Mechanism Of CT Gene LINC01977 Combined With RBM39 Mediating The Development Of Hepatocellular Carcinoma

Objective: To investigate the mechanism of action of CT gene(Cancer testis antigen)LINC01977 in promoting hepatocellular carcinogenesis through binding to RBM39 based on human tissues,hepatocellular carcinoma cell lines and immunodeficient nude mice.Methods: First,LINC01977 expression was examined in human normal tissues,and experiments were performed in human hepatocellular carcinoma and paraneoplastic tissues to clarify the expression of LINC01977 in hepatocellular carcinoma and paraneoplastic tissues,which were verified with normal hepatocytes and hepatocellular carcinoma cell lines.In vitro experiments,LINC01977 was knocked down in the corresponding hepatocellular carcinoma cells using shRNA,and the ability of clone formation,angiogenesis and migration invasion were verified by clone formation,angiogenesis and Transwell assays,and the ability of clone formation and migration invasion of the corresponding hepatocellular carcinoma cells was verified by overexpression of LINC01977.In addition,LINC01977 was overexpressed to observe the growth of the organoid.In vivo experiments were performed to validate the effect of LINC01977 on tumor proliferation and infiltration by constructing subcutaneous tumor and liver-lung metastasis models in nude mice.Finally,the LINC01977-binding protein was identified and the role of LINC01977 binding to this protein and of this complex in the development of hepatocellular carcinoma was verified.Results: In the present study,based on the results of human normal tissue samples,we found that LINC01977 was specifically expressed in testicular tissues,while it was largely absent in other normal tissues,and in human liver cancer tissues,the expression of LINC01977 was significantly higher in hepatocellular carcinoma tissues than in paraneoplastic tissues,and in hepatocellular carcinoma cell lines,the expression of LINC01977 was significantly higher in hepatocellular carcinoma cells than in normal cells.In the in vitro experiments,stable knockdown and overexpression cell lines were constructed using shRNA and overexpression plasmids.Two cell lines,HepG2 and MHCC97 H,were selected for knockdown and two cell lines,SMMC7721 and Hep3 B,were selected for overexpression according to the expression status of each cell.In vitro knockdown of LINC01977 in HepG2 and MHCC97 H cells using shRNA showed that clone formation,angiogenesis,and transwell assays were significantly inhibited.Overexpression of LINC01977 promoted clone formation and migration invasion in SMMC7721 and Hep3 B cells.In addition,overexpression of LINC01977 promoted organoid growth.Subcutaneous tumor and metastasis models were constructed in vivo using immunodeficient nude mice,and the results were consistent with those in vitro.Finally,we confirmed by RNA-pulldown and mass spectrometry experiments that LINC01977 specifically binds to RBM39,that RBM39 promotes hepatocellular carcinogenesis and development,and that LINC01977 in combination with RBM39 jointly promotes hepatocellular carcinogenesis and development.Conclusions: The expression of LINC01977 in human hepatocellular carcinoma tissues was significantly higher than that in paraneoplastic tissues,and in hepatocellular carcinoma cell lines,the expression of LINC01977 in hepatocellular carcinoma cells was significantly higher than that in normal cells.In addition,ex vivo experiments also confirmed that LINC01977 promoted clone formation,angiogenesis,invasive migration and tumor formation in hepatocellular carcinoma cells.Finally,it was confirmed that LINC01977 combined with RBM39 to promote hepatocellular carcinogenesis and progression.