The Effection Of CircVAPA/miR-130a-5p Axis And Shikonin On Migration And Invasion Of Breast Cancer

The purpose of this study was to investigate the role of circular RNA circVAPA in promoting the invasion and migration ability of human breast cancer cells through competitively binding to miR-130a-5p,and to study the intervention effect of shikonin in this process and its possible mechanism.This study provided a new molecular target for the diagnosis and treatment of metastatic breast cancer with integrated traditional Chinese and Western medicine,and also provided a theoretical basis for guiding the clinical use of shikonin in the treatment of breast cancer.The research is mainly divided into three parts:Part Ⅰ Expression and clinical significance of circVAPA in breast cancerObjective:To study the expression of circular RNA circVAPA in breast cancer tissues and cells,and explore its role in regulating breast cancer cell migration and invasion.Methods:(1)Circular RNA high-throughput sequencing technology(RNA-seq)was used to analyze the expression of circular RNA in frozen specimens from 3 cases of breast cancer and adjacent normal tissues.(2)The expression of circVAPA was detected by real-time quantitative PCR(RT-qPCR)in 29 pairs of breast cancer and adjacent normal tissues,and in human breast cancer MCF-7 and MDA-MB-231 cell lines.The back-splice junction of circVAPA was determined by sanger sequencing.(3)CircVAPA overexpression plasmid(p-circVAPA)was constructed and transfection of p-circVAPA was carried out to overexpress circVAPA in poorly aggressive MCF-7 breast cancer cells.Functional experiments were subsequently performed to explore the effect of circVAPA overexpression on the biological functions of MCF-7 cells.Meanwhile,highly aggressive MDA-MB-231 breast cancer cells were transfected with siRNA(si-circVAPA)to knock down the expression of circVAPA,and then functional experiments were performed to explore the impact of circVAPA low-expression on the biological functions of MDA-MB-231 cells.Results:(1)CircVAPA was highly expressed in breast cancre tissues,compared with that in adjacent normal tissues.Furthermore,the expression of circVAPA was significantly upregulated in lymph node positive tissues compared with lymph node negative tissues.(2)The expression of circVAPA was significantly upregulated in highly aggressive MDA-MB-231 cells,compared with poorly aggressive MCF-7 cells.(3)Overexpression of circVAPA promoted breast cancer cell migration and invasion and down-regulation of circVAPA inhibited breast cancer cell migration and invasion in vitro.Conclusions:The expression level of circular RNA circVAPA is positively correlated with the migration and invasion ability of breast cancer cells.Ectopic circVAPA regulated migration and invasion of breast cancer cells.Part Ⅱ Inhibitory effect of shikonin on the development of breast cancer in vitro and in vivoObjective:To study the role of shikonin in the proliferation,migration and invasion of breast cancer cell lines in vitro,and to explore its anti-tumor effect of breast cancer in vivo using animal models.Methods:(1)Different concentrations(1,2,4,8,16,32,64 μM)of shikonin were treated with human breast cancer MCF-7 and MDA-MB-231 cells and MTT cell proliferation assay was used to determine the inhibitory rate and IC50 of shikonin on breast cancer cells.Flow cytometry was used to observe the apoptosis rate of shikonin-treated MCF-7 and MDA-MB-231 cells after 24 h.The non-toxic dose of shikonin in both cells was determined.(2)EdU cell proliferation detection technology was employed to observe the proliferation inhibition effect of shikonin at different concentrations on breast cancer cells.Wound-healing and transwell assays were used to detect changes in breast cancer cell invasion and migration after shikonin treatment for 24 h.(3)A nude mouse model of human breast cancer cell MDA-MB-231 xenograft tumor was established.Nude mice were treated with shikonin at different doses,and the inhibitory and toxic side effects of shikonin on the growth of tumor in mice were observed.Results:(1)Shikonin had an inhibitory effect on the proliferation of cells MCF-7 and MDA-MB-231 breast cancers,and the effect was partly in a concentration and time dependent manner.Cell apoptosis rate was also positively correlated with shikonin concentration.(2)Shikonin at a concentration of 2,4 μM could significantly inhibit the invasion and migration of MCF-7 cells and at a concentration of 8 μM in MDA-MB-231 cells could significantly inhibit the ability of cells invasion and migration.