The Effect And Mechanism Of Mir-497 And Related Signaling Pathway On The Biological Function Of Triple Negative Breast Cancer

BACKGROUNDTriple-negative breast cancer(TNBC)is an aggressive tumor and difficult to treat.Therefore,it is essential to discover new therapeutic targets for TNBC treatment.The expression of miR-497 decreased in tissues and cells of various malignant tumors including breast cancer.However,the role and mechanism of miR-497 in TNBC are still unclear.We determined the target gene of miR-497 according to the prediction of biological information,and the3'-UTR of the YAP1 m RNA contains a miR-497 complementary binding site,suggested that miRNA-497 may play an important role in the regulation of YAP1 in TNBC cells.YAP1,a major downstream transducer of the Hippo pathway,is mostly identified as an oncogene.A lot of evidence have shown that dysregulation of YAP1leads to tumorigenesis,including TNBC.However,further understanding of the post-transcriptional control of YAP1 in TNBC remains elusive.OBJECTIVEThis study aimed to explore the potential function and mechanism of miR-497in TNBC.METHODSIn our study,the expression of miR-497 in collected 36 cases of TNBC tissues and matched non-cancerous breast tissues were determined by q RT-PCR.Moreover,we investigated the clinicopathological and prognostic significance of miR-497 in TNBC patients,clinical data of the 36 TNBC patients was collected and analyzed together with miR-497 levels.We detected miR-497 expression in the breast cancer cell lines MDA-MB-231?MDA-MB-468?MCF-7?SKBR3,and then compared the expression of miR-497 in cancer cell lines with MCF10A cells.To investigate the role of miR-497 in TNBC,we transfected MDA-MB-231?MDA-MB-468 cells with miR-497 mimics and negative control(NC),and the cells were subjected to colony formation assay?MTT assay?transwell assay.In addition to cell proliferation,we also investigated the role of miR-497 on cell cycle progression and cell apoptosis using flow cytometry analysis.To explore the mechanism of Yap1 in the process of mir-497 acting on TNBC,q RT-PCR and Western blot were used to detect the expression of Yap1 in 36 pairs of TNBC tissues and TNBC cell lines,and miR-497 mimics NC were transfected into MDA-MB-231 and MDA-MB-468 cells,the levels of YAP1 m RNA and protein were detected 48h after transfection.To determine whether miR-497 directly targeted YAP1,luciferase reporter assay was applied at last.RESULTSThe expression of miR-497 was significantly reduced in most TNBC tissues(27/36,75%)when compared with para-tumour tissues.The expression of miR-497was notably lower in TNBC patients with advanced stage(P<0.05)and lymph node metastasis(P<0.05).Kaplan-Meier analysis was used to evaluate the correlation between miR-497 expression and the final survival time of TNBC patients.The results suggested that the TNBC patients with miR-497 low expression had a shorter survival compared to those with miR-497 high expression(P<0.05).Moreover,the expression of miR-497 was significantly decreased in two TNBC cell lines(MDA-MB-231 and MDA-MB-468)compared to MCF-10A(P<0.01).The experiments carrying out in MCF-7(Lumina A type)and SKBR3(Her-2overexpression type)cells also showed the similar trend(P<0.001).Colony formation assay showed much less colony formation in miR-497 mimics group compared with NC group(P<0.01).MTT assay also demonstrated that overexpression of miR-497 significantly inhibited cell viability and cell proliferation in MDA-MB-231 and MDA-MB-468 cell lines compared to NC(P<0.05).These results verified that miR-497 inhibited proliferation of TNBC cells in vitro.To investigate whether miR-497 overexpression affects TNBC cellular migration,transwell assay was therefore conducted in TNBC cells,the MDA-MB-231 and MDA-MB-468 cell lines showed consistent trends in migration ability between miR-497 mimics group and NC.Cells penetrating the membrane significantly decreased at 24h after overexpression of miR-497 by mimics(P<0.001).This demonstrated that overexpression of miR-497 suppresses TNBC cell migration ability in vitro.Flow cytometry analysis indicated that up-regulation of miR-497increased the apoptosis rate in MDA-MB-231 and MDA-MB-468 cells compared with their respective NC cells.In addition,cell cycle analysis showed that the percentage of cells in G₀/G₁ phase was increased in miR-497 mimics group compared NC group in MDA-MB-231 and MDA-MB-468 cells.These results indicated that up-regulation of miR-497 could impact cell cycle distribution and induce apoptosis in TNBC cells.YAP1 mRNA and protein levels were obviously increased in TNBC tissues than that in normal tissues(P<0.001).The results also indicated that YAP1 m RNA and protein levels were dramatically higher in TNBC cells(MDA-MB-231,MDA-MB-468)when compared with MCF-10A cells.Results of q RT?PCR and Western blot analysis indicated that the YAP1 m RNA and protein levels in MDA-MB-231 and MDA-MB-468 cells were remarkably inhibited after miR-497 overexpression by miR-497 mimics.In Wild YAP1 group,luciferase activities decreased after adding miR-497 mimics in 293T cells,while no prominent differences were found in Mutant YAP1 group,suggesting that miR-497 specifically binds to the 3'-UTR of YAP1 m RNA.CONCLUSIONOur findings demonstrated that miR-497 is down-regulated in TNBC tissues and cells,and is able to inhibit cell proliferation and migration via direct regulation of the expression of YAP1,indicating that miR-497 could serve as a potential therapeutic target for TNBC.