Silencing UPAR Affects The Biological Function Of Human Hepatoma Cell Line SMMC-7721 And The Expression Of Integrin Alpha 5

Objective: Hepatocellular carcinoma(HCC)is the most common histological type of primary liver cancer.As a common malignancy,HCC has a significant contribution to the global burden of disease and mortality and is the second leading cause of cancer death worldwide.Urokinase plasminogen activator receptor(uPAR)is a member of the Lymphocyte antigen 6(ly-6)superfamily.uPAR is highly expressed in hepatocellular carcinoma and is closely related to the occurrence and development of hepatocellular carcinoma.This study investigated the effects of uPAR on the progression of liver cancer,explored the underlying molecular mechanisms,and further analyzed the relationship between uPAR and integrins.Methods: To explore the function of uPAR in hepatocellular carcinoma cell line SMMC-7721,we used uPAR-siRNA lentivirus to transfect liver cancer cells and down-regulate the expression of uPAR in hepatoma cell line SMMC-7721.The knockdown of uPAR at mRNA and protein levels was verified by real-time quantitative PCR(RT-PCR)and Western blot(WB),and the stably transfected cell lines were screened for expansion and culture.It was used as anuPAR-siRNA silencing group.At the same time,cells transfected with the negative control lentivirus were used as a negative control group(NC group),and cells without any treatment were used as a blank control group(BC group).After transfection,the proliferation ability of SMMC-7721 cells after silencing was verified by CCK-8 assay.The migration and invasion functions of the cells were verified by wound healing experiment,transwell migration and invasion assay respectively.Apoptosis and cycle were detected by flow cytometry.Clonal formation assays are used to detect the ability of cells to clone.In addition,to explore the interaction between uPAR and integrins,we examined the effect of knockdown of uPAR on integrin ?5 by RT-PCR and Western blot.SPSS 22.0software was used for statistical data,and comparison between groups was performed by one-way ANOVA.Results: The stable hepatoma cell line SMMC-7721 with down-regulated uPAR was successfully constructed.The results of RT-PCR and Western blot showed that the expression of uPAR mRAN and protein in uPAR-siRNA lentivirus transfection group was significantly lower than that in negative lentiviral transfection group and blank control group,and the silencing efficiency was 94.2%.The difference was statistically significant(P < 0.05).The CCK-8 assay for cell proliferation showed that compared with the other two groups,the uPAR-siRNA silencing group had a decreased proliferative capacity(P<0.05)within 48 h to 120 h,and the cell proliferation ability was inhibited.The results of wound healing experiments showed that the area of scratched blanks in the silencing group was larger than that in the other two groups at 24h(P<0.05).The Transwell migration experiment obtained similar results.The number of migrated cells in the uPAR-siRNA silencing group was 63.40±8.11,which was significantly lower than that in the BC group(162.40±11.13)and NCgroup(155.47±11.75).The difference was statistically significant(P<0.05),indicating that uPAR knockdown can significantly inhibit the migration function of SMMC-7721.The results of Transwell invasion assay showed that after transfection of uPAR-siRNA lentivirus,the number of invasive cells through Matrigel decreased,and the cell invasion ability was inhibited(P<0.05).In the colony formation experiment,the number of cloned cells in BC group,NC group and silence group were 387.67±12.90,365.67±15.04 and 121.67±7.64,respectively.The ability of clone formation in silencing group was inhibited,and the difference was statistically significant(P<0.05).The results of RT-PCR and Western blot showed that the knockdown of uPAR could down-regulate the expression of integrin ?5 mRNA and protein in SMMC-7721 cells,and the inhibition rate was 76.2%,the difference was statistically significant(P<0.05).Conclusion: The SMMC-7721 cell line silencing the uPAR gene was successfully constructed by lentiviral transfection.Down-regulation of uPAR levels can lead to a decrease in integrin ?5 and inhibit the proliferation,colony formation,invasion and migration of liver cancer cell SMMC-7721.Our study validated the role of uPAR in the progression of SMMC-7721 cells,suggesting that it may be a potential target for the treatment of liver cancer.