Analysis Of The Expression And Biological Functions Of MicroRNA-761 In Breast Cancer

Part 1 Expression And Biological Functions of MicroRNA-761 in Breast CancerPurpose MicroRNAs(miRNAs)constitute a group of endogenous,small,non-protein coding RNAs that are implicated in various physiological and pathological processes.This part was designed to examine the expression and functions of miR-761 in breast cancer.Methods56 pairs of primary breast cancers and adjacent noncancerous breast tissues from19 TNBC and 37 non-TNBC patients were included.Total RNA was extracted from tissues and cells and quantitative real-time PCR(q RT-PCR)was performed to detect miR-761 expression.A fragment containing the miR-761 precursor and its flanking sequences was obtained by PCR from human genomic DNA and cloned into a p CMV vector.For the establishment of stable miR-761-overexpressing clones,MDA-MB-231 cells were transfected with p CMV-miR-761 or an empty vector and selected in the presence of G418.To generate stable miR-761 knockdown clones,a lentiviral expression system was employed to deliver anti-miR-761 into breast cancer cells.In brief,293 T cells were transfected with 1 ?g pmiRZIP-761 and 4 ?g packaging vector using Lipofectamine 2000.Next,lentivirus-containing supernatants were collected 72 h after transfection and used to infect MDA-MB-453 cells.Transduced MDA-MB-453 cells were selected using puromycin(2 ?g/ml)for 2weeks to establish stable cell lines.The effects of miR-761 overexpression or knockdown on cell proliferation,colony formation,migration,and invasion were assessed.Female BALB/c nude mice(4-6 weeks old)were used for xenografting.MDA-MB-231 cells stably expressing miR-761 or containing an empty vector(1×106cells per mouse)were subcutaneously injected into the flanks of mice(4 mice per group).Tumor volumes were measured every week and 4 weeks after xenografting the mice were sacrificed and the tumors were weighed.For assessing lung metastasis,miR-761-overexpressing or control MDA-MB-231 cells(4×106)were injected intravenously into the tail vein.At 4 weeks after cell inoculation,the mice were sacrificed and the lungs were removed and subjected to pathological analysis.Results miR-761 was up-regulated in primary breast cancer tissues compared to its corresponding normal breast tissues(P=0.0098).Specifically,we found that miR-761 expression was significantly up-regulated in TNBC tissues compared to non-TNBC tissues(P=0.0136).Compared to non-malignant MCF10 A mammary epithelial cells,a significant(P<0.05)increase in miR-761 expression was observed in the breast cancer-derived cells,particularly in the TNBC-derived MDA-MB-453 and MDA-MB-468 cells.These cells showed significantly(P<0.05)higher miR-761 expression levels than the non-TNBC cells MCF-7 and T-47 D.Over-expression of miR-761 enhanced both the proliferation and colony formation capacities of MDA-MB-231 cells compared to untransfected control cells.In contrast,miR-761 expression knockdown resulted in a significant decline in the proliferation and colony formation capacities of MDA-MB-231 cells.Exogenous miR-761 expression significantly(P<0.05)increased the migration and invasion capacities of MDA-MB-453 cells,whereas miR-761 expression knockdown led to a significant inhibition of the migration and invasion capacities of these cells.Very similar results were obtained with MDA-MB-231 cells.MDA-MB-231 cells stably transfected with a miR-761 expression vector or an empty vector were injected subcutaneously into the flanks of nude mice after which tumor growth was monitored.miR-761over-expressing MDA-MB-231 xenografts yielded significantly(P<0.05)higher tumor volumes than the control xenografts.The average tumor weight in the miR-761over-expression group was found to be increased by 85% 4 weeks after inoculation compared to the control group(P=0.0241).Subsequent Western blot analysis revealed that the miR-761 over-expression group exhibited significantly lower TRIM29 levels(see below)than the control group(P=0.0163).We also injected miR-761over-expression and empty vector-transfected MDA-MB-231 cells into the tail vein of nude mice and assessed the occurrence of metastatic lesions in the lung 5 weeks after inoculation.We found that the number of lung metastatic nodules was significantly increased in the miR-761 over-expression group compared to the control empty vector group(P=0.0019).Conclusion miR-761 is upregulated in breast cancer,in particular TNBC.This miRNA can enhance breast cancer cell proliferation and invasion in vitro and tumor growth and metastasis in vivo.Part 2 MicroRNA-761 Promotes the Growth and Metastasis of Breast Cancer by Targeting TRIM29Purpose Although miR-761 shows the ability to promote breast cancer cell proliferation and invasion,the mechanisms are still unclear.This part was conducted to identify the direct target gene(s)of miR-761.Methods Bioinformatic analysis using the miRDB software tool(http://mirdb.org/miRDB/)revealed that tripartite motif-containing 29(TRIM29 may serve as a potential target of miR-761,since the 3'-UTR of its m RNA contains a putative target site for miR-761.A TRIM29 expression vector was generated by cloning the human TRIM29 open reading frame into a pc DNA3.1(+)vector.293 T cells were transfected with 0.2 ?g of the respective 3'-UTR reporter constructs,0.02?g p RL-TK and 50 n M miR-761 mimic or control miRNA.The p RL-TK vector,expressing Renilla luciferase,was used as internal control for luciferase activity.At24 h after transfection the cells were lysed and luciferase activities were measured using a dual luciferase assay kit.Firefly luciferase activity was normalized to Renilla luciferase activity.Western blotting analysis was performed to determine the effect of miR-761 on TRIM29 protein expression in breast cancer cells.Results TRIM29 may serve as a potential target of miR-761,since the 3'-UTR of its m RNA contains a putative target site for miR-761.There is a significant negative correlation between miR-761 and TRIM29 expression(r=-0.452,P=0.0126).Ectopic miR-761 significantly(P<0.05)decreased the activity of the reporter harboring the wild-type TRIM29 3'-UTR,whereas the activity of the reporter harboring the mutated TRIM29 3'-UTR was not altered by ectopic miR-761 expression.Consistent with the reporter assay,endogenous TRIM29 expression in MDA-MB-231 cells was significantly reduced after miR-761 mimic delivery,whereas transfection with anti-miR-761 significantly raised the expression of TRIM29.We performed rescue experiments with a TRIM29 variant lacking the 3'-UTR.When this plasmid was co-transfected into MDA-MB-231 cells together with miR-761 mimic,the miR-761-mediated down-regulation of TRIM29 was fully abolished.TRIM29over-expression impaired the proliferation and invasion of MDA-MB-231 cells induced by miR-761 mimic.Conclusion TRIM29 is a direct target gene of miR-761.miR-761 is negatively correlated with TRIM29 expression in breast cancer.TRIM29 down-regulation may account,at least partially,for the miR-761-induced aggressive phenotype of TNBC cells.