| BackgroundBreast cancer(BC)is the malignant tumor with the highest incidence in women,accounting for 7-10%of all malignant tumors in the whole body,which seriously threatens women’s health.It kills 458,000 people worldwide each year,making it the main cause of cancer-related deaths in women.The incidence of breast cancer has risen rapidly and continuously over the past few decades.It is estimated that there are approximately 1.38 million new cases worldwide each year,and this number is expected to continue to increase due to the aging population and the continuous improvement in survival rates.Fortunately,with the continuous in-depth research on the pathogenesis of breast cancer,many new targeted therapies have emerged,such as anti-Her-2 drugs trastuzumab,pertuzumab and endocrine therapy drugs Tamoxifen,aromatase inhibitors,etc.These drugs are designed to specifically target and inhibit different molecular targets,block the signal transduction of tumor cells or related cells,thereby inhibiting the growth and spread of tumor cells.Even so,there are still some breast cancers that lack curative effects on endocrine therapy and molecular targeted therapy.This sub-type of breast cancer accounts for 15%-23.8%of all pathological types of breast cancer,and is called triple negative breast cancer(TNBC).Triple negative breast cancer refers to the sub-type of breast cancer that does not express estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2/neu)genes,characterized by strong invasion,high recurrence rate,high distant metastasis rate and poor prognosis.Conventional chemotherapy is a standard strategy for the treatment of advanced TNBC,but its efficacy is not ideal.According to reports,34%of newly diagnosed TNBC patients relapse within 5 years after receiving adjuvant or neoadjuvant chemotherapy.In recent years,new therapeutic targets such as anti-PD-L1 antibodies and PARP inhibitors have been found based on other molecular biological characteristics.However,due to the heterogeneity of patients and the susceptibility to drug resistance,the existing therapeutic effect of existing treatment methods on TNBC is still not satisfying.The treatment effect of TNBC is still not so good.Therefore,it is of great clinical significance to find more effective tumor biomarkers and new targets for early screening,prognostic evaluation and treatment of TNBC.The SRY associated high mobility group(HMG)box(SOX)family has been reported to be involved in pathological and physiological processes of a variety of tissues,such as reprogramming and regeneration.So far,about 20 different SOX proteins have been identified,and they share a HMG domain with more than 80%sequence homology.SOX8,SOX9 and SOX 10 belong to the SOXE family.Studies have shown that the expression of SOX8 in non-small cell lung cancer is regulated by microRNA-124,and its high expression can promote the proliferation of tumor cells;the high expression of SOX8 in liver cancer also promotes the proliferation of tumor cells and is positively correlated with the high expression of β-catenin;SOX8 can also be used as a molecular marker to indicate the malignant prognosis of glioma.Searching the TCGA database has found that the expression of SOX8 gene in TNBC is higher than that of the normal control group.A further search of the TCGA and METABRIC databases has revealed that SOX8 is a functional oncogene that participates in maintaining the stem cell-like function of TNBC cells,and that ZEB1 is the transcriptional activation of SOX8 which enhances the expression of SOX8 by combining with the promoter.Therefore,SOX8 is expected to be used as a prognostic marker of TNBC.However,by searching the GEO(GSE76275)database,it is found that HORMAD1 and SOX8 in TNBC are significantly higher than those in non-TNBC,while SOX8 has no correlation with the prognosis,contradicting the previous conclusions.Therefore,it is necessary to further clarify the exact mechanism of SOX8 in TNBC.MicroRNA(miRNA)is a class of non-protein coding RNA molecules composed of 20-22 nucleotides that regulate gene expression by binding to specific targets.MiRNA expression dysregulation is related to a variety of diseases,and is considered to be a potential biomarker or key regulator of many cellular processes.miRNA is closely related to the occurrence and development of