression of P53 and MT1G in the si-Ctrl and si-SAP30 groups. (D) The protein expression of P53 and MT1G in the si-Ctrl, si-SAP30, si-MT1G and si-S+M groups. (E-G) CCK-8 and colony formation assays were used to analyse the effect of si-Ctrl, si-SAP30, si-MT1G and si-S+M on cell proliferation in 786-O and CAKI-1 cell lines. (H-I) Flow cytometry was used to detect apoptosis in the si-Ctrl, si-SAP30, si-MT1G and si-S+M groups in 786-O and CAKI-1 cell lines. *: P＜0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/5e21c0c7afed378b48b42738.png"},{"id":53759865,"identity":"cd9df7ac-2940-4731-a375-74be474aa097","added_by":"auto","created_at":"2024-03-29 19:43:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1474637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAP30 binds the MT1G promoter region to repress MT1G expression.\u003c/strong\u003e(A) Immunofluorescence was used to detect the expression location of SAP30 in 786-O and CAKI-1 cell lines. (B) mRNA of MT1G expression was obtained from TCGA. (C) Spearman correlation between MT1G methylation and mRNA expression in FIRC from TCGA. (D E) The mRNA and protein levels of MT1G were detected in the si-Ctrl, si-SAP30 and HDACi groups. (F) Immunohistochemical stained SAP30, MT1G and P53 protein in tumour tissues. (G-H) The correlation between levels of SAP30 and MT1G, P53 were analyzed. (I) The dual-luciferase reporter assay showed the activity of the MT1G promoter fragment. (J) ChIP-PCR analysis of the binding of SAP30 to the promoter of MT1G. (K) Exhibiting binding site base pair sequences with boxes. *: P＜ 0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/cd5a7e649a667493dc749da1.png"},{"id":53759612,"identity":"2ef390ef-610f-4fa3-a7e6-ae4483a7555f","added_by":"auto","created_at":"2024-03-29 19:35:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1264822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAP30 knockdown increases the sensitivity of ccRCC cells to sunitinib. \u003c/strong\u003e(A) The sensitivity of SAP30 to various drugs from the CTRP database. (B) The IC50 of sunitinib was detected in the 786-O and CAKI-1 cell lines. (C) WB analysis of the protein expression level of SAP30 induced by HDACi treatment at the indicated timepoints. (D-F) CCK-8 and EdU (green: EdU positive) assays were used to analyse the effect in the si-Ctrl, si-SAP30, sunitinib and si-S+sunitinib groups on cell proliferation in 786-O and CAKI-1 cells. (G-H) Flow cytometry was used to detect cell apoptosis in the si-Ctrl, si-SAP30, sunitinib and si-S+sunitinib groups in the 786-O and CAKI-1 cell lines. (I) The expression levels of apoptosis-related proteins (BAX and Bcl-2) in the si-Ctrl, si-SAP30, sunitinib and si-S+sunitinib groups. *: P＜0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/0022599238f8150a2a7c6902.png"},{"id":53759616,"identity":"f95f9209-0781-4da2-9ae2-41722c52b6f2","added_by":"auto","created_at":"2024-03-29 19:35:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1771834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnocking down SAP30 inhibits the progression of ccRCC in vivo. \u003c/strong\u003e(A-B) The subcutaneous tumors in the 12 nude mice and the separated tumors are displayed. (C-D) The volume and weight of tumor were measured in nude mice. (E) The tumor volume was measured at 6, 12, 24 and 48 days after tumors were formed. (F) The mRNA of MT1G was measured by qRT‒PCR in the si-Ctrl and si-SAP30 groups in vivo. (G) The protein expression levels of SAP30, MT1G and P53 were detected in the si-Ctrl and si-SAP30 groups in vivo. (H) Hypothetical model depicting the proposed mechanism by which SAP30 regulates proliferation and apoptosis of ccRCC cells through MT1G/P53 axis. *: P＜0.05, **: P＜0.01, ***: P＜0.001\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/9eab5a501be6127ce3dfd8b7.png"},{"id":54149929,"identity":"046b23fb-1d50-4221-a63a-4331e78e7330","added_by":"auto","created_at":"2024-04-05 10:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3868013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/93d93815-09dc-4193-b5c6-e15aec457994.pdf"},{"id":53759611,"identity":"5920662c-2755-4969-8140-c52eea980f17","added_by":"auto","created_at":"2024-03-29 19:35:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12502,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4164049/v1/215a029d290a4de43cd9cd81.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SAP30 promotes clear cell renal cell carcinoma proliferation and inhibits apoptosis through the MT1G/P53 axis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is one of the most common malignancies in the urinary system and accounts for nearly 5% of all malignancies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Of all the subtypes of RCC, clear cell renal cell carcinoma is the most common, accounting for more than 90% of cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although surgery is still the primary treatment modality for ccRCC in the early stages, more than 30% of patients have developed distant metastases before diagnosis and postoperative survival is not particularly satisfactory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, its poor prognosis is closely linked to its insensitivity to radiotherapy/chemotherapy and the lack of effective biomarkers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, it is particularly urgent for us to find suitable biomarkers.