MicroRNA-224 Promotes Cell Proliferation,Invasion,Migration And Drug Resistance Through Downregulation Of Caspase-9 In Triple-Negative Breast Cancer(TNBC)

Background and Objective:Breast cancer is one of the hereditary diseases responsible for high mortality rates among women worldwide.Despite the availability of advanced diagnostic tools and treatment strategies,the incidence of breast cancer is increasing every year and has become the leading cause of cancer-related death among women.Metastasis and local recurrence are the major causes of breast cancer treatment failure and patient death,and early prediction of metastasis can improve patient survival.Triple-negative breast cancer(TNBC)is a heterogeneous subtype of breast cancer,accounting for 10-20% of invasive breast cancers,but is the major subtype of death in breast cancer patients.Compared with other types of breast cancer,TNBC is more common in young women and exhibits more aggressive clinical behavior and unique metastatic pattern,with short-period recurrence,rapid progression,short survival,and poor prognosis.Therefore,the discovery of specific biomarkers associated with early diagnosis and prediction of metastatic prognosis and response to treatment is an urgent need.Micro RNAs(miRNAs)are a group of small,approximately 22 nucleotide long,non-coding RNA molecules that negatively regulate gene expression by preferentially binding to specific sequences on the 3'-untranslated region(3'-UTR)of their target messenger RNAs(m RNAs).Micro RNAs are key regulators of m RNA expression in both normal and abnormal biological processes,including cancer.miRNAs can be involved in cancer growth and metastasis as oncogenes or tumor suppressor genes and play key roles in cancer prognosis prediction,diagnosis,and therapeutic pathways.Mi R-224 is an important member of the Micro RNA family.Published studies have identified miR-224 is aberrantly expressed in several human tumor types and involved in tumor invasion,metastasis,and Drug resistance.miRNAs represent an emerging group of molecules that play a key role in disease development,and therapeutic strategies based on regulating the expression level of miRNAs and identifying their targets are promising approaches for miRNAs-based BC therapy.Apoptosis is a form of programmed cell death that plays an important role in embryonic development,immune system function,and maintenance of tissue homeostasis.Disturbance of the apoptotic pathway is also an important factor in tumorigenesis and its biological behavior.Caspases are a family of proteases that play a central role in the activation and propagation of apoptotic signals.Caspases-9 is particularly important in the endogenous pathway of apoptosis.It has been found that miRNA biogenesis is closely related with the function of caspases.Caspases can regulate miRNA biogenesis by cutting and inactivating key enzymes of miRNA production.On the other hand,many miRNAs regulate apoptosis by targeting apoptotic factors,including caspases,and negatively regulate their expression.However,the role and significance of the relationship between Micro RNA-224 and CASP9 in the development of breast cancer has not been reported.In present study,we detected the expression of miR-224 in breast cancer cells and analyzed the relationship between miR-224 and clinicopathological parameters of breast cancer as well as patient survival and prognosis.The effects of miR-224 expression on proliferation,migration,invasion,apoptosis,and drug sensitivity of TNBC cells were investigated by in vitro and in vivo experiments.Bioinformatics screening,luciferase assay,and Western blot were used to preliminarily explore the relationship and mechanism of targeted regulation between miRNA-224 and CASP9.This study is expected to provide new biomarkers for the treatment and prognosis of TNBC.Methods:(1)Fifteen cases for each group of TNBC,Luminal A/B and Her-2 subtype breast cancer tissues and paraneoplastic tissues with clear pathological diagnosis were collected,and the expression levels of miR-224 in different subtypes of breast cancer cell lines and tissues were detected by q RT-PCR.(2)Sixty-four patients with complete postoperative pathological,immunohistochemical diagnosis and clinical data of TNBC in The second hospital of Jilin Universityfrom January 2012 to December 2016 were collected.The miR-224 expression level was divided into high expression group and low expression group by the mean value,and statistical analysis was performed by comparing the clinicopathological characteristics of the patients and the level of miR-224.(3)In vitro experiments to assess the biological function of miR-224 in TNBC cells.The TNBC cell lines MDA-MB-231 and MDA-MB-468 were first transfected by anti-miR negative control(NC)and anti-miR-224.And then MTS assay was used to detect cell proliferation activity.Transwell assay was used to detect cell migration and invasion ability.