Effect Of MiR-429 On Proliferation And Migration Of Breast Cancer Cells And Preliminary Identification Of Its Target Genes

Objective MicroRNA(miRNA or miR)-429 is lowly expressed in some tumors and plays the role as tumor suppressor genes,but it is overexpressed in other tumors and acts as oncogenes.There were rarely studies on the expression of miR-429 in breast cancer.Studies have shown that the expression of miR-429 in breast cancer tissues is significantly lower than that in adjacent tissues.Qin et al.used Realtime PCR probe to detect the expression of miR-429 in human breast cancer cell lines.The results showed that miR-429 was expressed differently in human breast cancer cell lines.The miR-429 expression levels in BT20 and T47D cell lines were the highest,and the expression levels in BT474,SK-BR-3,MCF-7 and MDA-MB-231 cell lines were comparable,while those in Bcap37 cells were the lowest.Li et al.found that the expression of miR-429 was low in MDA-MB-231 and HCC1937 triple-negative breast cancer(TNBC)cells.There were some disputes about the target genes and biological functions of miR-429 in breast cancer.Qin et al.reported that miR-429 significantly promoted the proliferation and migration of Bcap37 cells.Li et al.found that mir-429-5p significantly inhibited the proliferation,migration and invasion of triple-negative breast cancer(TNBC)cells by down-regulating the expression of LIMK1 gene and protein.Ye etal.suggested that overexpression of miR-429 could inhibit the invasion and migration of breast cancer cells by regulating ZEB1 and CRKLproteins.miRNAs exert their biological functions mainly by binding to the y untranslated region(UTR)of the target genes completely or incompletely.Therefore,it is significant to study the interaction of miRNAs and their target genes in order to explore the mechanism of miRNAs in tumorigenesis.At present,the function and mechanism of miR-429 and its target genes in breast cancer are hot topics.This experiment aimed to predict possible target genes of miR-429 by bioinformatics methods,and summarized the biological functions of target genes based on GO analysis.Further more,investigated the expression and biological function of miR-429 in human breast cancer cell lines,and identify the target genes of miR-429.Methods?Human breast cancer MDA-MB-231,MCF-7 cells and human mammary epithelial MCF-10A cells were cultured in vitro.The relative expression of miR-429 in each cell line was detected by real-time quantitative PCR(qPCR).The cell line with the lowest expression of miR-429 was screened out.?CY3-labeled negtive control mimics and TransIntroTM EL Transfection Reagent were transfected into MDA-MB-231 and MCF-7 cells respectively,and the fluorescence intensity was observed at 24h,36h 48h,60h,72h after transfection respectively.The most efficient Transfection conditions were employed for subsequent transfection experiments.?Five groups were established as follow:miR-429 mimics,mimics NC,control,inhibitors NC,miR-429 inhibitors.The effects of miR-429 on proliferation and migration of human breast cancer MDA-MB-231 cells were determined by MTT assay and scratch assay.?Inputting(has-)miR-429 as a search term,target genes of miR-429 were predicted in Targetscan,miRanda and then we screened those genes that appeared in the two databases as the candiate.The predicted miR-429 target genes were annotated and enriched by C ytoscape 3.6.0 software.Thoes target genes involved in regulating bio logical behaviors such as proliferation,apoptosis,migration and invasion were picked out.Target genes related to breast cancer were screened by GCBI polygenic radar.The target genes that were verified to be regulated by miR-429 were excluded by miRTarBase,miRNACancerMap database and literature analysis.?qPCR and Western blot were adopted to detect the effect of miR-429 on the target genes at transcription and translation levels,and the target genes of miR-429 were initially confirmed.Results miR-429 was underexpressed in human breast cancer MDA-MB-231 and MCF-7 cell lines.After up-regulating the expression of miR-429,the proliferation of MDA-MB-231 cells was significantly inhibited(F=33.1060,P<0.05),and the migration ability was significantly attenuated(F=57.5900,P<0.05).As we supplemented the expression of miR-429 in MDA-MB-231 cells,the expression of the candidates such as LRP1(F=27.8400,P=0.0009),MYCN(F=25.9000,P=0.0002),PLK2(F=21.8900,P=0.0018)were significantly increased,while the expression of TIMP2,CREB1,DUSP1,MED13,GABPA,GDI2,UBE2B,EPS8,and TUBB(P>0.05)were not different.In addition,after elevated the expression of miR-429,there was no significant difference in the expression levels of CREB1(F=2.5570,P>0.05)and DUSP1(F=0.8425,P>0.05)protein but the relative expression of TIMP2 protein was decreased(F=33.7400,P<0.05)as well as the relative expression of LRP1 protein(F=6.2590,P<0.05).Conclusions Overexpression of miR-429 inhibits cell proliferation and migration in MDA-MB-231 cells.O verexpression of miR-429 may also inhibit the expression of TIMP2 and LRP1 proteins in MDA-MB-231 cells by post-transcrip tional translation.Thus,TIMP2 and LRP1 are preliminarily identified as the target genes of miR-429 while CREB1 or DUSP1 are not.