Effect Of Rorα Mirna In Proliferation Inhibition Of Diallyl Disulfide-induced Human Glioma Cell Line U251

Objective: Glioma is the most commont type of primary intracranial tumors. It has been reported that malignant glioma accounted for about 60% of gliomas. Although the currently available multi-modalitics of therapies, such as microsurgical operation, radiotherapy and chemotherapy, have been improved, the median survival time of patients with malignant gliomas only 12 months. Therefore, it is an urgently solved topic to raise the 5-year survival rate, explore new method to treat gastric cancer, and look for new effective anticancer drugs with low toxicity and inexpensive price. Diallyl Disulfide inhibits various kinds of cancer cell lines, and it becomes a very potential candicate anticancer drug. It was found that the relationship between the differential proteomic expression of RORαprotein in diallyl disulfide-induced human gastric cancer cells in our previous study. So RORαprotein can also participate in diallyl disulfide-induced human malignant glioma cells. We hope to supply a theoretical evidence for searching a new target of malignant glioma.Methods:1. MTT assay, cell average doubling time analysis and inverted microscope analysis were employed to confirm the DADS-induced cell growth inhibition in cell line U251.2. Immunocytochemistry and Western Blot were used to observed expression of RORαprotein in U251 cells by 35 mg·L-1 DADS.3. The pcDNATM6.2-GW/EmGFPmiR-RORαplasmid which contain the specific miRNA targeting RORαand the negative plasmid PcDNATM6.2-GW/EmGFP miR- negative were transfected respectively into human brain glioblastoma U251 cells by lipofectin medium. Expression of RORαprotein was anaylized by Western Blot.4. The impact on proliferation of DADS-induced U251 cells in vitro after inhibition of RORαwith miRNA was investigated by MTT methods and cell average doubling time analysis. Results:1.Apparently growth inhibition in U251 cell line treated with DADS could be seen and exhibited a dose-dependent model. Exposure of U251 cells to 15 mg·L-1 DADS for 96 h only yielded a ratio of 32.88%, whereas exposure to 30 mg·L-1 DADS increased the ratio up to 49.5%.2. Average doubling time retarded from 21.86 hours in normal cultured U251cells to DADS experimented U251 cells 36.69 hours (p<0.05).3. As exposed to inversion microscope, when 30 mg·L-1 DADS was added to culture medium for 24h, cell seeding was found to severely reduce the number of adherent cells, and cells tended to contract,floating and round. Cells'shape and size were with one accord. Their heteromorphism diminished and interstitial space accreted.4.Immunocytochemical stain indicated that positive expression of RORαincreased compared with the control, after U251 cells treated with 30 mg·L-1 DADS for 24 h.5.Western Blot showed that the RORαexpression was increased after U251 cells treated with 30mg·L-1 DADS for 24h and 48h, the gray scale value ratio of RORα/β-actin were 0.69±0.13 and 1.37±0.25 respectively, compare with 0.22±0.16 as a control (P <0.05). RORαexpression in cells treated by DADS for 48h was higher than that for 24h (P <0.05).6. The U251 cells transfected with pcDNATM6.2-GW/EmGFPmiR-RORαplasmid resulted in dramatic down-regulation of RORαprotein as demonstrated by western-blot analysis.7. The OD570 value of miR-RORα24h transfection group(0.77±0.1333)is higher than untransfected group(0.38±0.515)and miR-negtive group(0.39±0.0465)showed by MTT method (P<0.05), and after treated by DADS, OD570 value of miR-RORα24h transfection group(0.58±0.0864)was far lower than two other groups(0.21±0.0631)and(0.2±0.0702)(P <0.05) . The miR-RORα48h transfection group was effected by DADS the same as the miR-RORα24h transfection group.8. Average doubling time all delayed in every group by DADS. The miR-RORα transfection group retarded from 15.50 hours in normal cultured U251 cells to DADS experimented U251 cells 17.27 hours ; which was weaker than U251 group and lipid body transfected group by DADS(P<0.05).Conclusion:1. Apparently growth inhibition in glioblastoma cell line U251 treated with DADS could be seen.2.DADS can up-regulate RORαprotein in glioblastoma cell line U251.3. RORαexpression was obviously suppressed after pcDNATM6.2-GW/Em GFPmiR-RORα-miRNA transfected in U251 cells.4. Down regulation of RORαexpression by miRNA interference enhanced proliferation of U251 cells, and weaked DADS-induced growth inhibition in glioblastoma cell line U251, which suggested that RORαis involved in DADS-induced growth inhibition in U251 cells.