| Breast caner (BC) is a woman common cancer, recently, the therapy methods of BC have been constantly developed, the comprehensive treatment which include surgery, chemotherapy, radiotherapy, endocrine therapy, biologically targeted therapy and Chinese medicine treatment has become the mainstream model of BC therapy, the curative effect has improved continually, so BC has become one of the solid tumors with good outcome. Whereas, global incidence of BC has increased year by year, the morbidity of BC has become the number one of women malignancy in China, and has been trending younger lately, so BC has become the first killer of women. BC is a multi-phase developmental process that includes changes in multi-gene signatures, analysis of BC genome and proteome has showed that focusing on molecular heterogeneity within BC maybe available method to identify and develop novel therapeutics.MicroRNAs (miRNAs) are a group of naturally occurring small non-coding RNA molecules18-25nucleotides long. These miRNAs can regulate gene expression post-transcriptionally through binding to the3’-untranslated region (3’-UTR) of target genes to promote mRNA degradation or inhibition of protein translation. Thus, they play an important role in various biological functions, such as embryo development, cell proliferation and differentiation, and carcinogenesis. A great number of studies have demonstrated that miRNAs function as onco-or tumor suppressor genes and that their aberrant expression contributes to human diseases such as cancer. To date, extensive studies have reported aberrant expression of miRNA in breast cancer. Further investigation of miRNA involvement in breast cancer could help us better understand the molecular mechanisms responsible for breast cancer development and lead to novel strategies for effective control of breast cancer.The tumor suppressor gene miR-99a is frequently lost or expressed at reduced levels in various human cancers, involved in process of cell proliferation, differentiation and apoptosis. However, to date, there has been no study reporting the role of miR-99a in human breast cancer. Thus, this study aims to investigate in detail biological functions and mechanisms of miR-99a in breast cancer cells. Our findings could provide a potential therapeutic strategy for breast cancer.Part I The tissues and cells expression and significance of miR-99a in breast cancerObjective:To explore the expression pattern of miR-99a in BC tumor tissues and BC cell lines. Methods:The expression of miR-99a in paired BC tumor tissues and adjacent non-tumor breast tissues, normal human breast cells(HBL-100) and four BC cell lines (MCF-7、MDA-MB-231、MDA-MB-435s、 SKBr-3) were examined respectively by real-time PCR.Results:miR-99a was significantly down-regulated in BC tissues, as compared with that detected in matched non-tumor tissues, and miR-99a was distinctly down-regulated in BC cell lines (MCF-7、MDA-MB-231、 MDA-MB-435s、SKBr-3), as compared with human breast epithelial HBL-100cells.Conclusion:miR-99a expression has been shown to be significantly down-regulated in both human BC tissues and cells compared to matched non-tumor tissues and normal breast cells.Part II Effects of miR-99a on biological characteristics of human breast cancer cell linesObjective:Research the effects of miR-99a on cell proliferation and apoptosis in MCF-7and MDA-MB-231cells.Methods:l.The experiments were consists of three groups each cells: MCF-7-MOCK cell group、MCF-7-NC cell group、MCF-7-99a cell group; MDA-MB-231-MOCK cell group、MDA-MB-231-NC cell group、 MDA-MB-231-99a cell group.2.MTT assay was employed to measure cell proliferation of each cell group.3.Flow cytometry was employed to measure cell cycle of each cell group.4.The measurement of flow cytometry for Annexin/PI was used to assess the induction of apoptosis in each group.5.MDA-MB-231cells were