tumors.miR-139-5p is a kind of miRNA molecule with anticancer effect,whose expression is reduced in a variety of tumor tissues.Overexpression of miR-139-5p can however inhibit tumor cell proliferation,migration and invasion through a variety of target genes,and induce its apoptosis.Therefore,miR-139-5p has potential application prospects in the clinical diagnosis,treatment and prognosis of tumors.Studies have shown that miR-139-5p may be a reliable serum biomarker for colorectal cancer recurrence and metastasis;silencing miR-139-5p through histone methylation can promote the invasion of non-small cell lung cancer cells;miR-139-5p can target ZEB-1 to promote the proliferation,migration and invasion of breast cancer,pancreatic cancer,and cervical cancer cells.At present,there are few studies on the correlation between miR-139-5p and the mechanism of TNBC.Through searching the GSE database(GSE40049 and GSE19783),it is found that miR-139-5p is a factor predicting the recurrence of TNBC and is highly correlated with the prognosis of TNBC.There have been related reports on the regulatory relationship between miRNA and SOX family.For example,SOX5 is the target of miR-499 and SOX2 is the target of miR-145.However,there are few studies on the specific role of SOX8 and miR-139-5p in TNBC.In view of the role of SOX8 and miR-139-5p in tumor regulation,it is necessary to further explore the relationship between SOX8 and miR-139-5p in TNBC and find the regulatory mechanism of the two.ObjectivesThis study aims to explore the regulatory role of miR-139-5p on the pathogenesis and development of TNBC.It also aims to explore that the molecular mechanism of miR-139-5p regulates the expression of SOX8 by way of inhibiting the development of TNBC,so as to provide a new theoretical basis for studying the biomarkers and targets in the diagnosis and treatment of TNBC.Methods1.The expression pattern of SOX8 in TNBC cellsIn the experiment,two commonly used human TNBC cell lines(HCC1806 and BT549)were selected,and the relative expression of SOX8 at the mRNA and protein levels was detected by real-time fluorescent quantitative PCR and western blotting respectively.2.The effect of SOX8 gene on the biological behavior of TNBC cellsSOX8 overexpression plasmid or microRNA-SOX-8 specific short hairpin RNA was transfected into HCC1806 and BT549 cell lines to enhance or knockdown gene expression.The relative expression of SOX8 mRNA in transfected cells was detected by quantitative PCR to determine the efficiency of overexpression and interference.Then CCK-8,clone formation and Transwell assays were performed to determine the viability,clone formation ability and migration of HCC1806 and BT549 cells,and to evaluate the effect of SOX8 on the proliferation and migration of TNBC cells.3.The effect of SOX8 on TNBC cell apoptosis and stemnessThe apoptosis of HCC1806 and BT549 cells was analyzed by flow cytometry after SOX8 overexpression or interference.The self-renewal ability of these TNBC cells was identified by in vitro tumor sphere formation assay.4.The effect of overexpression of SOX8 on Wnt/β-catenin pathway activityNetwork software such as Targetscan and Starbase was used to predict the complementary binding site of SOX8 with the Wnt pathway receptor FZD7 through bioinformatics methods,and verify the effect of overexpression of SOX8 on the transcription of FZD7 through RT-PCR.5.The effect of miR-139-5p on the proliferation and migration of TNBC cellsQuantitative PCR was used to detect the expression of miR-139-5p in HCC1806 and BT549 cells and MCF-10A cells.The expression levels of miR-139-5p and SOX8 mRNA in these two TNBC cells were observed,and the relationship between miR-139-5p and SOX8 mRNA was revealed.The effects of miR-139-5p overexpression on cell proliferation and migration were studied by CCK-8,clone formation and Transwell assays.6.The effect of miR-139-5p on TNBC cell apoptosis and stemnessMiR-139-5p was overexpressed in HCC1806 and BT549 cells,respectively.The apoptosis rate was detected by flow cytometry,and the stemness was detected by tumor sphere formation in vitro.7.The interaction between miR-139-5p and SOX8The potential complementary binding sites of SOX8 3’UTR and miR-139-5p were predicted by bioinformatics using Targetscan,Starbase and other web tools.Subsequently,wild-type SOX8 