The Research On The Molecular Mechanism And Cell Biological Function Of LncRNA CASC2 In Cholangiocarcinoma

Objective: Based on the previous research results of LncRNA high-throughput sequencing of cholangiocarcinoma tissue specimens,the research group cultured cholangiocarcinoma cell lines in vitro,and explored the effect of LncRNA CASC2 on the proliferation,migration and invasion abilities of cholangiocarcinoma cells and the mitotic cycle of cells;Explored the mechanism of LncRNA CASC2,NF-κB and ERK1/2 pathways in the occurrence and development of cholangiocarcinoma,and to preliminarily verify the regulation mode of LncRNA CASC2 in cholangiocarcinoma,provided a theoretical basis for the development of early clinical diagnostic markers and molecular targeted therapy drugs,prognosis follow-up and monitoring of cholangiocarcinoma.Methods: 1.Cholangiocarcinoma cell lines HUH-28 and RBE were cultured in vitro,and the expression of CASC2 in cholangiocarcinoma cell lines was detected by q RT-PCR technique;2.The expression of CASC2 in cholangiocarcinoma cells was targeted knockdown by transfecting si-CASC2 with lipofectamine 2000,and the green fluorescence of the control group after transfection with FAM-si-NC(Carboxyfluorescein-labeled negative control si RNA)was observed by biological microscope to preliminarily judge the transfection effect,then the knockdown effect was verified by q RT-PCR;3.To study the effect of CASC2 knockdown on the proliferation of cholangiocarcinoma cells by CCK-8 assay;4.The effect of knockdown of CASC2 on the migration of cholangiocarcinoma cells was studied by Wound healing assay and Transwell assay;5.The effect of knockdown of CASC2 on the invasion of cholangiocarcinoma cells was studied by Transwell assay;6.To study the effect of knockdown of CASC2 on the mitotic cycle of cholangiocarcinoma cells by FACS cell flow assay;7.The effect of knockdown of CASC2 on the protein expression of extracellular signal-regulated kinase 1/2(ERK1/2)in nuclear factor κB(NF-κB)and mitogen-activated protein kinase(MAPK)pathway was detected by Western blot.Results: 1.The results of q RT-PCR experiments showed that compared with normal cholangiocarcinoma,CASC2 was highly expressed in cholangiocarcinoma Hu H-28 and RBE cell lines(p<0.05),which was consistent with our previous detection results in cholangiocarcinoma;2.Microscope showed that the transfection efficiency of HUH-28 and RBE after transfection of si-CASC2 could reach more than 80%;q RT-PCR results showed that CASC2 in HUH-28 and RBE transfected with si-CASC2 was significantly lower than that in control group(p<0.01);3.The results of CCK-8 showed that after knockdown of CASC2,the proliferation activity of cholangiocarcinoma cells HUH-28 and RBE was lower than that of the control group(p<0.05),that is,the proliferation activity of cholangiocarcinoma cells after knockdown of CASC2 was reduced;4.The results of cell scratch assay and Transwell assay showed that the migration rate of CASC2 knockdown was significantly lower than that of the control group(p<0.01),indicating that CASC2 knockdown could significantly reduce the migration ability of cholangiocytic cells;5.The results of Transwell invasion assay showed that after knockdown of CASC2,the number of cholangiocarcinoma cells penetrating matrigel decreased significantly compared with the control group,indicating that knockdown of CASC2 could significantly reduce the invasive ability of cholangiocarcinoma cells and the inhibition ability of HUH-28 cell line is the most significant(p<0.01);6.6.FACS cell flow experiments showed that compared with the control group,the number of cells in G1 phase of cholangiocarcinoma cells was significantly increased after knockdown of CASC2,while the number of cells in S phase was significantly decreased(p<0.01),indicating that knockdown of CASC2 can inhibit the mitosis of cholangiocarcinoma cells;7.Western blot results showed that after knockdown of CASC2,the protein expressions of NF-κB and ERK1/2 were significantly lower than those in the control group(p<0.01),indicating that knockdown of CASC2 could significantly inhibit the CASC2/NF-κB and CASC2/ERK1/2 signaling pathways activation.Conclusion1.LncRNA CASC2 can play a role of cancer-promoting factor by affecting the proliferation,migration,invasion ability and the G0/G1 phase of cell mitosis in cholangiocarcinoma cells;2.The mechanism that LncRNA CASC2 plays in cholangiocarcinoma is related to the activation of NF-κB and ERK1/2 signaling pathways;...