\u003c/p\u003e \u003cp\u003eSin3-related polypeptide p30 (SAP30) is an essential part of the SIN/HDAC histone deacetylase complex, which is widely expressed in human tissues, especially haematopoietic tissues [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and mainly functions as a transcriptional repressor [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SAP30 acts as a scaffold for SIN3, which acts as a repressor and corepressor of gene expression in the presence of binding proteins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Its biological functions include DNA and histone methylation regulation, nucleosome remodelling, histone deacetylase scaffolding, and N-acetylglucosamine transferase activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. SAP30 mainly performs transcriptional repression through two mechanisms. SAP30 binds to transcription factors such as YY1 and recruits HDAC1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. SAP30 binds directly to DNA sequences, bending them structurally to achieve transcriptional inhibition [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The effect of knocking out the SAP30 gene in yeast was reported to be similar to that of knocking out SIN3 and Rpd3, which have various functions, such as affecting cell cycle regulation, apoptosis, mitochondrial metabolism, DNA replication and repair [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, it can be inferred that SAP30 knockdown may also affect cell cycle regulation and apoptosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, SAP30 has rarely been studied in various tumors, especially the mechanism of action in the occurrence and development of ccRCC is still unclear.\u003c/p\u003e \u003cp\u003eHere, we show that SAP30 inhibits the expression of metallothionein-1G (MT1G), a small cysteine-rich protein that plays an important role in metal homeostasis and protection against heavy metal toxicity, DNA damage, and oxidative stress, at the transcription level [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. MT1G is expressed in a variety of tumors, and is involved in cell growth and proliferation as well as the EMT process [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been reported that MT1G increases the stability of P53 by inhibiting the P53 ubiquitinase MDM2 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MT1G can also interact directly with P53 to provide zinc ions to P53, thereby increasing P53 transcriptional activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, MT1G has been reported relatively little in kidney cancer.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that SAP30 affects the proliferation and apoptosis of kidney cancer cells through the MT1G/P53 axis in vivo and in vitro. This molecular mechanism has the potential to be a new clinical therapeutic target with value for further research.\u003c/p\u003e"},{"header":"2 Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines, cell culture and tissue specimens\u003c/h2\u003e \u003cp\u003eHuman renal cancer cell lines (786-O, CAKI-1 OS-RC-2, and CAKI-2) and human renal tubular epithelial cells (HK-2) were all purchased from the Cell Resources Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. All cell lines were cultivated in the recommended medium supplemented with 10% heat-inactivated foetal bovine serum (Gibco, Beijing, China) and 1% streptomycin/penicillin (Keygen, Nanjing, China) at 37\u0026deg;C and 5% CO2. RCC tissue specimens were obtained from 40 patients who were treated by radical nephrectomy or preserved kidney surgery at the Department of Urology, First Affiliated Hospital of Harbin Medical University between 2010 and 2019. All diagnoses were confirmed by histopathological examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Antibodies and reagents\u003c/h2\u003e \u003cp\u003eThe following antibodies were used for Western blotting: SAP30 (#LS-C676447, Lsbio), MT1G (#323730, US Biological), P53 (#60283-2-Ig, Proteintech), Bax (#50599-2-Ig, Proteintech), Bcl2 (#12789-AP, Proteintech), CCNB1 (#28603-AP, Proteintech), CCND1 (#60186-I-Ig, Proteintech), and CCNL2(#LS-C749918, LSBio). And the anti-SAP30 antibody for IHC was purchased from Lsbio(#LS-C676447). The anti-SAP30 antibody for ChIP-PCR was purchased from Abcam (#ab231804). Sunitinib was purchased from MCE (#HY-10255A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 siRNA and plasmid transfection\u003c/h2\u003e \u003cp\u003eThe siRNAs for SAP30 and MT1G were purchased from GenePharma (Suzhou, China), and their sequences are shown in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Transfection of the siRNA-SAP30 and siRNA-MT1G vectors was performed using jetPRIME (PolyPlus, Shanghai, China). The plasmids (pENTER-SAP30, pENTER-NC, pCMV3-SAP30 and pCMV3-NC) were purchased from Weizhen (Shandong, China) and SinoBiological (Beijing, China). Briefly, 1.2\u0026ndash;1.3 x10\u003csup\u003e6\u003c/sup\u003e cells were loaded in a 6 cm culture dish to prepare for transfection and collect cells for subsequent experiments after 47\u0026ndash;72 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quantitative real-time PCR (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from cells and tissues was extracted using TRIzol reagent (Ambion,USA). Reverse transcription kits (TianGen, Beijing, China) and PCR kits (SYBR Green) (TianGen, Beijing, China) were used to perform RT\u003cb\u003e‒\u003c/b\u003eqPCR according to the manufacturer\u0026rsquo;s instructions. The sequences of primers were provided in the \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. The results were analysed by the 2 -ΔΔCt method to quantify the fold change.