Upregluted RORα Inhibit Proliferation, Invasion And Migration Of Human Gastric Cancer MGC803Cells Induced By Diallyl Disulfide

Objective:Transgene expression and RNA interference were used to investigate the roleRORα in inhibition of proliferation, migration, and invasion of human MGC803gastric cancer cells induced by diallyl disulfide. The effect of overexpression ofRORα induced by DADS on the protein expression of Wnt/β-catenin pathway wasobserved to reveal the molecular mechanism of the inhibiting effect on proliferation,migration, and invasion of MGC803cells by DADS.Methods:The eukaryotic expression vector of human RORA gene (code the RORα protein)were constructed. The pcDNA3.1-RORα expression vector was transfected intoMGC803cells, and then screened stably transfected RORα/MGC803cells, whichoverexpressed RORα protein. The expressions of RORα mRNA and protein weredetected by Real-time PCR or Western blotting assay. The effect of RORαoverexpression on cellular proliferation, cell cycle, migration and invasion with orwithout DADS treatment in MGC803cells were detected by MTT, soft agar colonyformation, flow cytometry, wound healing and Transwell invasion chamber assays.The miRii-RORα/MGC803cells, which stably downexpressed RORα, wereestablished using RNA interference technology. The effect of RORα downexpressionon cellular proliferation, cell cycle, migration and invasion with or without DADStreatment in MGC803cells were detected by MTT, soft agar colony formation, flowcytometry, wound healing and Transwell invasion chamber assays. The mRNA andprotein expressions of Wnt/β-catenin pathway molecules β-catenin, Vimentin, Axin,c-Jun, MMP-9regulated by RORα with or without DADS treatment were tested byreal-time RT-PCR or Western blotting assay. Luciferease assay was used to assess the promoter activity of c-Myc with or without DADS treatment.Results:1. The successful construction of RORα/MGC803cells with stably overexpressedRORαRestriction endonuclease digestion and sequencing showed that the eukaryoticexpression vectors of human RORα (pcDNA3.1-RORα vector) were successfullyconstructed. The pcDNA3.1-RORα vector was transfected into MGC803cells. Realtime-PCR and Western blotting assays showed that the expressions of RORα mRNAand protein were stably increased in the RORα/MGC803cells, and suggested thatRORα/MGC803cells with stably overexpressed RORα was successfully constructed.2. Effect of RORα overexpression on the proliferation, migration and invasion inMGC803cells.MTT, soft agar colony formation and flow cytometry assays showed thatoverexpressed RORα inhibited the proliferation capability of RORα/MGC803cellsand induced G2-M arrest. DADS inhibited cellular proliferation in MGC803cells andpcDNA3.1/MGC803cells and induced G2-M arrest. However, the difference ofinhibition effect in RORα/MGC803cells was not significant with or without DADSstimulation.Wound healing and Transwell invasion chamber assays showed that themigration and invasion of RORα/MGC803were significantly decreased comparingwith MGC803cells and pcDNA3.1/MGC803. DADS inhibited the migration andinvasion in MGC803cells and pcDNA3.1/MGC803cells. However, the difference ofinhibition effect in RORα/MGC803cells was not significant with or without DADSstimulation.3. Effect of interference RORα expression on the proliferation, migration and invasioncapacity of MGC803cells.The miRii-RORα vector was transfected into MGC803cells. RT-PCR andWestern blotting assays showed that the expressions of RORα mRNA and proteinwere stably decreased in the miRii-RORα/MGC803cells, and suggested that miRii-RORα/MGC803cells with stably downexpressed RORα was successfullyconstructed.MTT, soft agar colony formation, flow cytometry, wound healing and Transwellinvasion chamber assays showed that the proliferation, migration and invasioncapacity of miRii-RORα/MGC803were significantly strengthened, suggesting thatthe down-regulated expression of RORα promoted the cellar proliferation, migrationand invasion in MGC803cells. DADS inhibited the migration and invasion inMGC803cells and miRii/MGC803cells. However, the inhibition effect of DADS oncellular proliferation, cell migration and invasion in miRii-RORα/MGC803cells wassignificantly weaker than which in MGC803and miRii/MGC803cells. These resultsindicated that the downexpression of RORα weakened the inhibition of proliferation,migration and invasion induced by DADS in MGC803cells, and suggested thatRORα may be involved in the inhibition of proliferation, migration and invasioninduced by DADS.4. Effect of DADS and regulated expression of RORα on Wnt/β-catenin pathwayRT-PCR and Western blotting showed that up-regulated RORα decreased themRNA and protein expression of several downstream molecules of Wnt/β-cateninpathway, such as Vimentin, Axin, c-Myc, c-Jun, and MMP-9, increased the mRNAand protein expression of TIMP3, and inhibit the promoter activity of c-Myc. Theseresults suggest that DADS may interfere with Wnt/β-catenin pathway throughup-regulated RORα protein expression, thereby inhibiting the cellular proliferation,migration and invasion in human gastric cancer.Conclusion:1. Overexpression of RORα may inhibit the proliferation, migration and invasion inMGC803cells, the inhibition effects of which is similar to DADS.2. Down-regulated expression of RORα can promote the proliferation, migration andinvasion in MGC803cells.3. The inhibition effects of DADS on the proliferation, migration and invasion inMGC803cells were weakened by the decreased RORα expression. 4. DADS could regulated the Wnt/β-catenin pathway through RORα, and then inhibitthe proliferation, migration and invasion in MGC803cells.