Study On The Expression And Significance Of SH3BGRL1 Gene In Cervical Cancer

Objective: To explore the relationship between protein and m RNA expression of SH3BGRL1 gene in cervical cancer cells and tissues,SH3BGRL1 gene and HPV(Human Papilloma Virus)infection;To investigate the effect of SH3BGRL1 gene on the proliferation,migration and cell cycle of cervical cancer cells,and exploring SH3BGRL1 gene inhibits the proliferation and migration of cervical cancer cells through Src-Akt-Erk signaling pathway.Methods:(1)The expression of SH3BGRL1 in normal cervical tissue and cervical cancer tissue was analyzed by The Human Protein Atlas(HPA),UALCAN and Gene Expression Profiling Interactive Analysis(GEPIA)databases.(2)The chip capture sequencing method was used to analyze the integration of HPV16 virus in the SH3BGRL1 gene.(3)Tissue microarray was constructed from normal cervical tissue and cervical cancer tissue,and immunohistochemical staining(IHC)was used to analyze the expression of SH3BGRL1 in tissues.(4)The collected cervical tissue samples were used to analyze the relationship between the gene and HPV infection by the Hybrid Capture 2(HC-2).(5)Western blot and q RT-PCR were used to detect the protein and m RNA expression of SH3BGRL1 gene in cancer cells.(6)The overexpression and RNA interference plasmids of SH3BGRL1 gene were designed and constructed respectively and transfected into He La and Si Ha cervical cancer cells.The control group(NC)was transfected with empty plasmids.(7)MTT assay and colony formation assay were used to detect the proliferation ability of He La and Si Ha cervical cancer cells after overexpression of SH3BGRL1 gene;MTT assay and colony formation assay were also used to detect the proliferation of He La and Si Ha after transfection of RNA interference plasmid.(8)Transwell assay and scratch assay were used to detect the migration ability of SH3BGRL1 overexpression cells He La and Si Ha;Transwell experiment was used to detect the migration of He La and Si Ha cervical cancer cells after gene interference.(9)Flow cytometry was used to detect the effect of overexpression of SH3BGRL1 gene on the cell cycle of cervical cancer He La and Si Ha.(10)Western blot detected protein expression levels of cell cycle-related molecules such as cyclin-dependent kinase 4(CDK4)and cyclin-dependent kinase 6(CDK6)after overexpression of the SH3BGRL1 gene.(11)Western blot was used to detect the expression of the total proteins and phosphorylated proteins of major molecules(Src,Akt,Erk)in the signal pathways related to proliferation and migration.Results:(1)HPA database analysis showed that SH3BGRL1 gene was relatively highly expressed in normal cervical tissue.UALCAN and GEPIA database analysis showed that SH3BGRL1 gene was highly expressed in normal cervical tissue and low expression in cervical cancer tissue(P<0.05).(2)By analyzing that these integration sites are all at the intron of the gene and far away from the exon,it is indicated that there may be no effect on the expression of the gene.Further analysis showed that the possible reason for HPV16 virus integration of this gene was due to microhomologous recombination.(3)Immunohistochemical staining showed that SH3BGRL1 was low expressed in cervical cancer tissues and high expressed in the normal cervical tissues(P<0.05).(4)HC-2 analysis showed that the expression of SH3BGRL1 gene might not be affected by HPV infection(P>0.05).(5)Western blot and q RT-PCR experiments showed that compared with Ha Ca T cells(a kind of immortalized keratinocytes),the protein and m RNA expression of SH3BGRL1 in three types of cervical cancer cells(Si Ha,He La,C33A)were significantly decreased(P<0.05).(6)MTT experiment and colony formation assay showed that when SH3BGRL1 gene was overexpressed,the proliferation of cervical cancer cells Si Ha and He La was inhibited(P<0.05);When SH3BGRL1 gene was decreased expression with RNA interference,the proliferation of cervical cancer cells Si Ha and He La was increased(P<0.05).(7)Transwell and scratch assay results showed that SH3BGRL1 gene overexpression the migration ability of Si Ha and He La cells was decreased(P<0.05).On the contrary,SH3BGRL1 gene down expression with RNA interference increased the migration of cervical cancer cells in transwell experiment(P<0.05).(8)The results of flow cytometry and Western blot showed that after overexpression of SH3BGRL1 gene,the cell cycle of cervical carcinoma He La and Si Ha was blocked in G0/G1 phase,while G2/M phase was significantly reduced and S phase had no significant change,thus inhibiting the cell proliferation(P<0.05);The expression levels of cell cycle related molecules CDK4 and CDK6 decreased significantly(P<0.05).(9)Western blot experiment results showed that SH3BGRL1 gene significantly inhibited the phosphorylation of Akt and Erk in the Src-Akt-Erk pathway and the phosphorylation of its upstream molecule Src,and the difference was statistically significant(P<0.05).Conclusion: SH3BGRL1 gene is lowly expressed in cervical cancer tissues and cells,and the expression is not related to HPV infection.The overexpression of SH3BGRL1 gene maybe inhibit the proliferation and migration of cervical cancer cells through Src-Akt-Erk signaling pathway.