The Roles And Mechanism Of MicroRNAs In Tumorigenesis And Chemoresistance Of Triple-negative Breast Cancer

Part I MiR-93 inhibits triple-negative breast cancer growth and chemoresistance by targeting E2F1 and CCND1Worldwide,breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death overwhelmingly affecting women health.Chemotherapy is the cornerstone of breast cancer systemic treatment,but in clinical practice,the development of chemoresistance usually leads to treatment failure.Studies have shown that microRNAs(miRNAs)regulate the expression of downstream target genes at the post-transcriptional level,and they are widely involved in multiple biological processes of many diseases including cancer.Moreover,the aberrant expression of miRNAs is closely related to the cancer initiation and progression.However,the clear mechanism of how miRNAs affect chemoresistance remains unknown.Chemotherapy is used as the primary treatment of myeloid breast cancer,a special tissue type of triple-negative breast cancer.To date,there are few researches focusing on the chemoresistance of myeloid breast cancer.Our previous work completed the high-throughput sequencing of parental and multi-drug resistant myeloid breast cancer cells,and established a miRNA library related to multi-drug resistance.In our present study,miR-93-5p(miR-93)was screened out of this miRNA library for its effective regulation of chemoresistance.Firstly,we detected the expression of miR-93 was significantly downregulated in chemoresistant breast cancer cells and in clinical breast cancer tissues.The MTT results showed that overexpression of miR-93 significantly inhibited the proliferation of breast cancer cells and the resistance to multiple chemotherapeutic drugs including paclitaxel.In contrast,miR-93 knockdown promoted cell proliferation and chemresistance.Moreover,the flow cytometry results showed that overexpression of miR-93 induced cell cycle arrest in G1/S phase and promoted cell apoptosis,whereas knockdown of miR-93 exihibited the opposite effects.Furthermore,we found that miR-93 significantly inhibited tumor growth of subcutaneous tumor in nude mice,and the inhibitory effect was more obvious in combination with paclitaxel.These results indicated that miR-93 could reduce the resistance of breast cancer to paclitaxel.Next,we predicted the downstream target genes of miR-93 in Target Scan database,and screened these predicted target genes by analysing TCGA database.Through qRT-PCR,Western Blot and Dual-luciferase reporter assays,we found that miR-93 notably decreased the expression of E2 F Transcription Factor 1(E2F1)and Cyclin D1(CCND1),and confirmed that miR-93 directly targeted E2F1 and CCND1.Moreover,we found that overexpression of E2F1 and CCND1 markedly promoted cell proliferation and PTX resistance,whereas knockdown of E2F1 and CCND1 exhibited opposite effects.In addition,the flow cytometry results showed that overexpression of E2F1 and CCND1 could antagonize miR-93-mediated cell cycle arrest and the increase of apoptosis.These findings indicated that E2F1 and CCND1 were dual targets of miR-93,and they notably promoted cell proliferation and paclitaxel resistance.Further mechanistic studies revealed that miR-93 downregulated the expression of E2F1 and CCND1,on the one hand,reducing the expression of proteins inRB-E2 F pathway and some cell cycle-related proteins to inhibit cell proliferation and cell cycle progression;on the other hand,inactivating the phosphorylation of AKT and affecting the expression of apoptosis-related proteins to promote cell apoptosis.Taken together,miR-93 regulated the chemoresistance of breast cancer cells via suppressing E2F1 and CCND1.In summary,our findings suggested that miR-93 notably inhibited the growth and chemoresistance of breast cancer,by dual targeting E2F1 and CCND1 thereby downregulating their expression.These findings can provide not only potential therapeutic targets for breast cancer patients,but also new ideas for the development of chemotherapy sensitizers in the future.Part II Exploring differentially expressed miRNAs specifically related to the prognosis of triple-negative breast cancerTriple-negative breast cancer is the most aggressive molecular type of breast cancer.Due to the lack of early diagnostic markers and effective therapeutic targets,the prognosis of patients with triple-negative breast cancer is unfavorable.MicroRNAs(MiRNAs)have a wide range of biological functions in cancers as ideal biomarkers.Our present study aims to screen out differentially expressed miRNAs(DEmiRNAs)that specifically affect the prognosis of triplenegative breast cancer.Through the analysis of TCGA database,the largest database of cancer information,we screened out 4 candidate miRNAs,miR-135b-5p,miR-9-3p,miR-135b-3p and miR-455-5p.These 4 miRNAs were differentially expressed between triple-negative breast cancer and peritumoral normal samples,but no difference was shown between non-triple-negative breast cancer and peritumoral normal samples.Meanwhile,these 4 candidate miRNAs were specifically related to the prognosis of triple-negative breast cancer patients,and high expression of miR-135b-5p,miR-9-3p or miR-135b-3p indicated good prognosis,whereas high expression of miR-455-5p indicated poor prognosis;but these miRNAs were not correlated with the prognosis of non-triple-negative breast cancer patients.Through MTT and Colony formation assays,we found that overexpression of miR-135b-5p,miR-9-3p and miR-135b-3p significantly inhibited the proliferation and colony formation of triple-negative breast cancer cells,while overexpression of miR-455-5p promoted cell proliferation and colony formation.Moreover,the wound healing and Transwell assays showed that overexpression of miR-135b-5p,miR-9-3p and miR-135b-3p,as well as knockdown of miR-455-5p notably attenuated the migration ability of triple-negative breast cancer cells.These results indicated that these 4 candidate miRNAs were closely related to the proliferation and migration of triple-negative breast cancer cells.The Weighted Gene Co-expression Network Analysis(WGCNA)is a bioinformatic method for exploring the correlation between gene modules and phenotypes of interest,and for further exploring hub genes in gene modules.Firstly,we used Target Scan database to predict the downstream target genes of these 4 miRNAs.Then,we performed WGCNA for the predicted target genes.As a result,we identified 4 hub genes which were highly positive correlated with triple-negative breast cancer type out of potential target genes of 3 miRNAs with good prognosis(miR-135b-5p,miR-9-3p and miR-135b-3p),that is FOXC1,BCL11 A,FAM171A1 andRGMA.Next,we performed Dual-luciferase reporter assays and verified that FOXC1 and FAM171A1 were the direct targets of miR-9-3p,andRGMA is the direct target of miR-135b-3p.In view of the high expression of FOXC1,FAM171A1 andRGMA in triple-negative breast cancer cells,we knocked down them by siRNA,and the results showed that cell proliferation and migration were significantly inhibited.In summary,we screened out 4 DEmiRNAs(miR-135b-5p,miR-9-3p,miR-135b-3p and miR-455-5p)that specifically affected the prognosis of triple-negative breast cancer,and 4 hub genes(FOXC1,BCL11 A,FAM171A1 andRGMA)that were closely related to triple-negative breast cancer.More importantly,we verified that FOXC1 and FAM171A1 were direct targets of miR-9-3p,andRGMA was the direct target of miR-135b-3p.Furthermore,we identified their effect on the proliferation and migration of triple-negative breast cancer cells.These findings can provide ideas for the development of specific prognostic biomarkers and potential therapeutic targets for patients with triple-negative breast cancer.