| Objective:To clarify the interaction between mi RNA-148b-3p and HOTAIR, seek the target sequences by which mi RNA-148b-3p and HOTAIR directively interact, and reveal that the low expression of HOTAIR mediated by mi RNA-148b-3p inhibits both proliferation and invasion of glioma.Method:Through the analysis of differential expression of lnc RNA in glioma in GEO gene expression library of Pubmed, determine whether there is differential expression of the HOTAIR in glioma, and whether the expression level and survival time are related. According to the survival time of patients, analysis whether differential levels of HOTAIR expression will influence prognosis. And then compare the expression level of HOTAIR in A172 and HA1800. Whether reducing expression of HOTAIR in vitro influence the cell proliferation rate, mobility and cell cycle is tested by MTT,FCM and Transwell test. Test the expression level of mi R-148b-3p of A172 transfected by si HOTAIR, and the expression level of HOTAIR of A172 transfected by mi R-148b-3p, analysis if the expression of both are relevant. The pmir GLO dual-luciferase report gene vectors with HOTAIR fragment containing mi R-148b-3p binding site is consructed to analysis how the overexpression of mi R-148b-3p effect of the pmir GLO-HOTAIR-WT. While binding-site mutated, the luciferase reporter gene activity is also tested. Rising the dosage of mi R-148b-3p in vitro, changes in cell proliferation, invasion and cell cycle in A172 are tested by MTT, FCM and Transwell test.Result:We analyzed the HOTAIR expression pattern through whole genome gene in NCBI’s GEO data(GSE4290) and found that, HOTAIR expression was significant higher in carcinoma tissues than in normal tissues(P<0.05), and Grade IV and III tumor tissues both demonstrated a significant increase in HOTAIR transcript levels compared with that observed in Grade II(P<0.05), although no significant difference was observed between Grade IV and III. The HOTAIR level was increased in A172 cells. Silencing of HOTAIR expression significantly reduced growth rates compared with that in cells transfected with a si RNA negative control(si-NC) and un-transfected cells. The results showed that the cell population in the G1 phase was increased but the S-phase population was decreased after HOTAIR gene silencing compared with the results seen for the si-NC cells. In vitro Matrigel invasion assay revealed that the invasiveness of A172 cells transfected with si-HOTAIR was suppressed compared with the control and si-NC(P<0.001). Real-time RT-PCR revealed that the expression level of mi R-148b-3p was markedly lower in A172 cells compared with HA1800 cells.The mi R-148b-3p mimic reduced the HOTAIR level in a dose-dependent manner, decreasing it by approximately 62% 72 h after transfection.In contrast, the mi R-148b-3p inhibitor increased the level of HOTAIR in a dose-dependent manner. Furthermore, we found that si-HOTAIR transfection significantly increased the expression of mi R-148b-3p in A172 cells. After co-transfection with the mi R-148b-3p mimic in A172 cells, we performed the Dual-Luciferase assay to determine the luciferase activity. The data shows that cells co-transfected with the constructs containing the wild-type HOTAIR sequence and the mi R-148b-3p mimic had much lower luciferase activity compared with that of those transfected with mutant HOTAIR. Transient transfection with the mi R-148b-3p mimic decreased proliferation of A172 cells by about 15% at 72 h compared with the mimic negative control group. The mi R-148b-3p mimic also increased the cell population in the G1 phase and decreased the S-phase population. Transwell invasion assay results clearly revealed that the mi R-148b-3p mimic decreased cell invasion to about 70%compared with the controls.Conclusion:our study shows that mi R-148b-3p plays a tumor-suppressive role by down-regulating HOTAIR in gliomas, and provides evidence for the import role of the interaction between lnc RNA and mi RNA in gliomagenesis. |
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