SF3A3 Affects The Proliferation And Apoptosis Of Colorectal Cancer Cells Through The PI3K/AKT/mROR Pathway

Background:Colorectal cancer（CRC）accounts for approximately 10%of cancer diagnoses and related deaths worldwide each year.It is the third leading malignancy worldwide and the second leading cause of cancer death.The SF3A3 gene is located on chromosome 1p34.3 and encodes subunit 3 of the splicing factor SF3a protein complex,with a molecular weight of60k Da.Splicing factors frequently undergo mutations and expression disorders in cancer.As an important part of the splicing body,SF3A3 has been rarely studied in cancer,especially colorectal cancer.Through GEO（Gene Expression Omnibus）database analysis and detection of clinical colorectal cancer tissue samples and human colorectal cancer cell lines,it is found that SF3A3 is significantly highly expressed in colorectal cancer,combined with SF3A3 in exercising spliceosome regulatory functions and some of its As reported in cancer,we speculate that SF3A3 may be a cancer-promoting factor in CRC.Since the specific molecular mechanism of its cancer-promoting effect is still unclear,this project aims to study the role and molecular mechanism of SF3A3 in CRC cell lines.Actively seek new molecular targets for the treatment and screening of CRC.Research methods:1.Analyze the expression level of SF3A3 in the colorectal cancer gene chip data of the GEO database,and detect the expression level of SF3A3 in colorectal cancer clinical samples by immunohistochemistry experiments,and detect the expression level of SF3A3in colorectal cancer cell lines by Western Blot and RT-PCR.2.After knocking down SF3A3 by si RNA in colorectal cancer cells and overexpressing SF3A3 by adenovirus transfection,CCK-8,plate clone formation,Ki-67 staining and flow cytometry were used to detect the proliferation and apoptosis of colorectal cancer cells.And scratch test and transwell test were used to detect the migration and invasion capabilities of colorectal cancer cells.3.The influence of SF3A3 knockdown and overexpression on PI3K/AKT/m TOR pathway in CRC cells was detected by Western Blot assayResults:1.The expression of SF3A3 in colorectal cancer1)GEO database analysis of the high expression of SF3A3 in CRCUse R（4.0.3）software to analyze the expression level of SF3A3 in the expression profile data of colorectal cancer tissues in the GSE21815 database collected in the GEO database,use the"limma"package for differential analysis,the"ggplot2"package to draw volcano maps,"The"gplots"package draws heat maps.The results showed that the expression of SF3A3 was significantly up-regulated in colorectal cancer tissues.2)High expression of SF3A3 in clinical colorectal cancer tissuesThe results of immunohistochemistry（IHC）showed that the expression of SF3A3 in colorectal cancer tissues was significantly higher than that in matched adjacent tissues.3)The expression of SF3A3 in colorectal cancer cell linesThe expression level of SF3A3 was detected in three human CRC cell lines（HCT116,SW620,SW480）and a normal human colorectal epithelial cell line（FHC）by Western Blot and RT-PCR.The results showed that the expression level of SF3A3 was the highest in SW620 cell line,followed by HCT116 cell line,and the lowest expression in FHC.Therefore,SW620 cells were selected as the colorectal cancer cell line that subsequently knocked down SF3A3,and HCT116 cells were selected as the colorectal cancer cell line that overexpressed SF3A3.2.The role of SF3A3 in the occurrence and development of colorectal cancer1)SF3A3 si RNA sequence screeningSynthesize three SF3A3 si RNA（si SF3A3-1,si SF3A3-2,si SF3A3-3）sequences and one negative control（NC）sequence,and transfect them into SW620 cells respectively.The results of RT-PCR and Western Blot experiments showed that the knockdown efficiency of si SF3A3-3 was the highest,and the SF3A3 protein expression level and SF3A3 m RNA expression level were significantly reduced.2)Detection of the efficiency of overexpression of SF3A3 adenovirusSF3A3 gene overexpression adenovirus and negative control adenovirus were transfected in HCT116 cells,and the transfection efficiency was detected by Western Blot experiment.The results showed that the SF3A3 protein expression level in the SF3A3 overexpression adenovirus group was significantly higher than that in the control group.3)The effect of SF3A3 knockdown on the function of colorectal cancer cellsCCK-8 experiment,plate clone formation experiment and ki-67 staining experiment were used to detect the effect of knocking down SF3A3 on the proliferation of SW620 cells.CCK-8 and plate clone formation results showed that SF3A3 knockdown significantly inhibited the proliferation of SW620 cells.The immunofluorescence staining method was used to detect the expression level of ki-67.The immunofluorescence results showed that the expression of ki-67 was significantly reduced after knocking down SF3A3,indicating that the cell proliferation ability was significantly inhibited.Use Annexin V/FITC to detect the effect of SF3A3 on SW620 cell apoptosis by flow cytometry.The results of flow cytometry showed that knocking down SF3A3 significantly enhanced the apoptotic ability of SW620 cells.4)The effect of SF3A3 overexpression on the function of colorectal cancer cellsThe results of CCK-8 experiment and plate clone formation experiment showed that adenovirus overexpression of SF3A3 in HCT116 cells significantly enhanced its proliferation ability.Using Annexin V/FITC to detect the effect of overexpression of SF3A3 in HCT116 cells on its apoptosis ability by flow cytometry,the results showed that the overexpression of SF3A3 in HCT116 cells significantly inhibited its apoptosis ability.3.The mechanism of SF3A3 regulating the proliferation and apoptosis of colorectal cancer cellsWestern Blot results showed that knockdown of SF3A3 significantly reduced the expressions of p-PI3K,p-AKT473 and p-m TOR in SW620 cells,and the expression of p-PI3K,p-AKT⁴⁷³,and the expression levels of p-PI3K,p-AKT⁴⁷³and p-m TOR were significantly increased after SF3A3 overexpression in HCT116 cells.It indicates that SF3A3 may regulate the PI3K/AKT/m TOR pathway,thereby affecting the proliferation and apoptosis of CRC cells.Conclusion:In summary,SF3A3 is a cancer-promoting factor in the occurrence and development of colorectal cancer,which can regulate the PI3K/AKT/m TOR pathway,thereby affecting the proliferation and apoptosis of CRC cells.