LncRNA CBR3-AS1 Regulates Of Breast Cancer Drug Sensitivity As A Competing Endogenous RNA Through The JNK1/MEK4-mediated MAPK Signal Pathway

Objective:Breast cancer is one of the malignant tumors that women are susceptible to,and its incidence is increasing year by year,which seriously endangers the health of women.Chemotherapy is an important mean of breast cancer treatment,but the drug resistance of tumors during chemotherapy has seriously affected the success rate of chemotherapy.Therefore,in-depth study of breast cancer drug resistance mechanism is a scientific problem that needs to be solved urgently.CBR3-AS1(CBR3 antisense RNA 1),also known as PlncRNA-1,is located on chromosome 21q22.12 and is an unspliced,polyadenylated transcript with a length of 1573nucleotides.It has been reported that CBR3-AS1 is highly expressed in cancer tissues such as prostate cancer,liver cancer and esophageal squamous cell carcinoma,and related to the poor prognosis of patients.Fang Z et al.found that CBR3-AS1 promoted the development and proliferation of prostate cancer by acting on androgen receptors.Yang Q et al.found that CBR3-AS1 regulated cell cycle,proliferation,autophagy and apoptosis through HER-2 pathway in prostate cancer.However,there is no report that CBR3-AS1regulates breast cancer resistance.The research results of this project will use the new perspective of ceRNA to reveal the new mechanism of breast cancer cell resistance,and clarify that CBR3-AS1 is transcriptionally activated by NRF2,and then dual-targeted regulation of MEK4 and JNK1mediated breast cancer resistance by ceRNA The research results of this topic will provide new ideas for elucidating the molecular mechanism of breast cancer resistance,and provide theoretical and experimental basis for discovering new target genes and action sites that can effectively reverse breast cancer resistance.Method:1.High-throughput cell chip Compare the expression of lnc RNA in breast cancer drug-sensitive cell MCF-7 and breast cancer drug-resistant cell MCF-7/ADR through high-throughput cell chip.2.Cell model construction The differential expression model of breast cancer cells CBR3-AS1 was constructed by transfection of CBR3-AS1 overexpression plasmid or si-CBR3-AS1 siRNA.miR-25-3p mimic or miR-25-3p inhibitor was added in breast cancer cell to establish miR-25-3p differential expression model;JNK1 inhibitor constructed a JNK1silence model.2.qRT-PCR and Western Blot qRT-PCR was used to detect miR-25-3p,JNK1/MEK4mRNA expression changes of breast cancer cells intervened CBR3-AS1,miR-25-3p or JNK1,CBR3-AS1;Western Blot was used to detect protein expression changes of JNK1,MEK4 and P-gp in the model.3.Detection of cell drug accumulation,drug toxicity and apoptosis The CCK-8 method was used to detect the changes in cell survival rate after intervention with CBR3-AS1 and treatment with adriamycin;flow cytometry was used to analyze intracellular drug accumulation,APC/7-changes of apoptosis after AAD double staining.4.RNA pulldown MCF-7 cells were transfected with CBR3-AS1 overexpression plasmid for 48 hours and then the cell suspension was extracted.Combine with streptomycin-labeled CBR3-AS1-wild and CBR3-AS1 antisense sequence probes and incubate overnight.After elution,qRT-PCR detected whether miR-25-3p can be pulled down and its expression changes.5.In vivo verification of transplanted tumors in nude mice.Healthy BALB/C athymic nude mice were inoculated subcutaneously into the right upper armpit of nude mice with 5×10~6cells to observe tumor growth,measure tumor size,weigh tumor,and draw transplanted tumors growth curve.RNA and protein were extracted from the tissues of transplanted tumors to perform qRT-PCR and Western Blot to analyze the expression changes of JNK1/MEK4 protein in the tumor tissues of nude mice in each group.6.In situ hybridization and immunohistochemistry In situ hybridization was used to detect the expression of CBR3-AS1 and miR-25-3p in breast cancer tumor tissue chips;immunohistochemistry was used to detect the expression of JNK1 and MEK4 in colorectal cancer tissues,and perform expression correlation analysis.7.Data analysis Use SPSS19.0 statistical software package for statistical analysis(SPSS Inc,Chicago,USA).All data were expressed as mean±standard deviation(x±SD),p<0.05indicated statistical significance.Comparisons between groups were performed by independent sample t-test or one-way analysis of variance(One-Way ANOVA);?~2 test was used to analyze the correlation between CBR3-AS1 expression and clinical pathological parameters of breast cancer;Kaplan-Meier was used to draw survival curves.Result:1.CBR3-AS1 is up-regulated in drug-resistant breast cancer cells,which is related to drug sensitivity and poor prognosis of breast cancer(1)By analyzing the differential expression profile of lnc RNA chip in adriamycin-resistant breast cancer cell MCF-7/ADR and its parental breast cancer cell MCF-7,it was found that CBR3-AS1 was highly expressed.The expression data of lnc RNA in various breast cancer cells was obtained through the CCLE database,and the IC50 value of ADR in these breast cancer cells was obtained from the GDSC database.The results of the combined analysis showed that the expression of CBR3-AS1 was positively correlated with the resistance of breast cancer cells to ADR(p<0.05,r>0.3).Four breast cancer cell lines were detected by qRT-PCR,and CBR3-AS1 was expressed in breast cancer cells.CBR3-AS1 was up-regulated with the increase of cell resistance(p<0.05),and the expression was most significant in MCF-7/ADR cells.