| Objective:Breast cancer is a common gynecological malignancy,and is one of the leading causes of cancer death in women.In order to discovery the function of lncRNA00536 in the proliferation and apoptosis of breast cancer lines,and the contribution of lncRNA00536 to breast cancer malignancy and the molecular mechanisms,the research includes four parts.1)Comprehensive analysis of the aberrantly expressed lncRNA-associated ceRNA network in breast cancer;2):Expression and clinicopathological characteristics of long non-coding RNA LINC00536/miR-204-5p/TGF beta-2 in breast cancer;3):Abnormal expression of LINC00536 in breast cancer and its effect on proliferation and invasion of breast cancer cells;4):Validation of the targeting effect of downstream molecules of LINC00536,mir-204-5p and TGFBR2,in breast cancer cells.Methods:1)A total 1222 RNA seq profiles from breast cancer patients were downloaded TCGA.2)A ceRNA network was constructed for breast cancer based on miRcode and miRTarBase.The top 10 lncRNA were selected using Cox regression analysis.3)Survival analysis was approximated based on KM(Kaplan-Meier)curve analysis.4)Download LINC00536/miR-204-5p/TGF?-2 single gene data from TCGA database.Use single gene analysis method to analyze the expression of breast cancer tissue and corresponding normal adjacent tissue specimens,download clinical data and analysis.5)The expression of LINC00536/miR-204-5p/TGF?-2 in 30 breast cancer tissues was detected by qRT-PCR.The correlation between LINC00536/miR-204-5p/TGF?-2 expression and age,tumor size,clinical stage,tumor location and TNM stage was validated by bivariate correlation analysis.6)Immunohistochemistry was used to detect the expression of TGF?-2 in breast cancer and adjacent normal breast tissues.7)Enzyme-linked immunosorbent assay(ELISA)was used to determine the expression of TGFBR2-related indicators from blood samples.8)Analysis of different metabolites by ~1H-NMR plasma metabolomics.9)Detection of target gene expression level:After routine culture of human breast cancer cell lines MCF-7,MDA-MB-231,MDA-MB-453,T-47D and SK-RB-3,the expression level of target gene LINC00536 was detected by qRT-PCR,and the up-regulated LINC00536 cell lines were screened.10)LINC00536 silenced T47D and MDA-MB-453 cell lines and LINC00536overexpressed MDA-MB-231 and MCF-7 cell lines,each group had six multiple holes,and detected three times.11)The proliferation of T47D,MDA-MB-453,MDA-MB-231 and MCF-7 cell line was detected by EdU Apollo?567 imaging kit in vitro.The nuclei and cytoplasm were stained by Hoechst staining and Apollo staining respectively.12)The proliferation,growth and wound healing ability of T47D and MDA-MB-453 cell lines were detected by scratch test and plate clone formation test.13)Bioinformatics analysis was used to predict the potential interaction between LINC00536and microRNA204-5p,microRNA204-5p and TGFBR2.14)Construction of recombinant reporter gene vectors pmirGLO-Wt3'UTR-LINC00536,pmirGLO-Mt3'UTR-LINC00536,pmirGLO-Wt3'UTR-TGFBR2 and pmir GLO-Mt3'UTR-TGFBR2.15)Western-blot was used to detect the expression of TGF-beta signaling pathway-related proteins in T47D cells.Results:1)We identified 1028 breast cancer related lncRNAs and 89miRNAs(fold change>2,P<0.05);among these,93 lncRNAs and 19 miRNAs were included in the ceRNA network.2)Subsequently,we choose 10 basic lncRNAs and analyzed overall survival.In addition,five lncRNAs(ADAMTS9-AS1,AL513123.1,C10orf126,LINC00536,WT1-AS)were found to be significantly associated with overall survival(log-rank P<0.05).3)The expression of LINC00536 in breast cancer tissues was higher than that in normal tissues.The expression of microRNA-204-5p in breast cancer tissues was lower than that in normal tissues.The expression of TGFBR2 in breast cancer tissues was down-regulated.There was no significant difference in sex and initial diagnosis of breast cancer patients,but race,nationality,age of diagnosis and stage of cancer were related to survival(P<0.05).In the regression univariate analysis of 