Effect Of Conditioned Medium Of Human Colon Cancer Cells On Activation Of Hepatic Stellate Cells And Its Mechanism

ObjectiveColorectal cancer is one of the most common malignant tumors worldwide,with the third in incidence and second in mortality.Liver metastasis of CRC is the main factor for the poor prognosis of patients.The stromal cells of the liver tissue microenvironment promote the development of the metastatic process.In the tumor microenvironment,tumor cells change the tumor microenvironment and promote tumor growth and metastasis by inducing hepatic stellate cells to gradually differentiate into cancer-related fibroblasts,but the mechanism is still unclear.PVT1 has been confirmed to be involved in the activation of primary hepatic stellate cells induced under hypoxia and other conditions,but whether PVT1 is involved in the activation of hepatic stellate cells in metastatic liver cancer has not been reported yet.The main purpose of this study is to reveal:(1)To study the hepatic stellate cell activation,biological function and protein function expression changes induced by the conditioned medium of colon cancer cells HCT116 and HT29.(2)Detect the effect of colon cancer cell conditioned medium on the expression of PVT1 in hepatic stellate cells,and study the effect of PVT1 gene on the activation and biological function of hepatic stellate cells.Methods1.We first prepare the conditioned medium of HCT116 and HT29 colon cancer cells and observe its effects on the proliferation,migration and activation of hepatic stellate cells LX2.CCK8 method detects the proliferation ability of LX2 cells under the intervention of conditioned medium,and Transwell detects the migration of LX2 cells in vitro ability.q RT-PCR was used to detect the gene expression changes of α-SMA,an activation marker of LX2 cells,and Western blot was used to detect the expression changes of α-SMA and tumor metastasis-related proteins IL6,TNFα and VEGF.2.Use q RT-PCR to detect the difference in PVT1 gene expression between LX2 cells co-cultured with HCT116 and HT29 cell conditioned medium and normal LX2 cells.A lentiviral vector was constructed to mediate RNA interference to silence the expression of PVT1 in hepatic stellate cells,and a negative control group(si-Ctrl group)was set up.Puromycin selection and stably transfected LX2 cells were used to verify the transfection efficiency of LX2 cells in the si PVT1 group by q RT-PCR.CCK8 and Transwell tested the effects of silencing PVT1 on the proliferation and migration of LX2 cells.The expression levels of the activation marker α-SMA m RNA in LX2 cells in the si PVT1 group and si-Ctrl group were detected.Result1.Colon cancer cells(HCT116,HT29)conditioned medium separately interfered with LX2 cells.Compared with normal LX2 cells,the proliferation and migration ability of LX2 cells was enhanced,the activation marker α-SMA m RNA and protein expression increased,and the inflammatory factors IL6,TNFα and The protein expression of angiogenesis factor VEGF increased,and the difference was statistically significant(P<0.05).2.Compared with the control group,the expression level of PVT1 in the LX2 cells of the intervention group was significantly higher.Silencing LX2 cells PVT1 can inhibit the proliferation and migration of hepatic stellate cells induced by the conditioned medium of colon cancer cells.The activation of LX2 cells is a sign of α-SMA.The m RNA expression level decreased,and the difference was statistically significant(P<0.05).Conclusion1.Under the induction of colon cancer conditioned medium,the expression of the activation marker αSMA of hepatic stellate cells LX2 increased,the proliferation and migration ability was enhanced,LX2 cells gradually transformed into CAFs,and the IL6,TNFα,TNFα,which are related to invasion and metastasis in LX2 cells The increased expression of VEGF protein may be related to the inflammatory microenvironment and angiogenesis of metastatic tumors.2.In the conditioned medium of HCT116 and HT29 cells,the expression of lnc RNA PVT1 in LX2 cells increased.After silencing the expression of PVT1,the expression level of αSMA of LX2 cells was down-regulated and inhibited the proliferation and migration ability of LX2 cells.The high expression of lnc RNA PVT1 may be the colon One of the mechanisms of hepatic stellate cell activation induced by cancer cells.