Study On Metastasis And Invasion Activity And Mechanism Of MiR-199a/b-3p Suppression On Breast Cancer Cells

MicroRNA (miRNA) are a recently discovered class of about 20-25 nucleotides in length of a small molecule RNA having post-transcriptional regulation of noncoding single-stranded, widely found in animals, plants, viruses, and other organisms. With the in-depth study of miRNA and miRNA discovery as a new regulatory factor that regulates cell proliferation, differentiation and apoptosis. In the human genome, miRNA genes gathered in more volatile areas and fragile sites associated with tumors, and its abnormal expression is closely related to the development of a variety of tumors. miR-199a/b-3p is highly conserved miRNA, abnormal expression in a variety of tumor liver cancer, ovarian cancer, renal cancer, etc., and through regulation of cell proliferation, apoptosis, migration and invasion force of change and participate in tumorigenesis development, and its expression level may serve as a variety of tumor diagnosis, classification and prognosis of pathological signs.The present study was designed to study miR-199a/b-3p expression in breast cancer cells, the study of miR-199a/b-3p expression and regulation effect on breast cancer cell proliferation, apoptosis, invasion and migration, and validation of analytical miR-199a/b-3p potential target in breast cancer genes, with a view to miR-199a/b-3p in tumor diagnosis, practice and application of treatment and early prevention and treatment to provide experimental evidence.Objective:Study the role of miR-199a/b-3p on breast cancer cell about impact and mechanism of proliferation, apoptosis, migration and invasion.Methods:(1) liposome transfected miR-199a/b-3p mimics the breast cancer cells; miRNA expression (2) qRT-PCR method to detect cells and target gene; (3) Western blot test protein levels; (4) MTT assay changes in cell proliferation; (5) flow cytometry cell cycle; (6) cells scratches and Transwell chamber migration and invasion assay; (7) luciferase reporter assay verification miR-199a/combined b-3p and 3’UTR of target genes; (8) SPSS 17.0 statistical software for data processing, statistical analysis between the experimental group using paired T test.Results:(1) High invasiveness of breast cancer cell lines, miR-199a/b-3p expression level reduced:Relative low invasiveness of breast cancer cell line MCF-7, miR-199a/b-3p in high invasiveness of breast cancer cell line MDA-MB-231, SUM149PT, CAL-120, HCC1395 expression levels were significantly reduced.(2) The role of miR-199a/b-3p inhibit breast cancer cell proliferation, migration and invasion:After breast cancer cell line MDA-MB-231 transfected with miR-199a/b-3p mimics, miR-199a/b-3p expression increasing, then cell proliferation, migration and invasion were inhibited. Regulated miR-199a/b-3p expression level, can significantly inhibit proliferation, inhibition of cell cycle progression in breast cancer cell line MDA-MB-231, can make proto-oncogenes Cyclin D1 decreased expression, p21 and p27 tumor suppressor gene expression increased.(3) PAK4 is a direct target gene of miR-199a/b-3p of:miR-199a/b-3p by mRNA 3’-UTR sequence complementary to the target gene PAK4 binding, pmir-PAK43 ’UTR luciferase activity group miR-199a/b-3p mimics incorporation group than in NC group was significantly lower (*P<0.05); with miR-199a b-3p expression increasing, mRNA expression and protein expression were inhibited PAK4, PAK4 and p-MEK pathway and p-ERK protein expression was down-regulated.(4) PAK4 gene silencing may inhibit breast cancer cell proliferation, migration and invasion:in MDA-MB-231 cells were transfected with PAK4 siRNA 48h later, PAK4 protein expression decreased, p-MEK and p-ERK protein expression were reduced, MDA-MB-231 cell proliferation levels decreased after 48h and 72h (**P <0.01), cell cycle inhibition, G1 arrest extension allows the expression of proto-oncogenes Cyclin D1 decreased, expression of tumor suppressor genes p21 and p27 increased, the number of migration and invasion cells significantly reduced. Meanwhile, the transfected gene overexpression PAK4 plasmid pcDNA3.1-PAK4 into the low invasiveness of breast cancer cell line MCF-7 in, PAK4 protein upregulation, p-MEK and p-ERK protein expression were lifting, MCF-7 cells proliferation was markedly increased (*P<0.05).(5) Overexpression of PAK4 can reply to miR-199a/b-3p suppression of breast cancer cells. Super PAK4 expression plasmid pcDNA3.1-PAK4 and miR-199a/b-3p mimics a total turn MDA-MB-231 cells, miR-199a/b-3p on breast cancer cell proliferation, migration and invasion inhibition joining after PAK4 overexpressing plasmid pcDNA3.1-PAK4 be partially recovered, and the difference was statistically significant (*P<0.05 or**P<0.01).Conclusions:(1) MiR-199a/b-3p in the high mobility of invasive breast cancer cell lines were significantly reduced;(2) Expression of miR-199a/b-3p recovery in breast cancer cells can inhibit cell cycle progression, cell proliferation, migration and invasion of the cancer cells;(3) PAK4 of mRNA 3’-UTR and miR-199a/b-3p combine complementary sequence is its direct target genes;(4) In breast cancer cells, inhibits expression of PAK4 may inhibit breast cancer cell proliferation, migration and invasion;(5) In turn miR-199a/b-3p of breast cancer cells upregulate the expression of PAK4 can make miR-199a/b-3p inhibition of breast cancer cells to be part of the reply;(6) MiR-199a/b-3p can exert inhibitory effects on breast cancer cells through regulation of target genes and signaling pathways PAK4.