Role Of Na⁺/k⁺ -ATPase α₁ Subunit In Regulation Of Hepatocellular Car Cinoma Cell Growth

Objective: To study the role of Na⁺/K⁺-ATPaseα1 subunit in the regulation of hepatocellular carcinoma cell growth, the proliferation activity and cell cycle of HepG2 cells were measured after treating with cinobufagin or knocking down the expression of Na⁺/K⁺-ATPaseα1 subunit.The expression of downstream signaling molecules were also determined in order to investigate the regulatory mechanism of Na⁺/K⁺-ATPaseα1 subunit in the growth of hepatocellular carcinoma.Method: (1)It was to construct the short hairpin RNA(shRNA) expressing vector shRNA-ATP1A1(ATP1A11, ATP1A12, ATP1A13) targeting human Na⁺/K⁺-ATPaseα1 subunit mRNA.The heptocellular carcinoma HepG2 cell line were transfected with vector shRNA-ATP1A1 for 24 hours.The Na⁺/K⁺-ATPaseα1 subunit expression level of HepG2 were detected by RT-PCR and immunocytochemistry. (2)The proliferation activity of ATP1A13 and cinobufagin on HepG2 cells were analyzed by MTT array, cell cycle were measured by flow cytometry. (3)Microscopic structure changes and ultranstructural feature of cells were observed by light microscopy and fluorescence microscope after DAPI staining. (4) The mRNA expression of CDK2, PCNA, cylinA, p21^CIP1, p53, c-src, Raf1, MEK1, MEK2 and c-Myc were determined by RT-PCR and real-time PCR. (5)The expression of CDK2,PCNA,CyclinA,p53,c-Src,total ERK,p-ERK and c-Myc protein were detected by western blot.Results: (1) The plasmid expression vectors were checked by enzyme digestion and sequencing, and they were successfully constructed. shRNA-ATP1A13 could significantly knock down the Na⁺/K⁺-ATPaseα1 subunit expression of HepG2 cells compared to the ATP1A11 and ATP1A12 group. Real-time PCR result showed that the knockdown effect of Na⁺/K⁺-ATPaseα1 mRNA expression level were in time dependent manner, and it decreased about 90 % after 72 hours.(2)ATP1A13 and cinobufagin both could inhibit HepG2 cell proliferation.The inhibition of ATP1A13 was in time dependent manner, and the inhibition of cinobufagin was in dose-time dependent manner.The median inhibitive concentration(IC50) of cinobufagin for 24, 48, 72 hours on HepG2 cells were (3.039±0.371), (0.758±0.102) and (0.133±0.028)μmol/L. (3) Cell cycle showed S phase arresting after transfecting with ATP1A13 for 48, 60 hours and treating with cinobufagin.The cells of S phase in the control group was (27.60±1.98) %.The cell population of S phase treated with ATP1A13 for 48, 60 hours were (39.4±2.04) % and (50.2±1.95) %.And the percents were (32.51±2.34) %, (40.28±1.89) %, (55.27±1.98) % and (67.28±1.92) % after treating with cinobufagin for 6, 12, 18, 24 hours which were markedly higher than that in the control group.The content of CDK2/CyclinA/PCNA complex in HepG2 cells decreased after treating with ATP1A13 and cinobufagin, and the mRNA and protein expression of p21CIP1 were significantly up-regulated.(3)The obvious morphological changes of cells treated with ATP1A13 and cinobufagin were detected by light microscopy.Some cells became round and swelling and had bad adherent ability after transfection for 24 hours.The cell morphology were irregular and some cells were floating after transfecting 36 hours.After transfecting for 48 and 60 hours,most of cells could have vacuoles phenomenon, and the normal contact inhibition of HepG2 cells disappeared and cell debris increased.Most of cells were death after treating with ATP1A13 for 72 hours. It could be seen that some cells became swollen and round when treating with 1μmol/L cinobufagin.With the effect of time, the swelling cells increased.The gap between cells became larger and cells became crushing and disordered after 24 hours'cinobufagin treating.HepG2 cells treating with ATP1A13 and cinobufagin were detected by fluorescent microscope after DAPI staining.Some cells could be seen with irregular shape, nuclear chromatin condensation and nuclear membrane rupture.With the prolongation of the cinobufagin and ATP1A13 treating, the morphology of HepG2 cells became more evident. (4)The mRNA expression of MEK1, MEK2, ERK and c-Myc decreased after transfecting with ATP1A13, and the expression of p53, c-src, Raf1 mRNA were significantly down-regulated.After treating with cinobufagin, the mRNA expression of Raf1, ERK, MEK1 and c-Myc were significantly up-regulated in HepG2 cells, and the expression of p53, c-src mRNA were decreased.(5)Compared with the control group, the expression of c-Src, ERK, p-ERK and c-Myc protein were down-regulated after transfecting.And treating with cinobufagin,it could down-regulate c-Src, p-ERK, c-Myc protein.The expression of p53 protein was significantly up-regulated after treating with ATP1A13 and cinobufagin.Conclusion: (1)The plasmid expression vectors coding for shRNA targeting Na⁺/K⁺-ATPaseα1 mRNA have been constructed successfully, and ATP1A13 could significantly know down the expression of Na⁺/K⁺-ATPaseα1 subunit.(2)ATP1A13 and cinobufagin could both inhibit HepG2 cell proliferation and induce cell cycle S phase arresting.The mechanism of S phase arresting in HepG2 cells may be the reduction of CylinA/CDK2/PCNA complex and up-regulation of p21^CIP1 expression level after treating with ATP1A13 and cinobufagin.(4)After treating with ATP1A13 and cinobufagin on HepG2, the expression of p53 protein were up-regulated.And the c-Src, p-ERK protein expression level were down-regulated which could down-regulate the expression of c-Myc protein.All of those could inhibit HepG2 cell proliferation.(5)The mRNA expression of Raf1/MEK1/2/ERK could be inhibited by ATP1A13.The down-regulation of ERK mRNA expression level could induce inhibition of HepG2 cell growth.(6)Konking down the expression of Na⁺/K⁺-ATPaseα1 and using Na⁺/K⁺-ATPase inhibitor could both treat tumor.But the effect mechanisms were not extractly the same.The studying of the mechanisms of knocking down the Na⁺/K⁺-ATPaseα1 subunit and Na⁺/K⁺-ATPase inhibitor may be a foundation for the research of anti-tumor therapy by RNA interference and drugs.