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western blotting analysis and immunohistochemical staining (IHC)\u003c/h2\u003e \u003cp\u003eProteins from nude mouse kidney cancer cells and tissue and tumor cell lines were extracted with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Quantified proteins were separated by SDS\u003cb\u003e‒\u003c/b\u003ePAGE and transferred to PVDF membrane. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with the corresponding antibodies. For the IHC, paraffin-embedded 4-\u0026micro;m-thick kidney cancer tissue sections were deparaffinized, subjected to antigen retrieval, blocked with 5% bovine serum albumin (BSA) for 2 hours at room temperature, and then incubated with antibodies against SAP30 (1:400) at 4\u0026deg;C. The next day peroxidase-conjugated polymer was added for 30 min. Finally, visualization was performed with DAB (Beyotime, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 CCK-8 cell proliferation assay\u003c/h2\u003e \u003cp\u003eWe used the CCK-8 assay (MCE, China) to analyse cell proliferation. Transfected cells were grown in 96-well plates (3000 cells/well) for 12 hours. Then 10% CCK8 solution was added at different time periods (0/24/48/72 h) according to the CCK8 kit manufacturer's protocol. The OD absorbance values were measured at 450 nm using a 96-well plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Colony formation assay\u003c/h2\u003e \u003cp\u003eCell colony formation capacity was assessed by a plate colony formation assay. Transfected 786-O, CAKI-1 and OS-RC-2 cells (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) were seeded in 6-well plates for 14 days to form colonies, which were subsequently fixed in paraformaldehyde and then stained with crystal violet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Hoechst staining\u003c/h2\u003e \u003cp\u003eA apoptosis of transfected cells was induced with H2O2. After fixation with paraformaldehyde, the cells were washed with PBS 3 times. Finally, the cells were stained with Hoechst dye (#C1017, Beyotime, China) for 5 min and photographed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Flow cytometry\u003c/h2\u003e \u003cp\u003eFirst, the transfected cells were trypsinized with EDTA-free trypsin and washed with cold PBS according to the manufacturer's instructions of the ANNEXIN V-FITC/PI Apoptosis Kit (Solarbio, Beijing, China). Second, apoptosis was examined in the dark with Annexin V-FITC/propidium iodide (PI) staining. Finally, cell cycle profiles were analysed by PI staining and flow cytometry according to the manufacturer's instructions of the Cell Cycle Detection Kit (Beyotime, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Chromatin immunoprecipitation assay (ChIP)\u003c/h2\u003e \u003cp\u003eThe chromatin immunoprecipitation assay with 786-O and CAKI-1 cells was performed according to the protocol for the SimpleChIP\u0026reg; Plus Enzymatic Chromatin IP Kit (Magnetic Beads). First, 1x10\u003csup\u003e7\u003c/sup\u003e cells were fixed with 1% formaldehyde and 1.25 M glycine was added to terminate the reaction. The cells were washed with cold PBS containing protease inhibitor and collected into a centrifuge for the next step. The last collected cells were sonicated in IP mixed medium and centrifuged at 14, 000 \u0026times; g for 30 min at 4\u0026deg;C. Protein A/G agarose beads were mixed with the above supernatant and centrifuged, and the chromatin in the supernatant was immunoprecipitated overnight with antibodies against SAP30 or IgG. The protein A/G agarose beads were then washed with mixed wash buffer, LiCl/detergent and TE buffer. The beads were heated overnight at 65\u0026deg;C in a buffer mixture containing 1% SDS, 0.1 M NaHCO3 to crosslink the beads in reverse. The beads were then treated with 1 ml of RNaseA for 15 min at 37\u0026deg;C and digested with 2 ml of proteinase K solution for 1 hr at 37\u0026deg;C. Finally, DNA was purified by extraction with LiCl, phenol/chloroform and ethanol, and the specific MT1G promoter region was amplified with DNA template. \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e lists the primer sequences designed to detect specific promoters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe SAP30 overexpressing plasmid pcDNA3.3-SAP30 and the plasmid pGL4.18WT\u0026thinsp;+\u0026thinsp;binding site (BS1, BS2 and BS3) containing the promoter region of MT1G were constructed. Binding site sequences for promoter reporter plasmid construction are listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. 