LncRNA MALAT1 Sponge MiR-200a-3p Regulates Gab1 Expression And Affects VEGF Autocrine In Hilar Cholangiocarcinoma Cells

Objective: To explore the regulation of lnc RNA MALAT1 sponge mi R-200a-3p on the expression of Gab1 in hilar cholangiocarcinoma cells,and then to regulate the autocrine of VEGF in hilar cholangiocarcinoma cells,so as to provide a theoretical basis for the clinical treatment of hilar cholangiocarcinoma.Methods: Human cholangiocarcinoma cell ICBD-1 and TFK-1 purchased from Aiyan Biotech(Shanghai)Co.,Ltd and Be Na Culture Collection,as well as normal human intrahepatic bile duct epithelial cell HUM-CELL-0035(Beijing Yuantang Shengxing Technology Co.,Ltd.)were selected in this study.The cells were cultured in10% FBS DMEM medium(24h after cell transfection in VEGF autocrine test,they were replaced with 2% FBS DMEM medium for further culture).1.The expression of VEGFR-2 and Gab1 in TFK-1 was detected by RT-PCR.2.Real-time PCR was used to detect the m RNA expression of Gab1 and VEGFR-2 in ICBD-1,TFK-1 and HUM-CELL-0035.3.The expressions of lnc RNA MALAT1 and mi R-200a-3p in ICBD-1,TFK-1 and HUM-CELL-0035 were detected by real-time PCR after cultured to the logarithmic growth stage.4.Three lnc RNA si RNA fragments and one independent interference sequence(negative control,NC)fragments were designed and transfected into logarithmic ICBD-1 and TFK-1.The expression of MALAT1 was detected by real-time PCR after 48 h and the highest interfering lnc RNA si RNA fragment was selected for transfection.The effects of lnc RNA MALAT1-si RNA on the expression of mi R-200a-3p and Gab1 in the two kinds of cells were detected by real-time PCR.5.After transfection of mi R-200a-3p mimic into ICBD-1 and TFK-1 at logarithmic growth stage for 48 h,real-time PCR was used to detect the effect of mi R-200a-3p mimic on Gab1 expression in the two kinds of cells.6.Gab1 wild-type overexpression vector(Gab1-WT)was designed and constructed.Gab1-WT and screened si Gab1 were transfected into logarithmic ICBD-1 and TFK-1,and the changes in the expression of Gab1 in the two kinds of cells were detected by western blot.7.si Gab1 and Gab1-WT were transfected into ICBD-1 and TFK-1,respectively for 24 hours,and then the culture medium was changed to 2% FBS for 24 hours.Western blot was used to detect the effect of Gab1 changes on VEGFR-2 in ICBD-1 and TFK-1 cells,respectively.8.Normal culture ICBD-1 and TFK-1,selected logarithmic growth phase cells transfected with si Gab1 and Gab1-WT for 24 hours,and cultured for 24 hours with low concentration of FBS.The cell culture supernatants of each group were collected,and the contents of VEGF-A and VEGF-C in the supernatants of each group were detected by ELISA.9.Immunofluorescence was used to detect the changes of intracellular distribution of Gab1 and p-VEGFR-2 and the effect of exogenous VEGF on tumor cells.Results:1.The expression of VEGFR-2 and Gab1 was also confirmed in TFK-1 by RT-PCR.Real-time PCR found that the expression of Gab1 and VEGFR-2 m RNA in ICBD-1 and TFK-1 cells was higher than that in HUM-CELL-0035.This is consistent with the previously reported expression of them in hilar cholangiocarcinoma.2.Real-time PCR was used to detect lnc RNA MALAT1 and mi R-200a-3p in two kinds of cells.Compared with HUM-CELL-0035,lnc RNA MALAT1 m RNA expression was increased in both cells,while mi R-200a-3p m RNA expression was decreased.3.The lnc RNA si RNA fragments with the highest interference efficiency were selected and transfected into two kinds of cells,and the effects of lnc RNA MALA T1-si RNA on the expression of mi R-200a-3p and Gab1 in the two kinds of cells were detected by real-time PCR.Compared with NC group,the expression of mi R-200a-3p m RNA in lnc RNA MALA T1-si RNA group was increased,while the expression of Gab1 m RNA was decreased in both groups.4.Real-time PCR was used to detect the effect of mi R-200a-3p mimic on the expression of Gab1 in the two kinds of cells.Compared with mimic-NC group,the expression of mi R-200a-3p m RNA in mi R-200a-3p mimic group increased while the expression of Gab1 m RNA decreased in both groups.5.Western blot was used to detect the effect of Gab1 changes on VEGFR-2 in the two kinds of cells.Compared with the NC group,the p-VEGFR-2 of the two kinds of cells in the si Gab1 group decreased,while compared with the Gab1-Vector group,the p-VEGFR-2 of the two kinds of cells Gab1-WT group increased.The change of Gab1 had no significant effect on the expression of VEGFR-2.6.Changes in VEGF-A and VEGF-C levels in cell supernatant were detected by ELISA.Compared with the NC group,the content of VEGF-A and VEGF-C in the supernatant of the two types of cells in the si Gab1 group decreased;Compared with the Gab1-Vector group,the content of VEGF-A and VEGF-C in the supernatant of the two kinds of cells in the Gab1-WT group increased.7.The intracellular distribution of Gab1 and p-VEGFR-2 was detected by immunofluorescence.Compared with Gab1-Vector group,the expression of Gab1 and p-VEGFR-2 in Gab1-WT group increased,p-VEGFR-2 moved to cytoplasm and nucleus,and nuclear aggregation of p-VEGFR-2 increased.8.The effect of exogenous VEGF on tumor cells was detected by immunofluorescence.When VEGF-A 50 ng was added to the cells of each group,VEGFR-2 phosphorylation increased in both Gab1-Vector and Gab1-WT groups after 5min,p-VEGFR-2 moved to cytoplasm and nucleus,and p-VEGFR-2 nucleus aggregation increased.Compared with the Gab1-Vector group,there were more VEGFR-2 phosphorylation and p-VEGFR-2 migration and nuclear accumulation in the Gab1-WT group.Conclusion:1.LncRNA MALA T1 sponge mi R-200a-3p regulates the expression of Gab1 in hilar cholangiocarcinoma cells.2.Hilar cholangiocarcinoma cells expressing VEGFR-2 have VEGF autocrine regulated by Gab1.Gab1 regulates VEGF autocrine by regulating the nuclear aggregation of p-VEGFR-2.3.3.Exogenous VEGF synergistically enhances the phosphorylation and nuclear aggregation of VEGFR-2 by autocrine VEGF.