Effect Of AQP9 Over-Expression On Hepatoma Cell Invasion And Metastasis And Its Possible Molecular Mechanism

Objective: To investigate the effect of aquaporin 9(AQP9) overexpression on hepatoma cell invasion and metastasis and its possible molecular mechanism.Methods:Part Ⅰ The expression level of AQP9 in four human hepatoma cell lines(Huh-7, Hep G2, SMMC-7721, and Hep3B) and a human normal hepatocyte line LO2 was detected by real-time fluorescence quantitative PCR and western blotting. We chose SMMC-7721 cell as cell model in vitro among the four HCC cell lines. Human hepatoma cell line SMMC-7721 cells were transfected with recombinant lentivirus LV-AQP9-GFP(LV-GFP empty vector as negative control) to establish AQP9 overexpressed SMMC-7721/LV-AQP9 recombinant cell models. The transfection efficiency of SMMC-7721 cells was observed under a laser scanning confocal microscope, and AQP9 was assessed by western blot assay. Cell swelling assay was used to detect the functional activity degree of AQP9 as a water channel in AQP9 overexpressed cell models. Scratch assay and Transwell migration assay were used to evaluate the differences of AQP9 over-expression on migration and invasion of SMMC-7721 cells.PartⅡ In vivo, recombinant hepatoma carcinoma SMMC-7721/LV-AQP9 and SMMC-7721/LV-GFP cells(the control group) were subcutaneously implanted in nude mice. The volume and growth rate of the xenograft tumors of 2 groups of nude mice were observed dynamically. Meanwhile, pulmonary metastasis of liver cancer model was established by tail intravenous injection in recombinant hepatoma carcinoma SMMC-7721/LV-AQP9 and SMMC-7721/LV-GFP cells, the transfer number of pulmonary surfactant cancerous tubercle of the 2 groups of nude mice was observed, and metastatic tumors in the lung tissue were observed by HE staining.Part Ⅲ Western blot was used to detect EMT related proteins(E-cadherin, N-cadherin and Vimentin) in SMMC-7721/LV-AQP9 and SMMC-7721/LV-GFP cells, to detect expression difference of PI3K/AKT pathway related proteins PI3 K, AKT, p-AKT and matrix metalloproteinase MMP2, MMP9. Protein expression of E-Cadherin, N-Cadherin and Vimentin in transplant tumor issues were detected by immunohistochemical staining.Result:Part Ⅰ Hepatoma cell lines Hep3 B did not express AQP9, AQP9 m RNA and AQP9 expression in SMMC-7721, Hep G2 and Huh-7 were significantly lower than in a human normal hepatocyte LO2 line(all P<0.05). Of which AQP9 m RNA and AQP9 expression in SMMC-7721 was the lowest. The infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was about 90% under the fluorescence microscope. Green fluorescent fusion protein AQP9-e GFP was found mainly on the membranes of SMMC-7721/LV-AQP9 cells through GFP fluorescence signal positioning. The protein expression levels in SMMC-7721/LV-AQP9 recombinant cells(AQP9 cells) were significantly increased compared to those in SMMC-7721/LV-GFP cells(GFP cells)( P < 0.01). The cell size of SMMC-7721/LV-AQP9 cells(AQP9 cells) was dramatically increased upon incubation in the hypotonic media compared to that of SMMC-7721/LV-GFP cells(GFP cells)( P<0.05). When AQP inhibitor Hg Cl2 was added to block water protein activity, the cell size of both AQP9 and GFP cells was not changed under hypotonic challenge. In the wound healing assays, we found that the GFP cells showed dramatic wound closure after 72(migration rate: 16.74%±1.40%), whereas the AQP9 cells showed minimal wound closure(migration rate: 6.38%±1.70%)(P < 0.01).In Transwell invasion assays, the number of AQP9 cells through the small room after 48 hours(57±8) showed significantly less invasion than the GFP cells(95±11)(P < 0.01).Part Ⅱ In vivo, the results of repetitive measurement deviation analysis of xenograft tumors v olume in both groups displayed that xenograft tumors volume in over-expression group was smaller and the growth rate was slower than those in the control group(F group = 79.161, P group = 0.000, F group×time = 18.481, P group×time = 0.002). HE staining in nude mice pulmonary metastasis models indicated that the volume of metastatic tumors in lung tissues was significantly smaller and the transfer quantity of cancerous tubercles on lung surfaces was significantly decreased in AQP9 group than that than that those in GFP group(P < 0.05).Part Ⅲ Western blot showed that after AQP9 cells had been overexpressed, the epithelial marker E-Cadherin, PI3 K and P-AKT protein expression was higher(all P < 0.05), phenotypic markers N-cadheri protein was lower(P < 0.05). There was no significant difference in vimentin, Akt, MMP9 and MMP2 expression compared to GFP cells(all P>0.05). IHC and density analysis showed that in AQP9 group E-cadherin expression of xenografted tumor was significantly increased(P<0.05)while N-cadherin and vimentin expression was significantly decreased compared to GFP cells( P < 0.01).Conclusion:Compared to normal liver cells, AQP9 in hepatocellular carcinoma is down-regulated and its over-expression suppresses hepatoma cell invasion and epithelial-to-mesenchymal transition. PI3K/AKT pathway may be involved in the regulation process. In addition, neither matrix metalloproteinase MMP2 nor MMP9 expression was affected by AQP9 over-expression.