293T Cells cultured in 6-well plates were transfected with overexpression plasmids or empty vectors. Luciferase activity was measured using a dual luciferase reporter system according to the manufacturer's instructions (E1910, Promega, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Detection of EdU incorporation\u003c/h2\u003e \u003cp\u003eCells (3 x 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded on coverslips in 6-well plates for 24 hours. Cells were then cultured in culture medium containing EdU for 2 h after 48 h of transfection. The cultures were subsequently stained with Azide 488 and Hoechst 33342. EdU-positive cells were observed under a fluorescence microscope and photographed. The EdU binding rate was expressed as the ratio of EdU-positive cells (green cells) to total Hoechst-labelled cells (blue cells).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Immunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eCells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were inoculated in 6-well plates with coverslips for 24 hours. First, after washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.05% Triton for 30 min, blocked with 5% BSA for 2 h, and incubated with antibodies against SAP30 overnight at 4\u0026deg;C. The next day, the type A fluorescent secondary antibody was incubated for 1 hour at room temperature. Finally, nuclear staining was performed with DAPI and subsequent imaging was performed with fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Xenograft tumor growth\u003c/h2\u003e \u003cp\u003e786-O cells (3 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/ml) were injected into the posterior ventral sides of 6-week-old male nude mouse, purchased from Charles River Laboratories (Beijing, China). After 6 days, visible tumors were visible, their volume was measured, and transfection was performed according to the instructions of the EntransterTM-In Vivo kit (Engreen, Beijing, China) and repeated every 3 days. The transfection was repeated every 3 days. Forty-eight days later, the nude mice were sacrificed, the tumors were removed, and their weights and volumes were measured. The protocol complied with the regulations of the Animal Ethics Committee of Harbin Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out by GraphPad software (version: 7.0). Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). A t test was used to assess statistical significance, and a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Result","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 SAP30 is highly expressed in ccRCC cells and tissues and correlated with an unfavourable prognosis\u003c/h2\u003e \u003cp\u003eTranscription factors play an important role in the regulation of cancer development [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, we first used the intersection of renal cancer differentially expressed genes in The Cancer Genome Atlas (TCGA) dataset and transcription factor ENCODE database to find the transcriptional suppressor SAP30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SAP30 was found to be highly expressed in kidney renal clear cell carcinoma (KIRC) on TCGA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We then examined the mRNA and protein expression levels of SAP30 in human ccRCC using samples from our own group. The PCR results showed that SAP30 was higher in tumor tissue than in normal tissue in 34 pairs of samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and was higher in other renal cancer cell lines compared to normal renal tubular cells (HK-2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, the western blotting results showed that SAP30 expression was higher in kidney cancer cell lines than in HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To further validate the differential expression of SAP30 in ccRCC, we performed immunohistochemical staining (IHC) in 40 pairs of tumor and adjacent noncancerous tissues, and SAP30 was significantly upregulated in tumor tissues versus adjacent nontumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Finally, we revealed that high SAP30 expression was associated with shorter OS times in ccRCC TCGA patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). In summary, SAP30 is highly expressed in ccRCC and is associated with a poor prognosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 SAP30 knockdown inhibits ccRCC cell proliferation and promotes cell apoptosis\u003c/h2\u003e \u003cp\u003eTo verify the biological functional significance of SAP30 in ccRCC, we knocked down its expression using its specific siRNAs in three different ccRCC cell lines (786-O, CAKI-1, OS-RC-2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The CCK8 assay showed that knockdown of SAP30 had an inhibitory effect on tumor cells in three cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consistent with this, the inhibition of proliferation was also reflected by the decreased colony formation under SAP30 silencing in ccRCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). We suspected that SAP30 may affect cell viability through