| Objective: Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer(PLC)with a poor prognosis.Urokinase plasminogen activator receptor(uPAR)is highly expressed in a variety of malignant tumors and plays an important role in tumor development.The previous research of our research group showed that uPAR expression is increased in liver cancer tissues,but its mechanism of action in HCC has not been fully clarified.This study will further verify the effect of overexpression of uPAR gene on the biological function of liver cancer cells,and explore the potential molecular mechanism between uPAR and integrin.Methods: First,in this experiment,gene overexpression technology was used to integrate the lentiviral vector carrying the overexpressed uPAR gene into hepatocellular carcinoma HepG2 cells,and the stable transfectants were screened and purified by puromycin for expansion and culture,established as the uPAR overexpression group(LV-uPAR).At the same time,the cells transfected with the negative control virus were used as the no-load control group(Control),and the cells without any treatment were used as the untreated group(Untreated).Quantitative Real-time PCR(RT-PCR)and Western blot(WB)were used to detect the expression of uPAR mRNA and protein levels in each experimental group after transfection to confirm whether uPAR overexpression was successful.Secondly,a series of functional experiments will be used to verify the effect of overexpression of uPAR on the function of hepatocellular carcinoma HepG2 cells.Among them,the CCK8 experiment was used to detect the cell proliferation ability;the Transwell migration and wound healing assay were used to detect the cell migration ability;the Transwell invasion test was used to detect the cell invasion ability;colony-formation assay was used to detect the cell clone formation ability;flow cytometry was used to detect the effect of overexpression of uPAR gene on HepG2 cell cycle and apoptosis.Finally,in order to explore whether there is interaction between uPAR and integrin β1,RT-PCR and WB analysis were used to detect changes in integrin β1 in HepG2 cells after overexpression of uPAR,and WB was used to detect the expression of focal adhesion kinase(FAK)Level.SPSS22.0 software is used for data statistics,and comparison between groups was performed by One-way ANOVA.Results: HepG2 cell line with stable overexpression of uPAR was successfully constructed.Cells transfected with lentivirus all emit GFP fluorescence and grow well,with transfection efficiency of more than 80%;RT-PCR and WB results showed that compared with untreated group and control group,uPAR mRNA and protein level was significantly increased,and the difference was statistically significant(P<0.01).CCK8 results showed that the cell proliferation ability of the LV-uPAR group was significantly higher than that of the other two groups at 48h(P<0.01);Wound healing assay showed that the wound healing rate of LV-uPAR group was lower than that of untreated and control groups during the period from 12 h to 24h(P<0.05);Transwell migration experiment found that the number of migrated cells in the untreated group,control group and LV-uPAR group were(180.20 ± 6.56),(192.67 ± 9.29)and(327.52 ± 3.61),the difference was statistically significant(P<0.01);Transwell invasion results showed that the number of LV-uPAR group passing through Matrigel gum was(316.00 ± 8.00),which was significantly more than that of the untreated group(259.28 ± 4.58)and the control group(271.33 ± 5.03)(P<0.01);in the clone formation experiment,the number of clones formed by cells in the untreated group,control group and LV-uPAR group were(225.33 ±11.06),(240.33 ± 5.86)and(409.33± 12.90)(P<0.01);the results of flow cytometry indicated that compared with the other two groups,the G0/G1 ratio in the LV-uPAR group was lower(P<0.05);but there was no significant difference in the number of apoptosis in each experimental group(P>0.05).Afterwards,RT-PCR and WB results showed that the expression levels of integrin β1 mRAN and protein in uPAR LV-uPAR group were significantly higher than the two control groups(P<0.01);In addition,the expression level of FAK protein also increased significantly,the difference was statistically significant(P <0.01).Conclusion: HepG2 cell line with stable overexpression of uPAR can be successfully constructed by lentivirus transfection.Overexpression of uPAR can promote the proliferation,migration and invasion ability of hepatocellular carcinoma cells,also,it promotes HepG2 cells to enter S phase from G0/G1 phase,but it has no obvious effect on the apoptosis outcome.In addition,overexpression of uPAR leads to increased levels of integrin β1,and its mechanism may be related to integrin β1-FAK signaling pathway. |
PDF Full Text Request