(2)Database analysis of high expression of CBR3-AS1 was related to poor prognosis of breast cancer patients.The mRNA expression profile and clinical information of breast cancer patients were obtained from the GSE20685 dataset of the GEO dataset.The results showed that among breast cancer patients received chemotherapy,patients with high expression of CBR3-AS1 had a poor prognosis.In the TCGA database,CBR3-AS1 was up-regulated in breast cancer tissues compared with adjacent normal tissues.Gene Set Enrichment Analysis(GSEA)showed that CBR3-AS1 was enriched in the ABC transporter signal transduction pathway.In addition,the expression of CBR3-AS1 was significantly correlated with the mRNA expression of p-glycoprotein(P-gp,ABCB1)and breast cancer resistance protein(BCRP,ABCG2).The log-rank test of the OS curve of breast cancer patients in TCGA also showed that,compared with the low expression of CBR3-AS1,the high expression of CBR3-AS1 was significantly correlated with poorer OS.(3)We evaluated the expression of CBR3-AS1 in 96 breast cancer samples from the First Affiliated Hospital of China Medical University through ISH.There was a significant correlation between the level of CBR3-AS1 and tumor size and pathological stage.The log-rank test of the overall survival curve of these breast cancer patients showed that the increased expression of CBR3-AS1 was significantly related to the poorer OS of breast cancer.(4)After intervention of CBR3-AS1,the phenotype of breast cancer cells changed.After overexpression of CBR3-AS1,the drug sensitivity of MCF-7 cell was significantly reduced(p<0.001);on the contrary,the drug sensitivity of cells was significantly increased after silencing CBR3-AS1(p<0.001).The results of the drug accumulation experiment showed that the drug accumulation in MCF-7 cell decreased after overexpression of CBR3-AS1,while the drug accumulation in the cells was significantly increased after silencing CBR3-AS1(p<0.05,p<0.01).Flow cytometry analysis found that after overexpression of CBR3-AS1,the percentage of apoptosis could be significantly reduced after using doxorubicin for 48 hours(p<0.01).The above results indicated that CBR3-AS1 was related to drug sensitivity and poor prognosis of breast cancer.2.The mechanism by which CBR3-AS1 regulates breast cancer drug sensitivity is through the MAPK signal pathway mediated by JNK1/MEK4(1)The direct binding effect of CBR3-AS1 and miR-25-3p.The qRT-PCR experiment showed that the expression of miR-25-3p was down-regulated in MCF-7/ADR cells.RNA pulldown experiment washed probe-agarose magnetic beads and collected RNA binding complex;qRT-PCR detected the expression of miR-25-3p and CBR3-AS1 probe binds more miR-25-3p compared with CBR3-AS1 antisense strand probe showing that CBR3-AS1 had a direct binding effect with miR-25-3p.(2)The expression of JNK1 and MEK4 changed after intervention of CBR3-AS1.The miRDB website predicted that miR-25-3p had binding sites with JNK1 and MEK4 mRNA3'UTR.Luciferase reporter gene experiment confirmed that miR-25-3p could bind to JNK1 and MEK4 mRNA 3'UTR.TCGA database analysis showed a significant positive correlation between the expression of CBR3-AS1 and JNK1 and MEK4 mRNA(P<0.05,R<0.1).(3)qRT-PCR analysis and Western blot analysis showed that after addition of doxorubicin,the overexpression of CBR3-AS1 increased the mRNA and protein expression of JNK1and MEK4 in the cells,while silencing CBR3-AS1 reduced the expression of CBR3-AS1.In addition,overexpression of miR-25-3p would reduce the expression of JNK1 and MEK4in MCF-7/ADR cells,while silencing the expression of miR-25-3p might increase the expression of JNK1 and MEK4 in MCF-7/ADR cells.(4)To verify the effect of CBR3-AS1 on cell drug sensitivity in nude mice transplanted tumors,MCF-7 cells were subcutaneously injected to prepare nude mouse transplanted tumor models.Overexpression of CBR3-AS1 resisted the inhibitory effect of ADR on tumor cells.The effect was reversed by the JNK1 inhibitor.In addition,JNK1 inhibitors could reverse the effect of CBR3-AS1 on increasing tumor size and weight.Western blot and immunohistochemical experiments showed that the overexpression of CBR3-AS1increased the expression of JNK1 and MEK4.These results indicated that CBR3-AS1promoted ADR resistance in breast cancer through JNK1/MEK4 in vivo.Conclusion:1.CBR3-AS1 is expressed in breast cancer drug-resistant cells,and can be used as an oncogene to enhance cell drug sensitivity and inhibit cell apoptosis.After analyzing the correlation between CBR3-AS1 and clinicopathological parameters and prognosis,it suggests that when CBR3-AS1 is highly expressed,the disease-free survival and overall survival of patients are shortened,and the prognosis is poor.2.Comprehensive analysis from a large sample database shows that CBR3-AS1 is negatively correlated with miR-25-3p expression,and is significantly positively correlated with JNK1/MEK4 expression.Experiments confirmed that CBR3-AS1 has a targeted binding effect with miR-25-3p,and miR-25-3p also has a targeted binding effect with JNK1/MEK4 3'UTR.3.The overexpression of CBR3-AS1 increases the mRNA and protein expression of JNK1and MEK4 in cells,while the overexpression of miR-25-3p offsets this effect.At the same time,the weakening of cell drug sensitivity caused by CBR3-AS1 can also be miR-25-3p offsets.