apoptosis, and consistent with this speculation, Hoechst staining revealed that SAP30 knockdown promotes cell apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). Moreover, flow cytometry apoptosis detection and a cell cycle assay indicated that knockdown of SAP30 enhanced cell apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) and blocked the G1 to G2 phase transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK) in three cell lines. Given the effect of SAP30 on the abovementioned related phenotypes, we also assessed changes in apoptosis and cell cycle related proteins after SAP30 knockdown. The expression of cell cycle-related proteins, such as CCNB1, CCND1 and CCNL2, was significantly downregulated upon SAP30 silencing in three cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). In addition, SAP30 knockdown also increased the expression of the apoptotic protein Bax and decreased the expression of the antiapoptotic protein Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Importantly, we found that P53 protein expression increased with SAP30 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM), indicating that SAP30 may be a key affecting cell proliferation and apoptosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, these data suggest that SAP30 may promote cell proliferation, inhibit apoptosis and affect cell cycle arrest by inhibiting the P53 signalling pathway in ccRCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Overexpression of SAP30 promotes ccRCC cell proliferation and inhibits cell apoptosis\u003c/h2\u003e \u003cp\u003eIn contrast, the SAP30-OE constructs were transfected into 786-O and CAKI-1 cells to ectopically overexpress SAP30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). CCK-8 and EdU assays showed that overexpression of SAP30 promoted the proliferation of renal cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), and flow cytometry verified that overexpression of SAP30 also inhibited apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In addition, overexpression of SAP30 reduced the proportion of cells in the G1-phase of the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These results indicated that overexpression of SAP30 could promote the proliferation and inhibit the apoptosis of tumor cells\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SAP30 affects the proliferation and apoptosis of tumor cells through the MT1G/P53 pathway in ccRCC\u003c/h2\u003e \u003cp\u003eTo dentify the targets of SAP30 causing the above phenotype, we obtained 251 targets of interest by intersecting the possible targets of SAP30 in the ENCODE database with the low expression differential base of kidney cancer in the TCGA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Then, GO term analysis of the 251 resulting target genes was performed, and growth-related metallothionein caught our attention (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Because studies have shown that MT1G can interact with P53 to promote the expression of P53 in hepatocellular carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we hypothesized that MT1G/P53 is required for the mechanism by which SAP30 affects proliferation and apoptosis in ccRCC. Subsequently, Western blot analysis showed increased protein expression of MT1G and P53 after knockdown of SAP30 in 786-O and CAKI-1 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We next knocked down MT1G and SAP30 simultaneously, and found that knocking down MT1G reversed the effect of SAP30 silencing on the P53 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggested that SAP30 regulates the expression of P53 protein through MT1G. Indeed, CCK-8 and colony formation assays showed that knockdown of MT1G restored tumor cell viability, partially reversing the inhibitory effect of SAP30 silencing on tumor cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Then, we used flow cytometry and found that knockdown of MT1G could also partially restore the number of apoptosis induced by SAP30 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The above findings suggest that SAP30 affects the proliferation and apoptotic phenotype of kidney cancer cells by inhibiting the P53 protein mainly through affecting MT1G.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 SAP30, as a transcription repressor, binds to the promoter of MT1G to inhibit its transcription\u003c/h2\u003e \u003cp\u003eGiven that SAP30 is a transcriptional repressor, we used immunofluorescence microscopy to assess SAP30 protein localization and found that it was mainly localized in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Subsequently, we analysed the expression level of MT1G of ccRCC based on TCGA database, and interestingly, MT1G was significantly downregulated in ccRCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, analysis of the TCGA database did not confirm a significant correlation between DNA methylation of MT1G and low expression of MT1G in renal cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Because SAP30 is part of the histone deacetylation complex and MT1G has been reported to be induced by histone deacetylase inhibitor (HDACi) treatment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], low expression of MT1G in renal cancer may be due to the regulation of deacetylation. As we speculated, either knockdown of SAP30 or the use of a histone deacetylase inhibitor (HDACi) has the potential to increase MT1G protein and mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To further validate the correlation between SAP30 and MT1G/P53 in clinical specimens, we assessed their expression in 40 tomour samples via IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), which implied that SAP30 expression was significantly negatively correlated with MT1G/P53 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These results suggest that SAP30 can suppress MT1G expression at the transcriptional level. Moreover, a luciferase reporter assay showed that SAP30 suppressed MT1G gene transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Next, to determine the ability of SAP30 to bind to the MT1G promoter region, chromatin immunoprecipitation (ChIP) assays were performed and showed that SAP30 knockdown reduced binding to the MT1G promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). The positions of SAP30-bingding sites were marked with colored boxes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). In summary, these data indicated that SAP30 binds to the MT1G promoter region and negatively regulates its transcription, thereby affecting downstream gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 SAP30 knockdown sensitized ccRCC cells to sunitinib.\u003c/h2\u003e \u003cp\u003eTo further verify the sensitivity of SAP30 to some small molecule inhibitors, we used the Cancer Therapeutics Response Portal (CTRP) database and found that SAP30 was associated with many targeted drugs, such as sorafenib, axitinib and sunitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Sunitinib is a first-line targeted drug for patients with advanced renal cancer with dual antiangiogenic and proapoptotic effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Given the above findings of the antiapoptosis role of SAP30 in ccRCC cells, we hypothesized that SAP30 might modulate the sensibility of ccRCC cells to sunitinib. Next, we treated the 786-O, CAKI-1 cell lines with a concentration gradient of sunitinib, and the half inhibitory concentrations (IC50) were found to be 10.62 uM and 12.64 uM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Consistent with our hypothesis, we treated renal cell carcinoma cell lines with 10uM sunitinib, and SAP30 protein expression increased with increasing treatment time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Compared to cells treated with sunitinib alone, 786-O and CAKI-1 cells expressing SAP30 siRNA were more sensitive to sunitinib treatment and showed stronger inhibition of cellular activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Similarly, flow cytometry apoptosis assays also showed that knockdown of SAP30 could increase the sensitivity of tumor cells to sunitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). To further identify the potential molecular mechanisms of SAP30 in its regulation of sensitivity to sunitinib in ccRCC cells, Western blot analysis revealed that the combination of si-SAP30 and sunitinib increased the expression of proapoptotic protein Bax and decreased the expression of antiapoptotic proteins Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Therefore, SAP30 knockout could make ccRCC cells more sensitive to sunitinib, at least in part, by inducing more apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Knocking down SAP30 suppressed tumor growth in a ccRCC xenograft mouse model.\u003c/h2\u003e \u003cp\u003eThe effect of SAP30 on ccRCC development was further proven in vivo based on a xenograft mouse model. 786-O cells were subcutaneously injected into the flanks of nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). When the tumors could be clearly observed, the sizes of the formed tumors were measured once every three days. Mice were then sacrificed and tumor weight and volume were measured. Compared with the si-ctrl group, the volume and weight of tumor were significantly reduced in the SAP30 knockdown group, and this reduction was reversed with knockdown of MT1G (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Notably, the regulation of MT1G and P53 expression by SAP30 was confirmed again in vivo. Consistent with the effect in vitro, RT‒PCR and Western blot analysis confirmed that the expression of MT1G and P53 was significantly increased after knockdown of SAP30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). The above in vivo experiments once again demonstrated that inhibiting the expression of SAP30 in ccRCC can suppress the progression of ccRCC by upregulating MT1G and P53.