Study On Mechanisms Of MiR-30c And MiR-181a Regulating Tumor Adriamycin Resistance And Development

Objective: Malignant tumors seriously endanger human physical and mental health and quality of life.In the world,the incidence and mortality of malignant tumors are the highest of all kinds of diseases,showing an upward trend year by year.It has become one of the diseases most burdened by human diseases.Tumors are affected by both genetic and environmental factors.The mechanism of their occurrence and development is not yet clear.Chemotherapy plays an important role in the treatment of malignant tumors and has been widely used in clinical treatment.However,its efficacy is often affected by endogenous or acquired multidrug resistance and reduces the sensitivity of chemotherapy drugs.Therefore,the search for malignant tumor chemotherapy resistance to development and development-related factors,figuring out its mechanism may become the key to the treatment of malignant tumors.MicroRNA(miRNA)is a class of non-encoded single-stranded RNA molecule that is encoded by endogenous genes,approximately 20 to 22 nucleotides in length.It is highly conserved during evolution,negatively by degrading the target gene mRNA and inhibiting translation by specifically binding to the 3'UTR region of the target gene mRNA in order to regulate downstream target gene expression.miRNA regulates more than 60% of protein coding genes and participates in almost all known cellular processes,including embryo development,cell proliferation and apoptosis,cell differentiation,and fat metabolism and a series of important life activities.However,functions such as cell proliferation and apoptosis often occur aberrantly in tumor cells.Therefore,it is indicated that the abnormal expression of miRNA is closely related to the occurrence and development of tumors.At the same time,it has been reported that the expression levels of many mi RNAs are closely related to the development of drug resistance in tumor cells.Abnormal expression of miRNAs leads to the loss or enhancement of mi RNA function,which leads to changes in the expression of target proteins.If such target proteins are involved in cell-to-drug reactions such as drug targets,drug transporters,apoptosis and repair and other related proteins,such proteins will lead to changes in the sensitivity of chemotherapy drugs.In this study,breast cancer and colorectal cancer,which are the second and thirdlargest fatalities in tumors,were selected for the study.MiR-30 c and miR-181 a weretaken as the starting point.The main research purposes included two aspects:(1)Study on the role of mi R-30 c in adriamycin resistance in p53 mutant breastcancer and participate in the regulatory mechanism(2)Study on the relationship between miR-181 a and the occurrence,developmentand prognosis of colorectal cancerThrough the above studies,it is proposed to provide a theoretical basis for theclinical treatment of adriamycin resistance in breast cancer and provide potentialmolecular markers for the prognosis of colorectal cancer.Methods:1.Bioinformatics analysis The Cancer Genome Atlas(TCGA)database was used to analyze the different expression of miRNA between tumor tissues and normal tissues.Different expression of mi R-30 c between p53 wild-type breast cancer tissues and p53 mutant breast cancer tissues were examined.TargetScan,RNA2 and RNAhybrid were used to predict miRNAs and binding energies targeted to the 3?-UTR region of REV1 and FANCF;the Gene regulation website predicts the presence of a transcription factor that binds to the miR-30 c promoter region.2.Cell model construction Wild type p53 cell model and mutant p53 cell model were constructed by transfecting p53 cDNA with liposome in p53 mutant cell line and p53R280 K plasmid in p53 wild type cell line respectively;Mi R-30 c mimic and miR-30 c inhibitor were constructed to over-expression and silence miR-30 c cell model;p53 shRNA plasmid was transfected into breast cancer cells to construct p53-silencing cell model.3.Detection of DNA damage repair Single cell gel electrophoresis and chromosome fragmentation analysis were used to detect DNA damage after treatment with doxorubicin in breast cancer cells;immunofluorescence staining was used to detect nuclear focus of ?