transfected with miR-99a eukaryotic expression plasmid CMV-miR-99a and control plasmid CMV-NC, stable transfectants were then selected by incubation with blasticidin and maintained in medium containing10ug/mL blasticidin, MDA-MB-231cells with stable expression of miR-99a or control were obtained, designated MDA-MB-231-99a and MDA-MB-231-NC, respectively.6.The expression of miR-99a in MDA-MB-231-MOCK, MDA-MB-231-NC and MDA-MB-231-99a were detected by RT-PCR.7.18nude mice were randomized into3groups and each group contained6:MDA-MB-231-MOCK cell group, MDA-MB-231-NC cell group、 MDA-MB-231-99a cell group, to establish the animal model of human breast cancer-bearing mice.8. After tumorigenesis, the volumes of planted subcutaneous tumors were measured per5days for drawing tumors growth curves. Nude mice were sacrificed to obtain tumor30days after injection, then compared the tumor weight in each group.Results:1.MTT assay shown that significantly reduced cell proliferation were observed in MCF-7-99a cells compared to MCF-7-MOCK cell group and MCF-7-NC cell group (P<0.05). There was no distinct different between MCF-7-MOCK cell group and MCF-7-NC cell group (P>0.05), as well as MDA-MB-231cells.2.Flow cytometry assay shown comparing to the MCF-7-MOCK (3.68±1.26%)、MCF-7-NC (4.61±1.77%)group, the sub-G1cell proportion in MCF-7-99a (19.37±2.92%) group was distinctly increased (P<0.05), there was no significant difference between MCF-7-MOCK and MCF-7-NC group(P>0.05). Compared to MDA-MB-231-MOCK (4.45±1.25%). MDA-MB-231-NC(5.10±0.99%), the sub-G1cell proportion in MDA-MB-231-99a (21.72±2.15%) group was distinctly increased as well(P<0.05), and there was no significant difference between MDA-MB-231-MOCK and MDA-MB-231-NC(P>0.05).3.Flow cytometry for Annexin/PI assay:the result showed that as comparing to the MCF-7-MOCK(3.82±0.84%), MCF-7-NC (4.05±0.94%) cell group, the percentage of apoptosis cells in MCF-7-99a(19.08±1.05%) was distinctly increased(P<0.05), and there was no markedly difference between MCF-7-MOCK and MCF-7-NC cell group(P>0.05). Comparing to the MDA-MB-231-MOCK(4.47±0.94%), MDA-MB-231-NC (4.96±0.87%) cell group, the percentage of apoptosis cells in MDA-MB-231-99a (21.82±1.60%) was distinctly increased as well(P<0.05), and there was no markedly difference between MDA-MB-231-MOCK and MDA-MB-231-NC cell group(P>0.05).4.The model mice of human breast cancer were successfully established, the tumor-formation rate was100%.5.The growth curves of planted tumor were mapped, as compared to MDA-MB-231-MOCK and MDA-MB-231-NC group, the tumor growth rate of MDA-MB-231-99a group was much slower (P<0.05). At the end point time, the average tumor volume of MDA-MB-231-99a group(544.248±49.12) was lower than MDA-MB-231-MOCK(1005.33±89.21) and MDA-MB-231-NC group(912.16±96.36)(P<0.05); the average tumor weight of MDA-MB-231-99a group(1.20±0.21)g was lower than MDA-MB-231-MOCK (1.04±0.25)g and MDA-MB-231-NC group (0.53±0.09)g (P<0.05).Conclusion:1.In breast caner MCF-7and MDA-MB-231cells, ectopic expression of miR-99a potently inhibits tumor cell proliferation, arrest cell cycle in sub-G1phase, and promotes apoptosis in vitro.2.1n human breast cancer-bearing nude mice, ectopic expression of miR-99a can effectively inhibit the growth of tumor in vivo.PartⅢ Prediction and confirmation of the downstream target gene of miR-99a and research regulating effect of target gene mTORObjective:Utilize bioinformatics to predict the probable target gene of miR-99a then to confirm it, and research regulating effect of the target gene.Methods:1.Predict the probable target gene of miR-99a according to a prediction by web-based microRNA targets prediction program.2.Constructed the mimics of sense miR-99a and Dual-Luciferase vector comprising the mTOR3’-UTRwt and mTOR3’-UTRmuto Transcription activity of miR-99a to the promoter series of target gene was