The Effect Of APQ9 On The Proliferation Of Hepatocellular Carcinoma And Its Mechanism In Anoxic Microenvironment.

Objective: To explore how AQP9 was involved in the hypoxia response of cancer cells induced by hypoxia microenvironment of hepatocellular carcinoma,and initially explored its possible mechanisms.Methods:PartⅠ The hypoxia model of SMMC-7721 and Huh-7 cells was successfully constructed by using hypoxia trigas incubator.In the hypoxia microenvironment,using q RT-PCR to detect the expression of AQP9 mRNA of SMMC-7721 and Huh-7 cells,by Western Blot to detect the expression of AQP9 protein of Huh-7 cells.The SMMC-7721 and Huh-7 cells were infected with recombinant lentivirus LV-AQP9(SMMC-7721-AQP9 and Huh-7-AQP9),LV-PWPI as a negative control(SMMC-7721-AQP9-si NC and Huh-7-PWPI),and then were infected with siRNA-AQP9(SMMC-7721-AQP9-si AQP9 and Huh-7-AQP9-AQP9),siNC as a negative control(SMMC-7721-AQP9-siNC and Huh-7-AQP9-NC).The infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 and Huh-7 cells was observed under a laser scanning confocal microscope,and the expression levels of AQP9 protein were detected by Western Blot,respectively.The proliferation and invasion of SMMC-7721-AQP9 and Huh-7-AQP9 cells were detected by CCK8 and transwell respectively.We used Western Blot to detect the expression of PI3K-AKT-mTOR signaling pathway relative proteins of SMMC-7721-AQP9,Huh-7-AQP9,SMMC-7721-PWPI and Huh-7-PWPI cells in hypoxic environment for 6h.The levels of IGF2 secreted by cells were detected by Elisa.The mRNA level of HIF-1α pathway relative proteins were detected by qRT-PCR,the localization of HIF-1α was detected by a laser scanning confocal microscope.PartⅡ The relative expression of HIF-1α and AQP9 in 25 HCC patients was detected by immunohistochemistry in 25 HCC patients.Part Ⅲ In vivo,recombinant hepatoma carcinoma SMMC-7721-PWPI,SMMC-7721-AQP9,Huh-7-PWPI and Huh-7-AQP9 cells were subcutaneously implanted in nude mice.The volume and growth rate of the xenograft tumors of 4 groups were observed dynamically.The expression levels of HIF-1α protein in tumor xenografts of the tumor-bearing mice were detected by immunohistochemistry.Results:PartⅠUsing qRT-PCR and Western Blot to detect the expression levels of AQP9 mRNA(F=435.693,P=0.00)and protein(F=586.788,P=0.00)in Huh-7 cells(P<0.05)and the expression levels of AQP9 mRNA(F=708.757,P=0.00)in SMMC-7721 were decreased in hypoxic microenvironment.In the hypoxia microenvironment,the over-expression of AQP9 inhibited the proliferation and invasion of SMMC-7721 and Huh-7 cells detected by CCK8 and transwell,which stronger than normoxia(P<0.05).In hypoxic microenvironment,the expression of pMTOR,pAKT and HIF-1α protein in SMMC-7721-AQP9 and Huh-7-AQP9 cells were significantly lower than SMMC-7721-PWPI and Huh-7-PWPI cells(P<0.05).The levels of IGF2 secreted by Huh-7-AQP9 and SMMC-7721-AQP9 cells were lower than Huh-7-PWPI and SMMC-7721-PWPI cells.The mRNA levels of VEGFA,CA9,TGM2,IGF2 and ENO1 gene in Huh-7-AQP9 and SMMC-7721-AQP9 cells were lower than Huh-7-PWPI and SMMC-7721-PWPI cells(P<0.05),the mRNA level of PFKFB3 was not obvious change(P>0.05).The expression of HIF-1α protein is mainly distributed in the nuclear.PartⅡ The expression of HIF-1α protein in the location with higher expression of AQP9 was lower than the location with higher expression of AQP9 detected by immunohistochemistry in 25 HCC patients.Part Ⅲ In vivo,AQP9 could inhibit the growth of tumor xengrafts of human hepatocellular carcinoma Huh-7 and SMMC-7721 cells.The expression levels of HIF-1α protein in tumor xenografts of the tumor-bearing mice transplanted with Huh-7-AQP9 and SMMC-7721-AQP9 cells lower than SMMC-7721-PWPI and Huh-7-PWPI cells(P<0.05).Conclusion: In hypoxic microenvironment,the increasing expression of AQP9 can inhibit the proliferation and invasion of SMMC-7721 and Huh-7 by inhibiting the expression of HIF-1α.Furthemore,the inhibitory effect of AQP9 on proliferation and invasion in anoxic environment is stronger than in the normal oxygen environment.