| Breast cancer has become the most common cancer in the world,and ERα-positive breast cancer accounts for nearly 80% of all breast cancer,which has become an important public health problem threatening women’s life and health.Erα-positive breast cancer often plays an anti-tumor role by inhibiting the function of ERα in clinical adjuvant endocrine therapy.Tamoxifen is the most used drug in endocrine therapy.Since the introduction of tamoxifen,the prognosis of ERα-positive breast cancer patients has been significantly improved.However,in recent years,about 40% of patients treated with tamoxifen have relapsed,showing tamoxifen resistance,which seriously affects the prognosis of ERα-positive breast cancer patients.As a new type of biomarker,miRNAs have attracted more and more attention.It has been found that a variety of miRNAs can be used for the diagnosis,prognosis,and drug resistance prediction of ERα positive breast cancer,and participate in the signaling pathway network by targeting regulatory genes,which become potential targets for the treatment of ERα positive breast cancer.However,there is still a lack of miRNA markers that can reflect the progression from ERα positive breast cancer to tamoxifen resistance.Therefore,this study has screened miRNA markers related to ERα positive breast cancer and tamoxifen sensitivity,which is of great value for the early diagnosis and precise treatment of ERα positive breast cancer,and helps to supplement and improve the pathological mechanism of ERα positive breast cancer.Objects:To screen miRNA markers of ERα-positive breast cancer and tamoxifen resistance,analyze the application value of miRNA markers in the diagnosis and prognosis of ERα-positive breast cancer and the prediction of tamoxifen resistance,and explore the effect of miRNA on ERα-positive breast cancer and tamoxifen sensitivity.In addition,the potential mechanism of miRNA involved in the pathological process of ERα positive breast cancer is described,to provide scientific basis for early diagnosis and precise treatment of ERα positive breast cancer.Methods:The research objects of this study included ERα positive breast cancer patients from the online databases TCGA(n=733)and METABRIC(n=996),and 15 pairs of ERαpositive breast cancer tissue and adjacent tissue samples collected by our research group.Plasma samples from 53 ERα-positive breast cancer patients and 53 matched healthy controls were collected.Firstly,based on the online database TCGA,the differentially expressed miRNAs in ERα positive breast cancer tissues and adjacent tissues,and tamoxifen-resistant and non-resistant breast cancer tissues were screened.The relationship between miRNA expression level and prognosis of ERα positive breast cancer was analyzed by Kaplan-Meier,univariate and multivariate COX regression.Univariate and multivariate logistic analysis were used to analyze the correlation between miRNA expression level and clinical characteristics of ERα positive breast cancer,and to screen miRNA markers related to ERα positive breast cancer and tamoxifen resistance.Based on the tissue and plasma samples collected by our research group and METABRIC database,the relationship between this miRNA marker and the occurrence,prognosis,and tamoxifen resistance of ERα positive breast cancer was verified.ERα positive breast cancer cells MCF7 and T47 D,tamoxifen resistant cells LCC2,ERα negative breast cancer cells BT549,HS578 T and MDA-MB-231 were used.MTS assay,colony formation assay,wound healing assay,invasion assay,ELISA and Western Blot were used to investigate the effects of miRNA on the proliferation,migration,invasion,epithelial-mesenchymal transition,apoptosis and tamoxifen sensitivity of ERα positive and tamoxifen resistant breast cancer cells.At the in vivo level,tamoxifen resistant breast cancer cell line LCC2 was used to construct the tamoxifen resistant breast cancer orthotopic transplantation tumor mouse model,and the effect of miRNA on the growth of tamoxifen resistant breast cancer in mice and tamoxifen sensitivity was detected.RT-q PCR,Western Blot,Ch IP-q PCR,and dual luciferase reporter gene assay were used to explore the regulatory relationship between miR-153 and ERα.Results:1.Screening of miRNA markers associated with ERα-positive breast cancer and tamoxifen sensitivityIn TCGA,the expression of miR-153 was lower in ERα positive breast cancer