(3)High-dose of shikonin(15 mg/kg/d)has obvious growth inhibitory effect on the tumor of nude mice with human breast carcinoma without significant toxic and side effects.Conclusions:The Chinese medicine monomer shikonin can not only effectively inhibit the proliferation,migration and invasion of breast cancer cells in vitro,but also can significantly inhibit the growth of breast cancer transplanted tumors in vivo,which has potential application value in the treatment of breast cancer.PartⅢ CircVAPA/miR-130a-5p regulatory axis promotes breast cancer invasion and metastasis and the effect of shikoninObjective:To study the specific mechanism of the effect of circular RNA circVAPA on breast cancer invasion and migration,and to explore the intervention of shikonin in the circVAPA/miR-130a-5p regulatory axis.Methods:(1)The Confocal FISH assay was performed to explore the localization of circVAPA in MDA-MB-231 cells.(2)MiRNA high-throughput sequencing technology(RNA-seq)was used to analyze the expression of miRNA in frozen specimens from 3 cases of breast cancer and adjacent normal tissues.Downstream potential miRNAs of circVAPA was predicted by using bioinformatics software.Candidate miRNA of circVAPA was identified by both prediction algorithms and miRNA-seq results.(3)The expression of miR-130a-5p was detected by RT-qPCR in 29 pairs of breast cancer and adjacent normal tissues,and in human breast cancer MCF-7 and MDA-MB-231 cell lines.(4)Dual-luciferase reporter assay was used to confirm adsorption relationship between circVAPA and miR-130a-5p.The transfection of miR-130a-5p analogue(mimics)was performed on MDA-MB-231 cells,and miR-130a-5p function was confirmed by the wound-healing and transwell migration and invasion assays.Finally,circVAPA was overexpressed to verify the biological function of miR-130a-5p whether could be inhibited through functional recovery experiments.(5)The expression of miR-130a-5p was detected by RT-qPCR in MDA-MB-231 cells before and after treating with shikonin.Transfection assays of the shikonin-treated MDA-MB-231 cells with miR-130a-5p inhibitors were used to confirm whether shikonin could play roles in breast cancer cells by upregulating miR-130a-5p.(6)The expression of VAPA was detected by RT-qPCR in human breast cancer MCF-7 and MDA-MB-231 cell lines and after overexpression and knockdown circVAPA.Kaplan-Meier survival analysis was performed to analyzed survival data of 1764 breast cancer patients.Results:(1)FISH assay confirmed that circVAPA was mainly present in the cytoplasm.(2)MiR-130a-5p was significantly downregulated in breast cancer tissues,compared with that in adjacent normal tissues.Furthermore,the expression of miR-130a-5p was significantly downregulated in lymph node positive tissues compared with lymph node negative tissues.(3)The expression of miR-130a-5p was significantly downregulated in highly aggressive MDA-MB-231 cells,compared with poorly aggressive MCF-7 cells.(4)Bioinformatics prediction results indicate that miR-130a-5p contains a binding site matching with circVAPA and has a strong binding possibility.Dual-luciferase reporter assay showed circVAPA could harbor miR-130a-5p.Meanwhile,both wound healing and transwell migration and invasion assays indicated that miR-130a-5p could inhibit the invasion and migration abilities of MDA-MB-231 cells.The opposite biological function of circVAPA and miR-130a-5p suggested that circVAPA could competitively bind miR-130a-5p to inhibit its ability to inhibit invasion and migration in MDA-MB-231 cells.(5)RT-qPCR results revealed that miR-130a-5p was up-regulated in shikonin treated MDA-MB-231 cells compared with MDA-MB-231 cells.At the same time,both wound healing and transwell migration and invasion assays confirmed that shikonin restored the ability of miR-130a-5p to inhibit invasion and migration of breast cancer cells by upregulating its expression.(6)Kaplan-Meier survival analysis showed that low expression of VAPA was closely correlated with poor disease-free survival(DFS)and knockdown or overexpression of circVAPA did not influence the expression of VAPA.Conclusions:CircVAPA could promote the invasion and migration of breast cancer cells by competitively binding to miR-130a-5p and shikonin could inhibit breast cancer invasion and migration by interfering with the circVAPA/miR-130a-5p regulatory axis.