Mir-4732-5p Promotes Breast Cancer Progression By Targeting TSPAN13

Breast cancer is one of the most common malignant tumors in women and its early diagnosis is an important clinical topic.microRNA(miRNA)is a small,non-coding single-stranded RNA and is a key regulator of human tumorigenesis and progression.miRNA content in a variety of body fluids and maintain a relatively stable,is currently the focus of research on biomarkers of malignant tumors.Our previous study found that miR-4732-5p differentially expressed in benign and malignant nipple discharge and was a potential tumor marker.However,its expression,function and molecular mechanism in breast cancer are still unknown.In this study,we use real-time quantitative PCR to examine the expression of miR-4732-5p in 67 pairs of breast cancer tissues and paired normal tissues,as well as in nine breast cancer cell lines and one non-tumorigenic cell line.The effect of miR-4732-5p on breast cancer cells was detected by cell proliferation,apoptosis and Transwell assay.The target genes of miR-4732-5p were identified by RNA sequencing,bioinformatics analysis,Western blot and luciferase assay.The results of this study suggest that miR-4732-5p may play a role as tumor suppressor gene in the stage of breast cancer,and promote tumor progression by targeting TSPAN13(tetraspanin 13)after the occurrence of breast cancer.Chapter ? Expression and functional verification of miR-4732-5p in breast cancer tissueObjective:1.To detect the expression of miR-4732-5p in 67 pairs of breast cancer tissues and adjacent normal tissues,as well as nine breast cancer cell lines and one non-tumorigenic cell line.2.To examine the effects of miR-4732-5p on breast cancer cells.Materials and methods:1.A total of 67 pairs of primary breast cancers and corresponding normal tissues were obtained from breast cancer patients at Qilu Hospital of Shandong University(Jinan,China)from 2015 to 2.017.The fresh specimens were immediately stored in liquid nitrogen until RNA was extracted.Written informed consent was obtained from each patient.The study was approved by the Ethics Committee of Qilu Hospital of Shandong University.2.In breast cancer and the corresponding paracancer tissue,the expression level of miR-4732-5p was detected by real-time quantitative PCR,and the relationship.between the expression level of miR-4732-5p and the clinicopathological parameters was analyzed.3,Nine breast cancer cell lines and one non-tumorigenic cell line were cultured,and the expression level of miR-4732-5p was detected by real-time quantitative PCR..4.The effect of miR-4732-5p overexpression on breast cancer cells was detected by cell proliferation,apoptosis and transwell assay.Results:1.The expression of miR-4732-5p in breast cancer tissues was significantly lower than that in normal tissues(P = 0.0140,paired t-test).2.miR-4732-5p expressed lower in all 9 breast cancer cell lines than in the non-carcinogenic MCF10A cell line.3.Overexpression of miR-4732-5p can promote the proliferation,migration and invasion of breast cancer cells.Compared with low metastatic cell lines(MCF-7 and T47D),miR-4732-5p expression levels in high metastatic cell lines(SK-BR-3,ZR-75-1,MDA-MB-453,BT549,MDA-MB-468,MDA-MB-231 and MDA-MB-157)were relatively higher.4.Compared with normal breast tissue,miR-4732-5p was down-regulated in the lymph node metastasis negative group(P<0.0001)rather than the positive group(P = 0.6838).5.miR-4732-5p was positively correlated with tumor size(one-way ANOVA,P =0.0080),Ki-67 index(P = 0.0394)and clinical stage(one-way ANOVA,P =0.0016).Conclusions:Compared with normal breast tissues,miR-4732-5p is down-regulated in breast cancer tissues,especially non-metastatic tissues.However,the expression level of miR-4732-5p is higher in metastatic breast Cancer tissues than:in non-metastatic breast cancer tissues.miR-4732-5p expression was positively correlated with lymph node metastasis,tumor diameter,clinical stage and Ki-67 expression.Over expression of miR-4732-5p can promote the proliferation,migration and invasion of breast cancer cells.This study provides a theoretical basis for miR-4732-5p to become a tumor marker of breast cancer.Chapter ? Screening,verification and functional prediction of miR-4732-5p downstream target geneObjective:1.To screen and verify the downstream target genes of miR-4732-5p.2.To analyze the possible function and prognostic value of TSPAN13.Materials and methods:1.Genome-wide mRNA sequencing was performed using MDA-MB-231 cell lines to screen down-regulated genes in the miR-4732-5p transfection group compared with the negative control group(multiple changes of>,2 and P<0.05).2.Among the 30 candidate genes,two genes(TSPAN13 and CDK18)were predicted by TargetScan to contain binding sites of miR-4732-5p in the 3'-utr region.3.Real-time quantitative PCR and Western blot were performed to determine whether miR-4732-5p could alter the mRNA and protein expression of TSPAN13.4.Luciferase assay was performed to determine whether TSPAN13 mRNA carried binding sites of miR-4732-5p.5.BioProfiling and PrognoScan database was used to explore the prognostic value of TSPAN13.Results:1.Compared with negative control transfected cells,mRNA and protein expression levels of TSPAN13 were significantly inhibited after miR-4732-5p transfection.2.The fluorescence intensity of miR-4732-5p co-transfected with wild-type 3 '-utr carrying TSPAN13 was significantly decreased,while that of the mutant 3'-utr without target sites co-transfected with miR-4732-5p had no significant effect on the fluorescence intensity.3.BioProfiling database analysis indicates that the lower expression of TSPAN13 indicates a lower survival rate of breast cancer patients(GSE250665,n = 507,P =0.000546),PrognoScan database findings indicate that patients with low TSPAN13 expression have a shorter survival time than patients with high TSPAN13 expression(GSE12276,n = 204,P = 0.001299).Conclusions:miR-4732-5p inhibits the expression of TSPAN13 in mRNA and protein levels by targeting the 3 '-utr region of TSPAN13.miR-4732-5p may play a role as a tumor suppressor gene in the stage of breast cancer,and after the occurrence of breast cancer,it promotes tumor progression by targeting TSPAN13.