tissues than in adjacent tissues,and the expression of miR-153 was lower in tamoxifen resistant breast cancer tissues than in non-resistant breast cancer tissues.The expression of miR-153 in tamoxifen resistant breast cancer cells LCC2 was significantly lower than that in non-drug resistant breast cancer cells MCF7(P<0.001).High expression of miR-153 in breast cancer tissues was an independent protective factor for the prognosis of patients with ERα positive breast cancer(HR=0.56,95%CI=0.31-0.99,P=0.047).The expression level of miR-153 in cancer tissues was correlated with T stage,histological type,and recurrence status in ERα positive breast cancer patients.The expression level of miR-153 in ERα positive breast cancer patients in T2/T3/T4 stage was lower than that in T1 stage(OR=0.61,95%CI=0.44-0.86,P < 0.05).P=0.005).The expression of miR-153 in ERα-positive breast cancer patients with invasive lobular carcinoma was higher than that in patients with invasive ductal carcinoma(OR=2.20,95%CI=1.17-4.15,P=0.015).The expression of miR-153 in breast cancer tissues of ERα positive breast cancer patients with recurrence was lower than that of patients without recurrence(OR=0.38,95%CI=0.16-0.91,P=0.029).2.Population validation of the association between miR-153 and ERα-positive breast cancerBased on the data of ERα positive breast cancer patients and healthy controls collected by our research group,it was found that the expression of miR-153 in the cancer tissues of ERα positive breast cancer patients was lower than that in the adjacent tissues,and the level of miR-153 in the plasma of ERα positive breast cancer patients was lower than that of healthy controls(P<0.01).ROC curve analysis showed that the level of plasma miR-153 could be used to distinguish ERα positive breast cancer patients from healthy controls(AUC=0.808,P<0.001).The plasma level of miR-153 in tamoxifen-resistant breast cancer patients was significantly lower than that in tamoxifen-non-resistant breast cancer patients(P<0.01).The expression level of plasma miR-153 was only related to the age of diagnosis in ERα positive breast cancer patients.The expression level of plasma miR-153 in ERα positive breast cancer patients aged >50 years was higher than that in patients aged ≤50 years,and the difference was statistically significant(OR = 5.7,95%CI = 1.12-28.9;P = 0.036).Based on METABRIC database analysis,it was found that the overall survival rate of ERα positive breast cancer patients with high expression of miR-153 in cancer tissue was significantly higher than that of patients with low expression(n=996)(HR=0.61,95%CI=0.47-0.81,P=0.002).The overall survival rate of breast cancer patients receiving endocrine therapy with high expression of miR-153 in cancer tissues was significantly higher than that of patients with low expression(n=779)(HR=0.61,95%CI=0.47-0.81,P<0.001).3.Effects of miR-153 on the function of ERα positive breast cancer cellsOverexpressed miR-153 significantly inhibited cytoactive,motility,invasion and epithelial-mesenchymal transition,and enhanced cell apoptosis of MCF7 and T47 D cell(P<0.05).Inhibition of miR-153 significantly enhanced the cytoactive,motility,invasion,and epithelial-mesenchymal transition of MCF7 and T47 D cells(P<0.05),and decreased the apoptosis of MCF7 and T47 D cells(P<0.05).4.Effects of miR-153 and tamoxifen on tamoxifen sensitivity in drug-resistant breast cancerWithout over-expression of miR-153,there were no significant differences in cytoactive,motility,invasion,epithelial-mesenchymal transition,and apoptosis between tamoxifen treated and untreated LCC2 cells(P>0.05).Overexpressed miRX153 directly inhibited the cytoactive,motility,invasion,and epithelial-mesenchymal transition of LCC2 cells,and promoted cell apoptosis(P<0.05).On the other hand,overexpressedmiR-153 significantly reduced the cytoactive,motility,invasion,and epithelial-mesenchymal transition of LCC2 cells treated with tamoxifen compared with untreated LCC2 cells(P<0.05),and significantly enhanced the apoptosis ability(P<0.05).5.Study on the regulation between miR-153 and ERαThe analysis results based on TCGA showed that the expression of miR-153 was negatively correlated with ESR1 in breast cancer tissues(r=-0.089,P=0.022).The plasma level of miR-153 was significantly lower in patients with ERα positive breast cancer than that in patients with ERα negative