| Objective: Retinoic acid-related orphan nuclear receptorα(RORα) may be involved in the regulation of tumor. Diallyl disulfide (DADS) could induce growth inhibition ,differentiation and cell cycle G2/M arrest in human gastric cancer cells, in which upregulated RORαexpression was involved. But its specific mechanism is not clear. In this study, after the specific miRNA targeting RORαwas constructed to interfer RORαexpression, it was observed that the impact on proliferation inhibition and cell cycle arrest induced by DADS was brought about from RORαexpression silence. Therefore, molecular mechanism was explored in inhibition of gastric cancer cells induced by DADS.Methods:1. The pcDNATM6.2-GW/EmGFPmiR-RORαplasmid which contain the specificmiRNA targeting RORαand the negative plasmid PcDNATM6.2-GW/EmGFPmiR-negative were transfected respectively into human gastric cancer SGC7901, MGC803 cells by lipofectin medium. Expression of RORαprotein was anaylized by Western Blot.2. In this study, cell counting, MTT assay and flow cytometry analysis were employed to observe the DADS-induced cell proliferation and cell cycle on cultured human SGC7901, MGC803 cells in vitro after inhibition of RORαwith miRNA.Results:1. The pcDNATM6.2-GW/EmGFPmiR-RORαplasmid was cuted by single enzyme and DNA sequencing analysis shows that constructing successful,fluorescence microscopy of transfected cells was observed within the green fluorescence. The SGC7901, MGC803 cells were transfected with pcDNATM6.2-GW/EmGFPmiR-R ORαplasmid resulted in dramatic down-regulation of RORαproteinas demonstrated by western-blot analysis(P<0.05).2. Cell counting indicated cell proliferation rate of miR-RORαtransfection groups increased, and population doubling time shortened (P<0.05), which showed knockdown of RORαenhanced proliferation of SGC7901, MGC803 cells. After treated with DADS in all groups, population doubling time increased which means that cell proliferation was inhibited. However, the effect in two miR-RORαtransfection groups were weaker than other groups after treatment with DADS(P<0.05), which suggest that knockdown of RORαinterfered function of growth inhibition by DADS.3. MTT showed that, cell proliferation ratio of miR-RORαtransfection group is higher than other groups(P<0.05), which showed knockdown of RORαpromoted growth. After treated with 15mg·L-1, 30mg·L-1 DADS for 24h respectively, cell proliferation rate decreased, was weaker than other groups in vitro. Growth inhibition ratio of miR-RORαtransfection groups 27.14% and 33.48%,were lower than untransfection groups 36.15% and 45.35%. It was the same result in DADS treatment for 48h, 72h and 96h. This displayed that gene silencing RORαweakened the growth inhibition effect induced by DADS.4. Flow cytometry analysis revealed that the percentage of G0/G1 phase cells cut down and percentage of S phase and G2/M phase cells raised up after transfected by miR-RORα. Meanwhile, the proliferation index(PI) was increased significantly ( P<0.05). All above showed that knockdown of RORαstimulated proliferation of SGC7901, MGC803 cells. But both percentage of cell cycle and PI had no change after transfected by negative vector. In the miR-RORαtransfection groups, the percentage of G2/M phase cells were lower than that of untransfection groups after treated with DADS, indicating that knockdown of RORαabated G2/M phase arrest by DADS .Conclusion: 1. RORαexpression was obviously suppressed after transfected by pcDNATM6.2-GW/Em GFPmiR-RORα-miRNA in SGC7901, MGC803 cells.2. Knockdown of RORαby miRNA interference enhanced proliferation of SGC7901, MGC803 cells, moreover, weakened DADS-induced growth inhibition and G2/M phase arrest in gastric cancer cell line SGC7901, MGC803, which suggested that up-regulation of RORαexpression is involved in molecular mechanism of DADS-induced growth inhibition in SGC7901, MGC803 cells.
|
PDF Full Text Request