analyzed by luciferase reporter gene expression analysis.3.Detect the relative levels of mTOR mRNA and protein after transfecting miR-99a mimics by RT-PCR and Western blot analysis to confirm the regulation mechanism of miR-99a on target gene.4.Transfected target gene cDNA without3’-UTR into MCF-7cells, then overexpressed miR-99a to detect the mRNA and protein of target gene by RT-PCR and Western blot analysis.Result:1.Bioinformatics analysis:predicted mTOR is one of the target genes of miR-99a by three online database TargetScan、miRBase、 Pictaralgorithms, mTOR was found to have a putative miR-99a binding sites within its3’-UTR by using Blast database.2.Dual-luciferase reporter gene assay shown miR-99a can combine with mTOR3’-UTR.3.MiR-99a down-regulated mTOR both in mRNA and protein level detected by RT-PCR and Western blot.4.MiR-99a can not down-regulated mTOR without3’-UTR both in mRNA and protein level detected by RT-PCR and Western blot. Inhibiting effect of miR-99a could be overcome.Conclusion:1.mTOR is the direct target gene of miR-99a combined with its3’-UTR region.2.MiR-99a down regulated mTOR both in mRNA and protein level.Part IV The tissues and cells expression and significance of miR-99a in breast cancerObjective:To explore the expression pattern of mTOR in BC tumor tissues and BC cell lines.Method:1.The expression of mTOR in paired BC tumor tissues and adjacent non-tumor breast tissues, normal human breast cells(HBL-100) and four BC cell lines (MCF-7、MDA-MB-231、MDA-MB-435s、 SKBr-3) were examined respectively by Western blot.2.Analyze the relationship among the expression of miR-99a and mTOR in BC tissue and cell lines.Results:1.mTOR was significantly down-regulated in BC tissues, as compared with that detected in matched non-tumor tissues, and mTOR was distinctly down-regulated in BC cell lines (MCF-7、MDA-MB-231、 MDA-MB-435s、SKBr-3), as compared with human breast epithelial HBL-100cells.2.A marked inverse correlation between mTOR expression levels and miR-99a levels in both BC tissues and cells was being observed.Conclusion:1.mTOR expression has been shown to be significantly down-regulated in both human BC tissues and cells compared to matched non-tumor tissues and normal breast cells.2.The expression of mTOR was negatively correlated with miR-99a in both BC tissues and cells.Part Ⅴ miR-99a-mediated inhibition of mTOR is involved in tumor suppression of breast cancer cellsObjective:Further confirm whether miR-99a-mediated mTOR inhibition confers antitumor activity in breast cancer cells. Methods:1.Inhibited the expression of mTOR in MCF-7by mTOR-siRNA, the experiment was consists of three groups: MCF-7-MOCK cell group、MCF-7-NC cell group. MCF-7-mTORsiRNA cell group, MTT assay was employed to measure cell proliferation of each cell group. The measurement of flow cytometry for Annexin/PI was used to assess the induction of apoptosis in each group.2.Transfected mTOR cDNA without3’-UTR into MDA-MB-231cells to up-regulated mTOR, the experiment were consists of three groups: MDA-MB-231-MOCK cell group、MDA-MB-231-NC cell group、 MDA-MB-231-mTOR cell group. MTT assay was employed to measure cell proliferation of each cell group. The measurement of flow cytometry for Annexin/PI was used to assess the induction of apoptosis in each group.3.Co-transfected mTOR cDNA without3’-UTR and miR-99a mimics into MCF-7cells to demonstrate miR-99a act as a tumor-suppressor via binding to mTOR3’-UTR. The experiment were consists of three groups:MCF-7-MOCK cell group、MCF-7-99a cell group、MCF-7-99a-mTOR cell group, MTT assay was employed to measure cell proliferation of each cell group. The measurement of flow cytometry for Annexin/PI was used to assess the induction of apoptosis in each group.Result:1.MTT assay shown that significantly reduced cell proliferation were observed in MCF-7-mTORsiRNA cells compared to MCF-7-MOCK cell group