3’-UTR(WT)or its mutant sequence(MUT)was fused on the reporter gene vector and co-transfected with miR-139-5p mimics,and dual luciferase reporter assay was carried out to confirm the direct interaction between miR-139-5p and WT or MUT SOX8 3’-UTR.8.Rescue assaySOX8 was meanwhile overexpressed in cells with miR-139-5p overexpression,and whether SOX8 overexpression could reverse the changes caused by miR-139-5p.Briefly,miR-139-5p mimics and SOX8 were transfected into HCC1806 and BT549 cells at the same time.The changes of cell viability,colony forming ability,migration,stemness and apoptosis were detected respectively.9.The effect of miR-139-5p and SOX8 on the growth of xenograft tumor in miceHCC1806 and BT549 cells were transfected with miR-139-5p mimics,pcDNA3.1-SOX8 and microRNA-SOX-8 specific short hairpin RNA alone or at the same time.The stably transfected cells were inoculated subcutaneously in the right forelimb of mice.The volume of xenograft tumor in subcutaneous xenografts of tumor bearing mice was recorded in different time periods.After 28 days of inoculation,xenograft tumors were removed and weighed to evaluate the effects of miR-139-5p and SOX8 on tumor growth of TNBC.10.Differential expression of miR-139-5p and SOX8 in TNBC cellsTNBC tissues were collected,and the kit was used to extract total mRNA in the tissues;the fluorescence quantitative PCR method was used to detect the mRNA content of miR-139-5p and SOX8 in the tissue,and their differential expressions were analyzed.Results1.The expression of SOX8 mRNA was up-regulated in TNBC cells.Compared with MCF-10A,the expression of SOX8 mRNA in HCC1806 and BT549 breast cancer cell lines was significantly up-regulated(P<0.05).2.SOX8 promoted the proliferation and migration of TNBC cells.The results of quantitative PCR showed that compared with the control group,the expression of microRNA-SOX-8 specific short hairpin RNA in HCC1806 and BT549 cells transfected with siRNA-SOX8 significantly decreased(P<0.05),while the expression level of SOX8 mRNA in overexpression group significantly increased(P<0.05).The results showed that the overexpression and interference efficiency of SOX8 gene were high,and that the transfected cell line was successfully constructed.The survival rate of HCC1806 and BT549 cells overexpressing SOX8 was significantly higher than that of control group(P<0.05).At the same time,after SOX8 overexpression,the relative clone number of the two kinds of cells increased(P<0.05),indicating that the clone ability of TNBC cells was enhanced.Transwell assay showed that SOX8 overexpression significantly increased cell migration(P<0.05).On the other hand,cells transfected with microRNA-SOX-8 specific short hairpin RNA showed opposite results.Therefore,SOX8 gene can promote the proliferation and migration of TNBC cells.3.SOX8 inhibited TNBC cell apoptosis and participated in maintaining cell stem.The apoptosis rate of HCC1806 and bt549 cells overexpressed with microRNA-SOX-8 specific short hairpin RNA was significantly lower than that of control group(P<0.05),while the apoptosis rate of cells transfected with siRNA-SOX8 significantly increased(P<0.05).Compared with the control group,the number of tumor cells in the SOX8 overexpression group significantly increased(P<0.05),while interference with SOX8 expression could weaken the ability of tumor cells to form spheres(P<0.05),indicating that SOX8 can inhibit the apoptosis of TNBC cells and participate in the maintenance of stem cell stem cells.4.Overexpression of SOX8 enhanced Wnt/β-catenin pathway activity.The overexpression of SOX8 in triple-negative breast cancer cells enhances the activity of the Wnt/β-catenin pathway,thereby enhancing the stemness of triple-negative breast cancer cells and aggravating their proliferation and migration behaviors.5.MiR-139-5p inhibited TNBC cell proliferation and induces apoptosis.The expression of miR-139-5p in HCC1806 and BT549 cells was significantly lower than that in MCF-10A cells(P<0.05).Over expression of miR-139-5p was observed in the two TNBC cells.Interestingly,overexpression of miR-139-5p resulted in a decrease in SOX8 mRNA expression,suggesting that miR-139-5p may be regulated by miR-139-5p.Further study on the effect of miR-139-5p on cell function showed that the cell proliferation and migration rate significantly