ADAM9 Regulates Radiosensitivity Of Hepatocellular Carcinoma Cells Through Autophagy

Objective: As one of the main treatment methods for primary hepatocellular carcinoma at present,radiotherapy has achieved considerable results in the treatment of most patients with liver cancer who are late staged and unable to accept radical treatment.However,due to the low tolerance of surrounding healthy liver tissues to radiotherapy,it is necessary to study the related mechanisms of regulating the radiotherapy sensitivity of liver cancer cells,improve the radiosensitivity and increase the survival rate of liver cancer cells,which has become valuable in the treatment of liver cancer patients.ADAM9 is a zinc dependent transmembrane protein which widely exists on the cell surface.Many studies have shown that the occurrence and development of hepatocellular carcinoma is related to ADAM9,but the connection between ADAM9 and radiosensitivity of hepatocellular carcinoma cells is not clear.In this study,we investigated the relationship between ADAM9 and radiosensitivity of hepatoma cells by infecting hepatoma cells overexpressing or low expression ADAM9 protein with virus,detected the expression of autophagy related proteins after X-ray irradiation,examined the ability of HCC cells’ proliferation and colony formation before and after the combination of autophagy activation dose or inhibitor which irradiated by X-ray,further explored what is the relationship between ADAM9 and the radiosensitivity of HCC.These studies provide cell-experimental support for finding new molecular targets that can improve the radiotherapy sensitivity of HCC.Methods: We constructed overexpression and knockout of ADAM9 protein’s lentiviral vectors,and transfected ADAM9 overexpression lentiviral vector into hepatoma cell line Huh-7 with low expression of ADAM9 protein,and compared it with empty group;Similarly,we transfected ADAM9 knockout lentivirus vector into MHCC97 H cell line,which overexpressed ADAM9 protein,and compared it with empty group.Verifying the effect of lentivirus packaging overexpression and knockout of ADAM9 protein in two hepatoma cell lines by Western blot.Irradiating the two cell lines and their control groups with 6MVX and 4Gy.Studing the colony forming ability of each group by plate clone formation experiment,and detecting the expression of autophagy related proteins LC3 and p62 by Western blot.Combining the MHCC97 H cells with autophagy activator,and the Hun-7 cells with autophagy inhibitor.Detecting the proliferation ability of cells in each group by CCK-8 assay。Result: 1.After overexpression of ADAM9 protein,the expression of ADAM9 protein in hepatoma cell line Huh-7 was higher than that in the blank control group;after knockout of ADAM9 protein,the expression of ADAM9 protein in hepatoma cell line MHCC97 H was significantly lower than that in the blank control group;2.Western blot results showed that compared with the control group,the expression of p62 and LC3-I protein in MHCC97 H cell line after knockout of ADAM9 protein was significantly increased,and the expression of LC3-II protein was obviously decreased;compared with the control group,the expression of p62 and LC3-I protein in Huh-7 cell line after overexpression of ADAM9 protein was decreased,and the expression of LC3-II protein was significantly increased;3.CCK-8 results showed that: compared with the control group,the proliferation of MHCC97 H cells in ADAM9 protein knockout group was significantly decreased(P < 0.05),and the proliferation of MHCC97 H cells increased after adding autophagy activator(P < 0.05);the proliferation of Huh7 cells in the ADAM9 overexpression group was significantly higher than that in the empty group(P < 0.05),and the proliferation of Huh7 cells in the ADAM9 overexpression group was decreased when combined with autophagy inhibitor(P < 0.05);4.The results of Plate clone formation experiment showed that the clone formation ability of ADAM9 knockout group was weaker than that of empty group,and the colony formation ability of ADAM9 knockout group was enhanced after combined with autophagy activator(P < 0.05);while the clone formation ability of ADAM9 protein knockout group was stronger than that of empty group,and the colony formation ability of ADAM9 protein knockout group was weakened after combined with autophagy inhibitor(P < 0.05).Conclusion: ADAM9 protein can reduce the radiosensitivity of HCC cells by inducing autophagy,ADAM9 may be a potential target for enhancing the radiosensitivity of hepatocellular carcinoma.