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe high mortality rate of renal cell carcinoma is mainly due to the difficulty of early diagnosis, recurrence after surgery, high metastasis rate and insensitivity to radiotherapy, so it is significant to explore effective molecular biological treatment targets [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Genetic regulation relies heavily on transcription factors [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which play important roles in a variety of diseases, including cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The cancer dependency map project (DepMap) found that transcription factors play a key role in tumor development and maintenance, suggesting that they may be potential therapeutic targets for cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies have identified SAP30 as a biomarker for many tumors, such as hepatocellular carcinoma, renal carcinoma, and basal cell carcinoma [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], but its biological function and mechanism of action are poorly understood. In our study, we found that SAP30 was highly expressed in ccRCC tissues through the TCGA database, and using our research group samples, we also found that SAP30 was highly expressed in ccRCC tissues, both mRNA and protein levels. In addition, high SAP30 expression was found to be associated with a poor prognosis. From detailed cell experiments to in vivo mouse models, we demonstrated that SAP30 plays an important role in promoting the proliferation of kidney cancer cells and inhibiting their apoptosis. Additionally, we also found that SAP30 can inhibit the P53 pathway. Overall, our data suggest that SAP30 facilitates the development of ccRCC and could be a potential therapeutic target for ccRCC.\u003c/p\u003e \u003cp\u003eTo further investigate the mechanism by which SAP30 promotes renal cancer cell growth and inhibits apoptosis, we used the ENCODE database and the TCGA database to identify targets for the downstream action of SAP30. We conducted a GO analysis of the target gene set we found. It was determined that MT1G may be a bridge between SAP30 and its effects on renal cancer cell proliferation and apoptosis. Dual-luciferase reporter assays and ChIP-PCR verified SAP30 as a transcriptional inhibitor of MT1G. The contribution of MT1G to P53 stability and activity has been validated in HCC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; we found that SAP30 can affect the expression of P53 through MT1G. Our findings show that the SAP30/MT1G/P53 signalling axis exerts an important effect on SAP30-induced ccRCC proliferation and apoptosis inhibition. However, our data analysis did not reveal which molecules of the HDAC family are recruited by SAP30 to exert transcriptional repression of MT1G. Moreover, our analysis also cannot exclude other signalling pathways of SAP30 in ccRCC that are parallel to the SAP30/MT1G/P53 axis. This requires us to perform into subsequent experiments. Small molecule inhibitors are one of the important means for the treatment of advanced renal cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To determine the sensitivity of SAP30 to small molecule inhibitors, we used the CTRP database and found that knockdown of SAP30 may increase the sensitivity of ccRCC cells to sunitinib which is a first-line targeted drug for patients with advanced kidney cancer. Subsequent phenotypic experiments also verified our hypothesis, and western blotting partially explained that this sensitization phenomenon may be achieved by inducing more apoptosis in tumor cells.\u003c/p\u003e \u003cp\u003eMT1G is one of eight functional (sub)isoforms of MT1 in the metallothionin family and is a small molecule protein rich in cysteine [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. MT1G has been studied in liver cancer, prostate cancer, oesophageal cancer, pancreatic cancer, and it has been found to be closely related to ferroptosis, tumor drug resistance, cell proliferation and apoptosis, and tumor cell stem stemness [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, there is particularly little evidence that MT1G is associated with kidney cancer. In our study we found that MT1G is expressed at low levels in ccRCC that and knockdown of MT1G can restore apoptosis and proliferation inhibition induced by knockdown of SAP30 in both cell lines and mouse models. Studies have shown that MT1G is necessary for SAP30-mediated regulation of tumor cell growth and apoptosis, which together regulate the development of ccRCC. Because SAP30 is part of the histone deacetylase complex, we demonstrated that HDACi can also inhibit the expression of MT1G at the mRNA and protein levels in kidney cancer cell lines. This also indirectly suggests that the low expression of MT1G in kidney cancer is at least partly regulated by histone deacetylation. It has been reported that low expression of MT1G in kidney cancer has no significant correlation with methylation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which indirectly supports our conclusions. It would be interesting to see if the low expression of MT1G in kidney cancer is mediated by other factors.