Screening,Validation And Functional Of Breast Cancer-differential MiRNA And Related LncRNA

ObjectiveBreast cancer is one of the most common cancers among women in the world.The latest data from China Cancer Center shows that the new cases of breast cancer in China are increasing year by year.The 5-year survival rate of early breast cancer patients can reach 90%,while the late 5-year survival rate is less than 40%.Breast cancer has become a major disease threatening women ’s physical and mental health.Therefore,it is important to improve the proportion of early diagnosis of breast cancer and standardize the diagnosis and treatment of breast cancer.This study collects the tissue and serum of clinically diagnosed breast cancer patients and the serum of normal physical examination personnel,studies the differential expression profiles of breast cancer miRNAs,and predicts its target lncRNA,and studies its functional mechanism through in vitro cell function experiments to provide new diagnostic and therapeutic prognosis for breast cancer potential biomarkers.Method10 cases of breast cancer patients with cancer tissues,adjacent tissues,serum and 10 normal human serum were collected.High-throughput sequencing and TargetScan,Clip-seq,TCGA and other databases were used to screen for miRNAs specifically expressed in breast cancer;The serum of 113 newly diagnosed breast cancer patients and 91 healthy control women were collected.RT-qPCR verification,screening differentially expressed miRNAs of interest,designing and synthesizing miRNA mimics and inhibitors,transfecting breast cancer cells MDA-MB-231 and T47D,CCK-8 detection of proliferation ability of breast cancer cell lines transfected with miRNA mimics and miRNA inhibitors;Transwell detection of migration ability of breast cancer cell lines transfected with miRNA mimics and miRNA inhibitors;detection of invasion ability of breast cancer cell lines transfected with miRNA mimics and miRNA inhibitors by invasion assay;using miRWalk,miRanda,RNAhybrid,Targetscan Four algorithms for target lncRNA prediction of the target miRNA,and calculate the score of each target lncRNA(that is,the total number of times the target gene can be predicted by different algorithms),and the intersection of highly reliable target lncRNAs(Score≥3 for high-reliability miRNA target lncRNA),combined with TCGA database,the most significant lncRNA was found by single factor COX and Log Rank analysis.The lncRNA was designed to synthesize the target lncRNA overexpression plasmid and the interference plasmid to construct a stably transfected breast cancer cell line T47D.The functional mechanism was studied by cell function tests such as CCK-8,Transwell and invasion assays.The positive correlation was found by lncRNA correlation analysis.The relevant regulatory genes were selected and the top 5 genes with the highest correlation were selected for experimental verification.Result1.Breast cancer differentially expressed miR-21-3p,miR-21-5p,miR-223-3p and miR-423-5p were obtained.2.Quantitative analysis of the expression levels of miR-21-3p,miR-21-5p,miR-223-3p and miR-423-5p in serum showed that the target miRNA was stably expressed in serum;the relative expression of miR-21-3p,miR-21-5p,miR-223-3p and miR-423-5p was up-regulated in breast cancer group compared with healthy control group,The expression of the previous sequencing results was consistent;miR-21-3p,miR-21-5p,miR-223-3p,P<0.001,the difference was statistically significant;miR-423-5p,P=0.284,the difference was not statistically significant.3.Experimental study on breast cancer cell function,we found that for breast cancer cell T47D,when up-regulated the expression level of miR-21-5p in cells,its proliferation,migration and invasion ability were increased;down-regulation of intracellular miR-21-5p expression level,its proliferative capacity,migration ability and invasion ability were all decreased.When the expression level of miR-223-3p was up-regulated,its proliferative capacity,migration ability and invasion ability were increased.When the expression level of miR-223-3p was down-regulated,its proliferation,migration and invasion ability were decreased.4.Combined with TCGA data analysis,the miRNA target lncRNA predicted the most significant long non-coding MAPT-AS1;through lncRNA correlation analysis,The positively related regulatory genes MAPT,MAPT-IT1,SPPL2C,KDM4B,and NXNL2 were used for experimental verification.5.Transfected long non-coding MAPT-AS1 overexpressing and interfering cell line T47D,the transfection efficiency was more than 90%by fluorescence microscopy,and RT-qPCR verified that the long non-coding MAPT-AS1 overexpression and interference stable breast cancer were successfully constructed.The cell line T47D was screened and the long non-coding MAPT-AS1 interference fragment shRNA3 with the highest interference efficiency was screened.Cell function experiments showed that the overexpression of long non-coding MAPT-AS1 cells increased the proliferation,migration and invasion ability,and the proliferation,migration and invasion ability of the interfering stably transfected cell lines were decreased.The long non-coding MAPT-AS1 co-expressing gene was verified,and the expression levels of MAPT,MAPT-IT1 and NXNL2 after transfection of shRNA3 interference fragment were decreased,which was consistent with the expression trend of long non-coding MAPT-AS1.ConclsionmiR-21-5p and miR-223-3p play a role as oncogenes in the development of breast cancer,and can promote the proliferation,migration and metastasis of breast cancer cells;their common target lncRNA-MAPT-AS1 is highly expressed in breast cancer patients.It is verified that the co-expression genes MAPT,MAPT-IT1 and NXNL2 are consistent with the expression of long non-coding MAPT-AS1;The interaction mechanism between miRNA,lncRNA and mRNA plays a key role in the development of tumors.For the study of miRNA-lncRNA-mRNA regulatory networks,this will help to develop better breast cancer diagnosis and treatment strategies.