reduced after overexpression of miR-139-5p(P<0.05).6.MiR-139-5p induced TNBC cell apoptosis and inhibits of tumor sphere formation.The apoptosis rate of miR-139-5p overexpression group was significantly higher than that of control group(P<0.05),and the ability of tumor sphere formation of miR-139-5p group was lower than that of SOX8 knockdown group(P<0.05).Therefore,it is concluded that miR-139-5p may play a biological role by reducing the expression level of SOX8 and play a role in controlling cell growth.7.SOX8 was the direct target of miR-139-5p.Bioinformatics method predicted that SOX8 3’-UTR had complementary binding sites with miR-139-5p.Double luciferase reporter analysis showed that miR-139-5p mimics co transfection significantly reduced the luciferase activity of wild-type SOX83’-UTR cells(P<0.05),but had no effect on the luciferase activity of mutant sequences,indicating that miR-139-5p can target SOX8 and inhibit its translation.8.MiR-139-5p induced phenotypic changes of TNBC cells by targeting SOX8.Overexpression of miR-139-5p or knockdown of SOX8 in HCC1806 and BT549 cells significantly reduced the viability,colony formation and migration ability of the two TNBC cells,and the difference was statistically significant(P<0.05).It should be noted that the proliferation and migration rates of miR-139-5p and SOX8 overexpression groups were significantly higher than those of the above groups(P<0.05),which was close to the cells before transfection.On the other hand,compared with the blank control group,miR-139-5p transfection or SOX8 knockdown could increase the apoptosis rate(P<0.05),but the ability of tumor cells to form spheres significantly reduced(P<0.05).However,the apoptosis rate of miR-1-9-5p and SOX8 overexpression group was lower than that of transfection group,and the number of tumor spheres significantly increased.Therefore,up-regulation of SOX8 expression in cells can eliminate the effect of miR-139-5p overexpression on cell phenotype to a certain extent,which further confirms that miR-139-5p can inhibit the growth and migration of breast cancer cells by downregulating the expression of SOX8.9.MiR-139-5p inhibited the growth of xenograft tumor in mice by targeting SOX8.The growth rate of xenograft tumor in mice inoculated with HCC1806 and BT549 cells with miR-139-5p overexpression or siRNA-SOX8 was significantly suppressed compared with the control group,at different time points after inoculation(P<0.05),while the volume of transplanted tumor derived from cells overexpressing miR-139-5p and SOX8 increased(P<0.05).In addition,28 days after inoculation,the weight of xenografts showed a similar trend.Thereafter,miR-139-5p inhibits the growth of TNBC tumor and acts as a tumor suppressor gene in animals.This effect is achieved by downregulating SOX8.10.There were differentiated expressions of SOX8 and miR-139-5p in TNBC cells.Compared with adjacent tissues,SOX8 mRNA content in TNBC lesion tissue increased and miR-139-5p content decreased significantly.The difference between the two groups was statistically significant(p<0.05);the fluorescence intensity carried by the miR-139-5p fluorescence probe in lesion tissues significantly reduced,significantly different from that of adjacent tissues.ConclusionMiR-139-5p can inhibit the growth and migration of triple-negative breast cancer cells and induce cell apoptosis.It acts as an important regulator in the pathogenesis of breast cancer.This effect is achieved by directly targeting down-regulation of SOX8 expression.miR-139-5p interacts directly with the 3’-UTR site of SOX8 to prevent SOX8 transcription.When the content of miR-139-5p decreases,the SOX8 3’-UTR site is released,which enhances the transcription and translation of SOX8,activates the Wnt/β-catenin signaling pathway,and makes triple-negative breast cancer cells proliferate and migrate,as well as the significant improvement of the invasion capacity.Therefore,inhibiting the proliferation and migration of triple-negative breast cancer cells by interfering with the SOX8 gene or up-regulating the expression of miRNA-139-5p can slow the progression of the disease to a certain extent.This research can provide new ideas for the diagnosis and molecular targeted therapy of triple-negative breast cancer. |
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