\u003c/p\u003e \u003cp\u003eIt is well known that the TP53 gene encoding the p53 protein has an anticancer effect; in the process of cancer suppression, P53 binds to DNA response elements to induce multiple biological processes, such as cell cycle arrest, apoptosis, DNA repair, autophagy and ferroptosis [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Because P53 is a zinc ion-dependent transcription factor [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and the P53 ubiquitinase MDM2 can affect its stability [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], when we knocked down SAP30, the P53 protein level was upregulated, and this upregulation was partially restored by knocking down MT1G. However, we cannot completely rule out SAP30 adjusting P53 in other ways. Collectively, our results suggested that P53 functions in accordance with SAP30 and MT1G to regulate ccRCC development.\u003c/p\u003e \u003cp\u003eAltogether, our data highlight the critical role of SAP30 knockdown in inhibiting cell proliferation and enhancing apoptosis in inhibiting ccRCC progression. Our results also illustrated that the SAP30/MT1G/P53 axis affects the proliferation and apoptosis of ccRCC cells in vitro and in vivo, so this axis may become a future therapeutic target for ccRCC.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our current study shows that the transcriptional repressor SAP30 promotes the proliferation and inhibits the apoptosis of ccRCC cells through the MT1G/P53 pathway. SAP30 binds to the MT1G promoter region and represses MT1G expression at the transcriptional level. Subsequently, it reduces the MT1G interaction with P53 and decreases P53 protein levels by promoting MDM2-mediated P53 degradation. Ultimately, it promotes tumor cell proliferation and inhibits tumor cell apoptosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted at the Department of Urology, First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China, which was authorized by the Institutional Review Board of Harbin Medical University, and the subjects provided informed consent for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the TCGA database and ENCODE database, as well as the Department of Urology, the First Affiliated Hospital of Harbin Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original documents will be provided later.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original draft was prepared and written by Wei Xue. Wei Guo and Shuwen Wang were responsible for the data processing. Yu Dong and Zitong Yang helped to review and revise all the diagrams, and Zhinan Xia helped to review and revise the article. Cheng Zhang designed the experiment and conducted the management project. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded partly by the National Natural Science Foundation of China (Grant No. 81872084)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Jemal A, Cancer statistics (2020) CA Cancer J Clin. 2020;70(1):7\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21590\u003c/span\u003e\u003cspan address=\"10.3322/caac.21590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh JJ, Purdue MP, Signoretti S et al (2017) Renal cell carcinoma. 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Cancer Res 82(7):1313\u0026ndash;1320. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-21-2381\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-21-2381\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Transcription factor, P53, SAP30, MT1G, ccRCC","lastPublishedDoi":"10.21203/rs.3.rs-4164049/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4164049/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSin3-associated polypeptide p30(SAP30) is an important component of the SIN/HDAC histone deacetylase complex that act as a scaffolding and facilitates target gene binding. SAP30 is highly expressed in a variety of tumors, however; its role in renal cell carcinoma is still unclear. In our study, we found that SAP30 was upregulated in tissues of renal clear cell carcinoma (ccRCC), and high SAP30 expression was associated with a poor prognosis. According to relevant studies, SAP30 may be associated with the growth, proliferation and apoptosis of renal cell carcinoma cells, and GO analysis of SAP30 downstream regulatory target genome showed that SAP30 repressed the expression of MT1G, a P53-binding protein. Mechanistically, SAP30 inhibits MT1G expression at the transcriptional level, reducing the ability of MT1G to deliver to zinc ions to P53, thus reducing P53 activity, and the downregulation of MT1G also attenuates the inhibition of MDM2, thereby reducing the stability of P53, which ultimately promotes the development of renal cell carcinoma. In summary, our study shows that SAP30 inhibits the P53 pathway by inhibiting MT1G, suggesting that SAP30 and MT1G may become markers of renal cell carcinoma prognosis and therapeutic targets.\u003c/p\u003e","manuscriptTitle":"SAP30 promotes clear cell renal cell carcinoma proliferation and inhibits apoptosis through the MT1G/P53 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 19:35:03","doi":"10.21203/rs.3.rs-4164049/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":"2028fee6-718b-4c56-be28-d78aa5c0f6e6","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-05T10:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 19:35:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4164049","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4164049","identity":"rs-4164049","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}