-H2 AX and detect DNA damage after treatment with doxorubicin in breast cancer cells.4.qRT-PCR and Western Blot qRT-PCR detected miR-30 c expression in breast cancer cells after ADR treatment,overexpression of p53 and intervention of miR-30c;Western Blot analyzed REV1 and FANCF protein levels in breast cancer cells and nude mice transplant tumors after overexpression and silencing miR-30 c.5.Luciferase reporter assays and chromatin immunoprecipitation assays By luciferase reporter assays,we determine whether miR-30 c binds directly to the 3'UTR regions of REV1 and FANCF mRNA and detect direct binding of p53 to the miR-30 c promoter region.The chromatin immunoprecipitation assay detects whether the p53 and miR-30 c promoter regions bind directly in vivo.6.Nude mouse experiments 4-6 week-old female athymic BALB/C nude mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.,both at the China Medical University No Specific Pathogen Free(SPF)Experimental Animal Center Aseptically caged.The tumor models of MCF-7 and MDA-MB-231 breast cancer cells in nude mice were established.The tumor volume and tumor weight were measured after intratumoral injection of miR-30 c agomir.7.Cell proliferation and drug susceptibility testing CCK-8 was used to detect the sensitivity of breast cancer cells to doxorubicin.8.In situ hybridization and immunohistochemistry In situ hybridization was used to detect the expression of miRNA in tumor tissue.The expression of REV1,FANCF and p53 in breast cancer tissues was detected by immunohistochemistry.9.Data analysis SPSS 20.0 statistical software was used for statistical analysis(SPSS Inc,Chicago,IL,USA)Data were expressed as mean ± standard deviation(x ± s)of three independent experiments.One-Way ANOVA was used for comparison between groups and pairwise comparison using Turkey's post hoc Test method for statistical analysis;Pearson 2 and Fisher's test to compare the expression of miR-30 c and miR-181 a with clinicopathological parameters.Kaplan-Meier Methods and log-rank tests were used to assess survival probabilities.Multivariate Cox regression models were used to assess the association between multivariate and prognosis.p<0.05 indicated significant difference with statistical significance.Results:1.mi R-30 c increases doxorubicin drug sensitivity by inhibiting DNA damage repair in breast cancer cells(1)Doxorubicin resistance to p53 mutant breast cancer cells was associated with high expression of FANCF and REV1.p53 mutant breast cancer cells were more resistant to ADR than p53 wild-type cells(p<0.05),suggesting that p53 mutant breast cancer cells were more resistant to ADR than p53 wild-type cells.After ADR treatment with p53 wild-type and p53 mutant cells,DNA damage in p53 wild-type cells was significantly greater than p53 mutant cells(p<0.05)and expression of REV1 and FANCF was significantly higher in p53-mutant cells(p<0.05),suggesting p53 mutant breast cancer cells were closely related to doxorubicin resistance and high expression of FANCF and REV1.(2)miR-30 c inhibits REV1 and FANCF expression by targeted inhibition of the 3?-UTR region of REV1 and FANCF.Bioinformatics predicted that miR-30 family simultaneously targets the 3?-UTR region of REV1 and FANCF and overexpresses miR-30 family in breast cancer cells.After the overexpression of miR-30 c,the down-regulation of REV1 and FANCF mRNA and protein levels were most significant(p<0.01).Luciferase reporter assay confirmed that miR-30 c directly bound to REV1 and FANCF mRNA 3?-UTR regions(p<0.01),suggesting that miR-30 c inhibits REV1 and FANCF expression through targeted inhibition of the 3?-UTR region of REV1 and FANCF.(3)miR-30 c increased ADR antitumor effect in vivo and in vitro,inhibiting tumor growth.The expression of miR-30 c in p53 wild type cells was significantly higher than that in p53 mutant cells(p<0.05).After ADR treatment,the expression of miR-30 c in p53 wild-type cells increased(p<0.05)while the expression of p53 mutant cells had no significant changes,suggesting that there was a positive correlation between miR-30 c expression and ADR sensitivity.After overexpression of miR-30 c in p53 mutant cells,the sensitivity to ADR was significantly increased(p<0.05).After silencing miR-30 c in p53 wild-type cells,the sensitivity to ADR was significantly decreased(p<0.05).In nude mice,over-expression of mi R-30 c significantly slowed the growth of transplanted tumors in nude mice,and,compared with the ADR alone group,ADR treatment at the same time overexpressed 30 c more significantly inhibited tumor growth(p<0.05).And in 30c-overexpressing xenografts,the expression of REV1 and FANCF decreased significantly(p<0.01).The above results suggested that miR-30 c enhanced the efficacy of ADR therapy in vivo by inhibiting the expression of REV1 and FANCF.(4)miR-30 c affected breast cancer cell DNA damage repair by inhibiting the expression of REV1 and FANCF.In the TCGA database,there are more DNA mutations in patients with high expression of miR-30 c in patients with high expression of miR-30 c breast cancer,suggesting that miR-30 c promotes DNA damage and increases gene instability(p<0.05).Overexpression of mi R-30 c promoted ADR-induced chromosome breaks in breast cancer cells compared to the NC group(p<0.01).Single cell gel electrophoresis experiments confirmed that miR-30 c overexpression inhibited DNA repair compared with the NC group and promoted ADR-induced DNA damage in breast cancer cells(p<0.05).After miR-30 c overexpression,both ADR treatment and mir-H2 AX nuclear focus were significantly increased compared to the mi R-30 c alone group,indicating that mi R-30 c promoted ADR-induced DNA damage(p<0.01).2.p53 activates miR-30 c transcriptional regulation REV1 and FANCF expression(1)p53 can bind directly to the promoter region of miR-30 c.We predicted that there was a binding site in the promoter region of p53 and miR-30 c through the bioinformatics website.