(4)In vivo experiments to assess the biological function of miR-224 in TNBC.Six nude mice were randomly divided into two groups,a control group and a miR-224 inhibition group,and xenograft tumors were planted orthotopically.After 30 days of xenograft,the tumors were removed and the weight and volume of the tumors in the experimental group and the control group were measured and compared.Immunohistochemical staining was performed afterwards.(5)The target genes of miR-224 were preliminarily screened by bioinformatics software,and further analyzed by KEGG and GO analysis.The dual-luciferase reporter gene system and Western blot were used to further verify the regulatory role and mechanism of miR-224 and the target gene CASP9.(6)Drug sensitivity experiments investigated the effect of miR-224 on the chemotherapeutic drug sensitivity of breast cancer cells,and the effect of miR-224 on apoptosis was detected by flow cytometry.Western blot was employed to investigate the mechanism of miR-224 targeting CASP9 in regulating chemotherapeutic drug sensitivity.Results:(1)Compared with normal breast epithelial cells,miR-224 was overexpressed in most breast cancer cell lines and tissues(p < 0.05),and the highest level of miR-224 expression was found in TNBC(p < 0.01).(2)Analysis of the relationship between different miR-224 expression levels and clinicopathological factors in patients with TNBC revealed that high miR-224 expression was associated with histological grade(p=0.007),lymph node metastasis(p<0.001),clinical stage(p=0.005),Ki-67 expression(p=0.004)and vascular involvement(p<0.001)was positively correlated.Survival analysis showed that patients in the high miR-224 expression group had significantly shorter OS,with a median survival time of only 60 months(p<0.01).Cox regression analysis suggested that high miR-224 expression(p=0.037)was an independent risk factor for poor prognosis in TNBC patients.(3)In vitro experiments were conducted to verify the effect of miR-224 on the biological functions of breast cancer cells.The results showed that the expression of miR-224 was decreased significantly in MDA-MB-231 and MDA-MB-468 cells after transfecting with anti-miR-224.Down regulation of miR-224 significantly reduced the proliferation,migration and invasion of TNBC cells by MTS and Trans-well assay.(4)In vivo experiments to verify the effect of miR-224 on the biological function of breast cancer cells.The results showed that the volume and weight of the xenograft tumors of nude mice in the anti-miRNA-224 group were significantly smaller than those of the control group.Further immunohistochemical staining of xenograft tumor tissues revealed that down-regulation of miR-224 expression could suppress mammary tumor cell proliferation(p < 0.05)and angiogenesis(p < 0.01).(5)CASP9 was identified as a potential target of miR-224 in TNBC by bioinformatics techniques and GO and KEGG analysis.The direct target relationship between miR-224 and CASP9 was further verified by luciferase assay and Western blot detection.The downstream cascade reaction of CASP9 and the activities of Caspase-3and Caspase-7 were also detected,and the activities of Caspase-3 and Caspase-7 were found to be enhanced.(6)Drug sensitivity experiments revealed that anti-miRNA-224 could increase the sensitivity of breast cancer cells to 5-FU treatment.Flow cytometry and Western blot assay showed that anti-miR-224 combined with 5-FU could induce apoptosis of cancer cells by elevating the expression of CASP9.Up-regulation of miR-224 exert antiapoptotic effect and involved in tumor progression and Drug resistance.Conclusion:(1)Compared with normal breast epithelial cells,miR-224 was overexpressed in most breast cancer cell lines and tissues,and the highest level of miR-224 expression was found in TNBC.(2)High miR-224 expression was positively correlated with histological grade,lymph node metastasis,clinical stage,Ki-67 expression,vascular involvement,and survival status;miR-224 overexpression was associated with OS shortening in TNBC patients and was an independent risk factor for poor prognosis.(3)Downregulation of miR-224 significantly inhibited cell proliferation,migration,and invasion.(4)CASP9 is a potential target of miR-224 in TNBC cells.In TNBC,miR-224 is overexpressed and directly regulates apoptosis by downregulating CASP9,which plays an important role in breast cancer development,invasion,migration and Drug resistance.In summary,miR-224 is involved in tumor formation,invasion,migration and drug sensitivity in triple-negative breast cancer through down-regulation of CASP9.Our results suggest that the miR-224/CASP9 axis plays an important role in the progression of TNBC,and Micro RNA-224 may be an important potential therapeutic target for triple-negative breast cancer.