1081breast cancer patients,race,nationality,age of diagnosis and initial diagnosis were related to the expression level of LINC00536(P<0.05).In the regression univariate analysis of1070 breast cancer patients,race,ethnicity,age of diagnosis and preliminary diagnosis were related to the expression level of microRNA-204-5p(P<0.05).In the regression univariate analysis of 1090 breast cancer patients,the expression of TGFBR2 was correlated with race,age of diagnosis,initial diagnosis and tumor stage(P<0.05).4)The results of qRT-PCR showed that the expression of LINC00536 in breast cancer tissues was significantly higher than that in normal tissues(P<0.05).The expression of microRNA-204-5p/TGFbeta-2 in breast cancer was down-regulated(P<0.05).The expression of LINC00536/miR-204-5p/TGFbeta-2 had no significant difference with age,tumor size,clinical stage,tumor location and TNM stage(P>0.05).5)Immunohistochemical results showed that TGFBR2 positive cells,cytoplasmic staining and extracellular staining in breast cancer tissues were lower than those in normal breast tissues(P<0.05).6)In the breast cancer group,the levels of TGFBR1 and Smad2increased,and the levels of TGFBR2,Smad3,and Smad4 decreased significantly(P<0.05).7)Compared with the normal group,the breast cancer group had elevated pyruvate,citric acid,cholesterol,glycine,acetoacetate,methylamine,dimethylamine,asparagine,leucine,and?-glucose(P<0.05).8)The expression of LINC00536 was detected in MDA-MB-231,T47D,MCF-7,MDA-MB-453 and SK BR-3 cell lines,and was significantly increased in T47D and MDA-MB-453 cells dicreased in MDA-MB-231,MCF-7.9)The results of CCK8 assay showed that the absorbance of T47D and MDA-MB-453 cells in si-LINC00536 group was significantly reduced compared with the blank group and the control group,and the difference was statistically significant with the duration of time(P<0.05).After the expression of LINC00536 was up-regulated,the viability of breast cancer MDA-MB-231 and MCF-7 cells increased significantly compared with the control group(P<0.05).10)T47D and MDA-MB-453 cells were stained with EdU Apollo<567.The number of positive stained cells in si-LINC00536group was significantly lower than that in blank group and control group(P<0.05).EdU cell experiment results showed that compared with the control group,the number of positive cells of breast cancer MDA-MB-231 and MCF-7 cells in the LINC00536overexpression group increased significantly(P<0.05).15)Wound healing test results showed that in T47D and MDA-MB-453 cells,the Wound healing ability of si-LINC00536 cells was significantly lower than that of blank group and control group(P<0.05).11)The results of plate cloning formation experiment showed that the number of clones in T47D and MDA-MB-453 cells in si-LINC00536 group was significantly lower than that in blank group and control group(P<0.05).12)Bioinformatics analysis speculated the potential interaction between miR-204-5p and LINC00536 and TGFBR2seed region has specific binding sites.13)Double luciferase reporter assay showed that TGFBR2 was a target gene of microRNA-204-5p,and there was a binding site between microRNA204-5p and LINC00536.14)The abnormally high expression of LINC00536 in breast cancer cells can down-regulate the expression of miR-204-5p and exert its biological effect by affecting the expression of TGFBR2.Conclusion:According to the bioinformatic analysis,93 lncRNAs,19 microRNAs and 27 RNA were screened to form a breast cancer-related ceRNA network.Five lncRNAs(ADAMTS9-AS1,AL513123.1,C10orf126,LINC00536,WT1-AS)were found to be significantly associated with overall survival(log-rank P<0.05).Downregulation of LINC00536 in vitro can inhibit the growth of breast cancer cells and reduce the ability of cell invasion and migration.LINC00536 regulates the expression of TGFBR2 by negative regulation of miRNA-204-5p,thus exerting its biological function.LINC00536 can promote the growth of breast cancer and is expected to become a new target for gene therapy of breast cancer. |
PDF Full Text Request