and MCF-7-NC cell group (P<0.05). Flow cytometry for Annexin/PI assay:the result showed that as comparing to the MCF-7-MOCK(3.95±0.89)%, MCF-7-NC (4.27±0.84)%cell group, the percentage of apoptosis cells in MCF-7-mTORsiRNA (16.90±1.14)%was distinctly increased (P<0.05), and there was no markedly difference between MCF-7-MOCK and MCF-7-NC cell group(P>0.05).2.MTT assay shown that significantly increased cell proliferation were observed in MDA-MB-231-mTOR cells compared to MDA-MB-231-MOCK cell group and MDA-MB-231-NC cell group (P<0.05). Flow cytometry for Annexin/PI assay:the result showed that as comparing to the MDA-MB-231-MOCK (4.25±0.07)%, MDA-MB-231-NC(4.43±0.15)%cell group, the percentage of apoptosis cells in MDA-MB-231-mTOR (2.19±0.22)%was distinctly decreased as well (P<0.05), and there was no markedly difference between MDA-MB-231and MDA-MB-231-NC cell group (P>0.05).3.MTT assay shown that significantly decreased cell proliferation was observed in MCF-99a-MOCK group compared to MCF-7cell group, but cell proliferation of MCF-7-99a-mTOR cell group was increased compared to MCF-99a group(P<0.05). Flow cytometry for Annexin/PI assay:the result showed that as comparing to the MCF-7-MOCK(4.15±1.09)%, the percentage of apoptosis cells in MCF-7-99a (15.85±1.83)%was distinctly increased (P<0.05), but the percentage of apoptosis cells in MCF-7-99a-mTOR group(8.63±1.19)%was marked decreased compared to MCF-7-99a(P<0.05).Conclusion:1.In breast caner MCF-7cells, inhibited expression of mTOR potently inhibits tumor cell proliferation and promotes apoptosis similar to the function of miR-99a in BC cells.2.Over-expression of mTOR promotes tumor cell proliferation and inhibits apoptosis in BC MDA-MB-231cells opposite to the function of miR-99a in BC cells.3.Co-transfection of miR-99a and mTOR without3’-UTR promotes tumor cell proliferation and inhibits apoptosis in BC MCF-7cells, rescued the suppress tumor function of miR-99a in BC cells.4.MiR-99a inhibits mTOR expression in BC via binding to mTOR3’-UTR to execute suppress tumor function.Part Ⅵ Effect of miR-99a to expression of mTOR downstream signal moleculeObjective:Explore the effect of miR-99a to expression of mTOR downstream signal molecule.Method:1.Over-expressed miR-99a in MCF-7cells to detect the expression level of mTOR downstream signal molecule4E-BP1, S6K1and phosphorylated4E-BP1, S6K1protein by Western blot assay.2.Inhibited expression of mTOR in MCF-7cells to detect the expression level of4E-BP1, S6K1and phosphorylated4E-BP1, S6K1protein by Western blot assay.3.Co-transfected mTOR cDNA without3’-UTR and miR-99a mimics into MCF-7cells to detect the expression level of4E-BP1, S6K1and phosphorylated4E-BP1, S6K1protein by Western blot assay.Result:1.Levels of p-4E-BP1and p-S6K1proteins were both markedly decreased after miR-99a mimics transfection in MCF-7cells (P<0.05). Total4E-BP1and S6K1protein showed no change (P>0.05).2.Knockdown of mTOR expression using mTOR siRNA also distinctly reduced levels of p-4E-BP1and p-S6K1proteins (P<0.05), however, total4E-BP1and S6K1protein showed no change (P>0.05).3.Re-expression of mTOR distinctly increased levels of p-4E-BP1and P-S6K1proteins (P＜0.05). Total4E-BP1and S6K1protein showed no change (P>0.05).Conclusion:1.MiR-99a inhibited expression of mTOR and inhibited phosphorylation process of4E-BP1and S6K1protein meanwhile to decreased expression of p-4E-BP1and p-S6K1proteins in BC cells.2.Silence of mTOR induced decreased expression of p-4E-BP1and p-S6K1proteins in BC cells as the same function of miR-99a.3.The inhibitory effects of miR-99a mimics on p-4E-BP1and p-S6K1could be negated by re-expression of mTOR.4.MiR-99a inhibits mTOR expression in BC via binding to mTOR3’-UTR to influence phosphorylation process of4E-BP1and S6K1protein and then suppress tumor proliferation, promote cell apoptosis ultimately. |
PDF Full Text Request