Circular RNA THBS1 Promotes Breast Cancer Metastasis Via Sponging MiR-129-5p To Regulate SOX4 Expression

Objective: Tumor metastasis is a common clinical disease,and it is also a difficult problem in the treatment of breast cancer.The mechanism of tumor metastasis still needs further study.The purpose of this study is to explore the relationship between the expression of circRNAs(Circular RNA)and the migration and invasion and prognosis of breast cancer cells,and hope to provide effective target for future treatmentMethods: 1.CircRNAs microarray analysis was used to screen out the related circRNAs,and was verified by RT-qPCR(Real-time quantitative polymerase chain reaction,real time fluorescence quantitative polymerase chain reaction).2.The overexpression and siRNA of circular RNA THBS1 were constructed and respectively transfected into MCF-7 and MDA-MB-231 cells.The effects of circular RNA THBS1 on the ability of migration and invasion in breast cancer cells were detected by scratch and Transwell.3.Predict and analyze the circRNA-miRNA regulatory network,to find miRNAs which could combine with the circular RNA THBS1 through bioinformatics software,such as miRanda and RNAhybird,then to further verify whether it could bind to miR-129-5p by using the dual luciferase reporter system.4.To explore the function of miR-129-5p in breast cancer,we transfected the miR-129-5p mimics and inhibitors into breast cancer cells,and utilized the scratch and Transwell to detect the ability of migration and invasion in the breast cancer cells effected by miR-129-5p.5,using bioinformatics software such as Targetscan to predict the potential downstream target genes of miR-129-5p,then transfected miR-129-5p mimics and inhibitor to intervene cells,and the RT-qPCR method was used to detect the expression of SOX4(SRY-box 4)mRNA.The dual luciferase reporter system was used to verify the relationship between miR-129-5p and downstream target gene SOX4.6,using the database of TCGA(The Cancer Genome Atlas,cancer database)to draw the Kaplan Meier survival curve to verify the correlation between the expression level of miR-129-5p and SOX4 and the patient's survival time.Results 1.CircTHBS1 related to metastasis was selected by Microchip analysis,further,that the expression level of circTHBS1 in the MDA-MB-231 cell line was significantly higher than that in the MCF-7 cell line verified by using RT-qPCR.Besides,the FISH(fluorescence in situ hybridization)found that circTHBS1 was mainly located in the cytoplasm.It probably plays the role of regulating miRNA.2.Transfection the over expression of circTHBS1 and si-circTHBS1 to MCF-7 and MDA-MB-231 cells,PCR was used to detect expression level and transfection effect.The results showed that the migration and invasion ability of MCF-7 cells was increased obviously after transfected with circTHBS1 overexpression,and the migration and invasion ability of MDA-MB-231 cells were obviously weakened after si-circTHBS1 transfection.It was concluded that circTHBS1 can promote the migration and invasion of breast cancer cells.3.The biological information software(such as miRanda,RNAhybrid,etc.)predicted that miR-129-5p was the target binding miRNA of circTHBS1.At the same time,the TCGA database analysis found that the expression of miR-129-5p in breast cancer tissue was lower,compared with paracancerous tissue.The Kaplan Meier survival curve also showed that the prognosis of the patients with low miR-129-5p expression was poor.The results of the double luciferase reporter gene showed that the fluorescence ratio of the co transfected wild type(WT)/miR-129-5p mimic group was significantly lower than that of the transfected mutant(MUT)/miR-129-5p mimic and NC group,and had statistical significance.At the same time,the expression level of miR-129 5p was detected by RT-qPCR.The results also showed that the expression level of miR-129 5p was negatively correlated with the expression level of circTHBS1.It can be deduced that circTHBS1 can adsorb and regulate the expression of miR-129-5p.4.The miR-129-5p mimics and inhibitors were transfected into MDA-MB-231 and MCF-7 cells.Compared to the control group,the miR-129-5p expression level in MDA-MB-231 cells was significantly higher after transfection of miR-129-5p mimics,and the migration and invasion ability of MDA-MB-231 cells was weakened after transfection with miR-129-5p mimics.The expression level of miR-129-5p in MCF-7 cells after transfection with inhibitors was lower than that in the control group,simultaneously significantly enhanced the migration and invasion ability of MCF-7 cells.The results showed that miR-129-5p inhibited the migration and invasion of breast cancer cells.5.Using bioinformatics software to predict target genes of miR-129-5p,we found that SOX4 is the downstream target gene of miR-129-5p.RT-qPCR found that miR-129 5p was negatively correlated with the expression of target gene SOX4.At the same time,the results of the double luciferase reporter gene showed that the fluorescence ratio of the co transfected SOX4-3 '-UTR wild type /miR-129-5p mimic group was significantly lower than that of the transfected /miR-129-5p mimic and the NC group.Therefore,we believe that SOX4 is the target gene of miR-129-5p downstream regulation.Conclusions 1,circTHBS1 can promote the migration and invasion of breast cancer cells.2.CircRNA-miRNA regulatory network prediction analysis miR-129-5p is a combination of circTHBS1 targeted miRNA,and its expression is negatively regulated by circTHBS1.3.Bioinformatics software shows that SOX4 is the downstream target gene of miR-129-5p,and its expression is negatively regulated by miR-129-5p.4,circTHBS1 may regulate the expression of target gene SOX4 in cells through adsorption and miR-129-5p,thus affecting the migration and invasion ability of breast cancer cells.5.Inhibition of circTHBS1 expression is likely to improve the prognosis of patients with breast cancer.