(2)p53 activates miR-30 c transcription by binding to the miR-30 c promoter region.Luciferase reporter assays confirmed the direct binding of p53 to the miR-30 c promoter region,and subsequent CHIP experiments confirmed direct binding of p53 and miR-30 c promoter regions in vivo.ADR activated p53 wild-type cell p53 expression,meanwhile miR-30 c expression was significantly increased(p<0.05),while p53 mutant cells had no significant changes(p>0.05).After overexpression of p53 cDNA,the expression of miR-30 c in MCF-7 cells and MDA-MB-231 cells was significantly increased(p<0.05).This suggested that p53 activates miR-30 c transcription by directly binding to the miR-30 c promoter region.(3)p53 affects REV1 and FANCF expression through activation of miR-30 c transcription.After overexpression of p53 in breast cancer cells,the expression of REV1 and FANCF were significantly decreased(p<0.01).After treatment with ADR,the expression of REV1 and FANCF in p53 wild type cells was decreased(p<0.01).There was no significant change in p53 mutant cells(p>0.05).MiR-30 c inhibited the effect of p53 wild-type cells on REV1 and FANCF after silencing.Consistent with the changes in cell levels,the expression of REV1 and FANCF in the MCF-7 tumors treated with ADR decreased(p<0.01)but the protein expression in MDA-MB-231 cells did not change significantly(p>0.05).3.Low expression of miR-30 c in tissues of patients with p53-mutant breast cancer and is closely related to poor prognosis.In the TCGA database,miR-30 c was significantly higher in normal tissues than in breast cancer tissues(p<0.05),and high expression of miR-30 c was closely associated with longer survival in breast cancer patients(p<0.001).This suggested that high expression of miR-30 c predicted a good prognosis,and TCGA database analysis of p53 wild-type breast cancer patients showed that miR-30 c was significantly higher than p53 mutant patients(p<0.001),compared with p53 wild-type tissue chip p53 mutations.In breast cancer tissues,p53 was highly expressed,miR-30 c was lowly expressed in p53-mutant breast cancer tissues(p<0.01),and REV1 and FANCF were highly expressed in p53-mutant breast cancer tissues(p<0.001,p<0.05),miR-30 c was negatively correlated with the expression of REV1 and FANCF in breast cancer tissues(p<0.01,p <0.05).The expression level of miR-30 c was correlated with clinical stage,lymph node metastasis and triple negative status of breast cancer cells.There was a significant negative correlation,and miR-30 c could significantly affect the prognosis of breast cancer patients as an independent factor.4.miR-181 a is highly expressed in human colorectal cancer and is associated with poor prognosis(1)miR-181 a was highly expressed in colorectal cancer tissues(p<0.01).Of the 329 samples,302(91.9%)were positive for mi R-181 a in colorectal cancer.In contrast,miR-181 a expression was positive in only 6(13.3%)of 45 para-cancerous tissue samples.(2)The expression of miR-181 a was significantly correlated with tumor diameter,liver metastasis,peritoneal dissemination,and stage of tumor clinical stage(p<0.001,p=0.004,p=0.018,p=0.003).There was no correlation between miR-181 a expression and other clinical case parameters of colorectal cancer.(3)The prognosis of patients with high expression of miR-181 a is poor.The high expression of miR-181 a in patients with colorectal cancer undergoing adjuvant chemotherapy was significantly associated with shorter overall survival(OS)and disease-free survival(DFS)compared with colorectal cancer patients who did not receive adjuvant chemotherapy(p<0.001,p<0.001).(4)Cox multivariate regression analysis suggested that miR-181 a is an independent prognostic risk factor for OS and DFS in colorectal cancer patients(p<0.001,p<0.001).(5)The high expression of miR-181 a in the TCGA database was significantly associated with the overall survival(OS)of patients with colorectal cancer(p=0.014).The expression of miR-181 a was significantly increased in advanced T-stage tumor size and N-stage lymph node metastasis(p=0.033,p=0.015).Conclusions:1.p53 mutant breast cancer cells resistance to adriamycin is closely related to the high expression of FANCF and REV1.2.miR-30 c inhibits the expression of REV1 and FANCF in order to DNA damage repair in breast cancer cells and increases doxorubicin drug sensitivity.3.p53 activates miR-30 c transcriptional repression of REV1 and FANCF expression by binding to the promoter region of miR-30 c.4.It is suggested that the p53/miR-30c/FANCF/REV1 pathway may be a novel mechanism of drug resistance to adriamycin in breast cancer cells,and mi R-30 c may be a new target for reversing the resistance to doxorubicin.5.miR-181 a is highly expressed in human colorectal cancer tissues and predicts poor prognosis.