breast cancer(P<0.01).The expression of miR-153 in ERα positive breast cancer cells LCC2,MCF7 and T47 D was significantly lower than that in ERα negative breast cancer cells BT549,HS578 T and MDA-MB-231(P<0.001).After estrogen deprivation for 1 to 3 days,the expression of TFF1 in LCC2,MCF7 and T47D cells was significantly decreased(P<0.05),while the expression of miR-153 was significantly increased(P<0.05).However,treatment of LCC2,MCF7 and T47 D cells with 1n M estrogen for 6,12,24 and 48 hours significantly increased the expression of TFF1(P<0.05)and decreased the expression of miR-153(P<0.05).Ch IP-q PCR assay confirmed that there were six binding sites between ERα and miR-153 in the promoter region(P<0.05).After exploring the regulatory effect of miR-153 on ERα,it was found that the expression of ESR1 and TFF1 was significantly down-regulated after overexpression of miR-153 in LCC2,MCF7 and T47 D cells(P<0.05).After miR-153 inhibition in LCC2,MCF7 and T47 D cells,the expression of TFF1 was up-regulated in all three cell lines,while ESR1 expression was significantly up-regulated only in LCC2 cells(P<0.05),indicating that miR-153 regulates the activity of ERα in ERα positive breast cancer cells.The target genes of miR-153 were predicted by online databases Target Scan and miRDB.After intersecting with the genes involved in endocrine resistance through ERα pathway,8 candidate target genes of miR-153 were predicted.GRB2(HR=1.41,95%CI=1.01-1.97,P=0.040)and IGF1R(HR=1.48,95%CI=1.04-2.10,P=0.028)were associated with tamoxifen resistance.Moreover,the expression of IGF1 R in tamoxifen resistant breast cancer cell LCC2 was higher than that in non-drug resistant cell MCF7(P<0.01),while the expression of miR-153 in LCC2 cells was lower than that in MCF7 cells(P<0.001).Dual luciferase reporter gene assay confirmed that miR-153 could bind to the 3’UTR region of IGF1 R in 293 T cells(P<0.01)and MCF7 cells(P<0.01).Overexpressed miR-153 significantly retained the expression of IGF1 R in LCC2,MCF7 and T47 D cells(P<0.05),while inhibition of miR-153 significantly accelerated the expression of IGF1R(P<0.05),which confirmed the target gene of miR-153.The expression levels of IGF1 R,ERα,p-Ser118 ERα and p-Ser167ERα were significantly decreased after miR-153 was overexpressed in LCC2,MCF7 and T47D cells(P<0.05).However,after simultaneous overexpression of miR-153 and IGF1 R,the protein expression levels of IGF1 R,ERα,p-Ser118 ERα and p-Ser167 ERαin LCC2,MCF7 and T47 D cells were significantly higher than those in the miR-153 overexpression group(P<0.05).These results indicated that miR-153 could regulate the phosphorylation of ERα by targeting IGF1 R.6.miR-153-ERα feedback loop mediates tamoxifen resistance in ERα-positive breast cancerThe expression levels of ERα encoding gene ESR1(P<0.001),ERα activation marker gene TFF1(P<0.001)and TFF1/ESR1(P<0.001)in tamoxifen resistant cell LCC2 were significantly higher than those in non-tamoxifen resistant cell MCF7.In addition,the protein expression levels of IGF1 R,ERα,P-Ser118 ERα and P-Ser167ERα in LCC2 cells were higher than those in MCF7 cells.These results suggest that ERα phosphorylation is associated with tamoxifen resistance,and miR-153-ERαfeedback loop mediates tamoxifen resistance in ERα-positive breast cancer.Conclusion:1.The expression level of miR-153 in ERα positive breast cancer tissues and plasma is decreased,and the overall survival of ERα positive breast cancer patients is shortened.miR-153 can be used as a potential marker for the prognosis of ERα positive breast cancer.2.High level of miR-153 can inhibit the cytoactive,motility,invasion,and epithelialmesenchymal transition of ERα positive and tamoxifen resistant breast cancer cells,promote cell apoptosis,and inhibit the growth of tamoxifen resistant breast cancer in mice.3.The expression level of miR-153 is decreased in tumor tissues and plasma of tamoxifen-resistant breast cancer patients.High level of miR-153 can enhance the sensitivity of drug-resistant breast cancer cells and tumor-bearing mice to tamoxifen.4.ERα regulates the expression of miR-153,and miR-153 participates in the phosphorylation of ERα by regulating its target gene IGF1 R.ERα-miR-153-IGF1RERα feedback loop mediates the occurrence and development of ERα positive breast cancer and tamoxifen resistance. |
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