Glycogenes Mediate The Invasive Properties And Chemosensitivity Of Human Hepatocarcinoma Cells

Objective: This study aimed to identify the alterations of glycogene involved intumor invasion and drug resistance in MHCC97-H and MHCC97-L humanhepatocarcinoma cell lines, which have high, low metastatic potential, respectively. Tofurther predict and diagnose human hepatocarcinoma invasion and drug resistance, andprovide a new therapeutic strategy and target point of human hepatocarcinoma.Methods:(1) Real-time PCR was used for quantification of glycogenes. The difference(i.e.,>3-fold higher) of glycogenes were analyzed in MHCC97-H and MHCC97-Lcells. Characteristic alteration of glycogenes in invasion and drug resistance ofhuman hepatocarcinoma is explored firstly.(2) The present study clearly showed the lectin binding profiles of cell surfaces inMHCC97-H and MHCC97-L cell lines by flow cytometry consisting of8FITC-lectinswith distinctive binding specificities.(3) We silenced the expression levels of glycogenes MGAT3and MGAT5byusing RNA interference approach, which were over-expressed in MHCC97-L andMHCC97-H cells. To examine whether the targeted down-regulation of MGAT3inMHCC97-L cells and MGAT5in MHCC97-H cells affected the invasive ability, weperformed an in vitro ECMatrix gel analysis. Drug sensitivity was measured using anMTT assay. The effect of altered expression of glycogenes to humanhepatocarcinoma invasion and drug resistance was also observed.(4) Further analysis of the N-glycan regulation by way of tunicamycinapplication or PNGase F treatment, CD147and integrin β1were analyzed byway of western blot, Coimmunoprecipitation was carried out to detect whetherglycosylation interfered the interaction of CD147with the integrin β1. To investigatewhether N-glycans are related to drug sensitivity of tumor cell, the antitumor activity of 5-fluorouracil was examined in vivo and in vitro.Results:(1) Using real-time PCR for quantification of glycogene, we found that10genesout of62were differentially expressed between MHCC97-H and MHCC97-L cells.High expressions of glycogene are corresponding with high fluorescenceintensity of lectins in both cell lines.(2) Western blot analysis revealed a significant decrease in MGAT3and MGAT5expression at protein levels after transfection. Knockdown of MGAT3expressionpromoted MHCC97-L cells invasion and increased resistance to5-fluorouracil in vitro.The silencing of MGAT5in MHCC97-H cells inhibited invasion and increasedsensitivity to5-fluorouracil in vitro.(3) Western blot analysis revealed a significant alteration in CD147and integrinβ1expression at protein levels after the N-glycan regulation in MHCC97-H orMHCC97-L cells. Glycoprotein levels of CD147and integrin β1in MHCC97-H cellswere much higher than those in MHCC97-L cells. Analysis of the N-glycan regulationby tunicamycin application or PNGase F treatment in MHCC97-H cells showed showedpartial inhibition of Nglycan glycosylation, decreased invasion and increased sensitivityto5-fluorouracil both in vitro and in vivo.Conclusion:(1) The expression of glycogens and glycan profiling were different inMHCC97-H and MHCC97-L human hepatocarcinoma cell lines, which have high, lowmetastatic potential, respectively. These characteristic alterations are associatedwith invasion and drug resistance in human hepatocarcinoma.(2) The altered expression of glycogenes and modification ofN-glycosylation in human hepatocarcinoma correlate with invasion and drugresistance, and have significant implications for the development oftreatment strategies.