The Preliminary Study About The Prohibitin Protein And Paclitaxel Resistance In Ovarian Cancer

ObjectiveTo silence the expression of Prohibitin (PHB) gene, which is up-regulated in paclitaxel-resistant ovarian cancer cell lines(SKOV3/Taxol-25), by RNA interference (RNAi) to investigate the effect of PHB on proliferation and apoptosis of paclitaxel-resistant ovarian cancer cell line by MTT (methyl thiazolyl tetrazolium) and Flow Cytometry assay to comfirm the PHB act as a candidate multi-drug resistant related gene and provide the theoretical and experimental basis for the treatment of reverse paclitaxel-resistance in ovarian cancer.Methods1. In this research, Western blotting and Real Time-PCR were used to further confirm the expression of PHB in protein and mRNA expression level, which was identified in our group’s former results as an up-regulated differential gene related to paclitaxel-resistance by comparative proteomic profiling of SKOV3/Taxol-25and SKOV3cell lines.2. Three specific small hairpin interfering RNAs(shRNA) with PHB-complementary sequences were designed and inserted into the same plasmid vectors; Three screening shRNA plasmids with green fluorescin sequence were constructed and named as pshRNA/PHB136, pshRNA/PHB248, pshRNA/PHB427. Three specific small interfering RNAs (shRNA) were respectively transiently transfected into SKOV3/Taxol-25cell lines by use of standard approach of lipofectamin2000(Invitrogen).3. Transfection efficacy was determined by fluorescence transfection visualized with fluorescence microscopy; To further comform the efficacy of interference of shRNA, total RNA and Protein were extracted48hours after transfection and subjected to Real Time-PCR assay and Western blot, respectively. High efficacy of interference fragment in three shRNA plasmid as well as the scrambled shRNA plasmid were selected.4. Cell proliferation of PHB transiently silencing SKOV3/Taxol-25cell line and the control were determined by MTT assay; Apoptosis index of PHB transiently silencing SKOV3/Taxol-25cell line and the control were determined by flow cytometry.Results1. Western blot and Real Time-PCR results revealed that the PHB in protein and mRNA expression levels were significantly higher in SKOV3/Taxol-25cell line than SKOV3cell line.(P<0.05)2. The efficacy of transient transfection of targeted pshRNA/PHB plasmid into SKOV3/Taxol-25cell were confirmed by green fluorescence protein expression observed in fluorescence microscopy; The highlightest green fluorescence protein expression was about48hours after transfection. These suggest the transfection was successful. The specific three shRNA fragments targeting PHB displayed significant suppression of PHB in the protein level of expression by Western blot in transfected cells. The silencing efficency of shRNA/PHB136and shRNA/PHB427were better than PHB248, especially after the transfection for48h, were confirmed by Western blot. In protein level of expression PHB in shRNA/PHB136and shRNA/PHB427transfected cell lines (0.652±0.224)%.(0.598±0.026)%were significantly higher than the negative control (0.84±0.039)%in quantitative grayscale scanning analysis(P<0.05). There were no significantly difference in three experimental group(P>0.05). To select the fragments, it was efficenciest suppression, for later in the experiment. The Real Time-PCR result showed that, after the recombinant plasmid shRNA/PHB136and shRNA/PHB427expressing PHB-targeted shRNA was transfected for48h, the PHB mRNA of the recombinant plasmid shRNA/PHB136and shRNA/PHB427was obviously reduced than the NC group. There was significantly difference among PHB1-shRNA、PHB3-shRNA and the NC group by2-△△Ct method. There was no significantly difference between shRNA/PHB136and shRNA/PHB427. 3. PHB influenced cell proliferation and apoptosis:proliferation of shRNA/PHB136and shRNA/PHB427scilencing cells were inhibited compared to the NC shRNA transfected cells by MTT assay after0,24,48,72and96hours. The proliferation of shRNA/PHB136and shRNA/PHB427cell significantly inhibited comparing with the control (P<0.05); Apoptosis rate of PHB-shRNA knockdowned SKOV3/Taxol-25cells was significant higher than the control by the detection of flow cytometry (p<0.05). There was no significantly difference between PHB1-shRNA and PHB3-shRNA in apoptosis rate.ConclusionsTransient PHB shRNA plasmid transfection can effectively inhibit both protein and mRNA expression levels of PHB in paclitaxel-resistant ovarian cancer cell lines; downregulating or silencing PHB may inhibit the proliferation of paclitaxel resistant cells and induce cell apoptosis. It indicates that high level expression of PHB may associate with drug resistance in ovarian cancer; Silencing of PHB may increse sensitivity to paclitaxel resistant ovarian cancer cell.