ZEB2Contributes Multi-drug Resistance Of Small Cell Lung Cancer And Is Regulated By MiR-200b

BackgroundThe national lung cancer association’s latest survey showed the incidence of lung cancer,as one of the most harmful cancers in the world, occupied the first spot among various malignant tumors in our country.According to the morphology under microscope, lung cancer can be divided into small cell lung cancer(SCLC), adenocarcinoma, squamous cell carcinoma and large cell lung cancer. The latter three are collectively referred to as non-small cell lung cancer (NSCLC). Incidence of SCLC accouts for about15%of that of primary lung cancers. It is poorly differentiated and easy to hematogenous metastasis at early stage, which leads to poor prognosis. Although sensitive to chemotherapy initially, SCLC develops multi-drug resistance (MDR) soon, which resulted in failure of chemotherapy. Moreover,MDR can result to high recurrence rate of SCLC, which leads poor clinical prognosis. Therefore, MDR is one of the problems to be conquered in the basic research and clinical treatment of SCLC.The snail superfamily of Zinc-finger transcription factors are composed of Snail, E12/FA7ZEB1, ZEB2and Slug. Snail superfamily play significant roles in the important biological progresses, such as cell differentiation, proliferation.apoptosis, and so on. Abnormal expression of snail gene family may lead to the abnormal morphology in ontogeny and formation process of tissue and organ, and can lead to prostate cancer, breast cancer, gastric cancer, lung cancer and other malignant tumors. Research have shown that snail family genes also can adjust the resistance and apoptosis of tumors. Study find that ZEB2widely expresses in rat and human organization, especially the heart and nerve tissue. By combining with E-cad gene promoter of the E-box sequence, ZEB2restrain the the transcription of E calcium sticky protein, cell keratin (cytokeratin), sticky protein, the MAC-1and bridge grain protein. Downregulation of those protein expression especially E calcium protein, plays important roles of epithelial interstitial transformation (epithelial mesenchymal transition, EMT). Study find that the zinc finger E-box-binding homeobox2(ZEB2) gene is an important member of the snail gene family, closely related to tumorigenesis, development, metastasis and prognosis of many tumors. The research show that ZEB2is involved with the resistance to epidermal growth factor inhibitors in bladder cancer and cisplatinum in ovarian cancer. However, at present there is no related reports whether ZEB2genes are involved in small cell lung cancer cell apoptosis and resistance.MicroRNAs (miRNAs) are small single-stranded noncoding RNA (18-24nt) that regulate gene expression by interfering with mRNA stability or protein translation. miRNAs are highly evolutionarily conserved, and widely involved in growth and development of plants and animals,cell differentiation,proliferation and apoptosis. hormone secretion and oncogenesis. Study find that miRNAs are closely linked to resistance of tumor. Down-regulation of miR200c can increase resistance to doxorubicin in the breast cancer cells. Up-regulation of miR-125b can increase resistance to chemotherapy drugs in acute promyelocytic leukemia. Up-regulation of miR-224can increase resistance to methotrexate in colorectal cancer. Research demonstrate that the loss of heterozygosity in miR-128b often occurs in non-small cell lung cancer (NSCLC) tissues.The epidermal growth factor receptor is a target gene of miR-128b which can regulate of its expression levels in NSCLC, which relates to the sensitivity to gefitinib and survival rate of patients. We adopted miRNA chip comparative analysis of the miRNAs expression difference between small cell lung cancer drug resistant cells H69AR and non-resistant cells H69.We found61miRNAs expressed abnormally,24miRNAs including miR-375and miR-200b, expressed high,and37miRNAs,including miR-100and miR-224,appeared low expression. All the results of miRNA chip were provided by real-time quantitative PCR.MiR-200family contains miR-200a, miR-200b, miR-200c, miR-141and miR-429.Many research identified,by targeting two important cell transcription repressors Zinc finger E-box-binding homeobox1/2(ZEB1/2),miR-200b family regulate epithelial-mesenchymal transitions and mesenchymal-epithelial transitions so as to result in tumorigenesis,development,metastasis and drug resistance.miR-200b is an important member in miR-200family. Bioinformatics software indicates that there are over7000targets of miR-200b.Christoffersen detected that miR-200b and ZEB2mRNA common expressed in rats brain via situ hybridization technology and the mRNA expression level of ZEB2and protein decreased when downregulated miR-200b.Our initial DNA chip technology analysis showed ZEB2gene expressed higher in SCLC drug-resistant H69AR cell lines. To further definite the relationship between ZEB2and the drug resistance of small cell lung cancer, in this experiment we assumed to reduce ZEB2expression in H69AR cells through RNA interference technology. The ZEB2mRNA and protein level were detected before and after transfection of SiRNA. Using the cell count kit-8(cell counting kit-8, CCK8) method and flow cytometry,we analyzed changes of drug sensitivity、apoptosis and cycle with ADM、DDP and VP-16. Moreover, we analysed SCLC tissues to explicite the relationship between ZEB2expression and the clinical pathological features of patients and prognosis.We used bioinformatics software (MiRanda and Targetscan software) to predict and found that ZEB2is one of the target genes of200b. MiR-200b locates between ZEB2cluster, which locates in romosome2q22.3, and it pairs partly with the3’-UTR mRNA of ZEB2and so as to affect its transcription after the translation process. By miRNA microarray technology, we found that miR-200b in H69AR significantly low expressed, indicating a reverse relationship of the expression of ZEB2, which prompt that miR-200b may targeting regulated ZEB2. The regulated relationship between miR-200b and ZEB2was to further defined in this research.ObjectivesTo analyze the differently expressed genes in small cell lung cancer cell line H69and its multi-drug resistant cell line H69AR and select the most differentiated expressed ZEB2gene for further study. To analyze its effect on chemoresistance of small cell lung cancer. To analyze the regulation of miR-200b on ZEB2expression. To provide new theoretical basis for clinical treatment for SCLC。Materials and Methods1、Modulation of chemoresistance of small cell lung cancer by ZEB2.ZEB2siRNA was transfected into cells to inhibit ZEB2expression in SCLC multi-drug resistant cell strains H69AR, then chemosensitivity to adriamycin (ADM), cisplatin (DDP) and etoposide (VP-16) were analyzed by cell counting kit-8(CCK8) method. Apoptosis and cell-cycle induced by the three drugs were analyzed by flow cytometric analysis.2、Regulation of ZEB2by miR-200b.(1)After transfection of miR-200b mimic, ZEB2mRNA and protein expression was verified by using real-time quantitative PCR and Western Blot.(2)We first constructed psiCHECK-2vector containing ZEB23’-untranslated region (UTR)(psiCHECK-2-ZEB2-3’UTR), and then co-transfected with miR-200b mimic or inhibitor into H69AR cell. Luciferase activity of ZEB2was determined by using dual luciferase reporter gene assay.3、Effect of miR-200b on multi-drug resistance of small cell lung cancer.According to microRNA microarray, miR-100mimic was transfected into cells to increase miR-200b expression. Chemosensitivity to the above three drugs were determined by using CCK-8assay.4、Relationship between ZEB2expression and survival of small cell lung cancer. We collected68cases of paraffin-embedded small cell lung cancer tissue. They were sectioned serially and immunohistochemical stained. Relationship between ZEB2expression and clinical features of patients with small cell lung cancer were analyzed.5、Statistical analysis.All results were treated by SPSS13.0statistical software. The experiments were repeated independently for at least three times. The selected charts were a result of repeated experiments. Data was showed as mean±standard deviation. Difference between samples in quantitative real-time PCR、western blotting、apoptosis and cycle experiment、luciferase reporter gene assay was used one-way ANOVA analysis if data homogenity of variance,and F test/Welch method if data heterogeneity of variance. LSD/Dunnett ’s T3methods were used for multiple comparisons. Difference in CCK8results were analyzed by two factors factorial design of variance and SNK method for multiple comparisons. Before comparison, data homogeneity of variance was first examined using F test. If the case of heterogeneity of variance, the approximate variance F test/Welch method was used. Fisher’s accurate analysis was used to evaluate the relations between ZEB2expression and the clinical and pathological features. Kaplain-Meier analysis was used to evaluate the relationship between ZEB2expression and survival rate. A p value<0.05was considered significant.Results1、Reduction of ZEB2expression in H69AR cell increased its drug sensibility.(1) ZEB2-642reduced the ZEB2expression significantly in H69AR cell.We designed and synthesized three pairs of siRNA oligonucleotides targeting ZEB2, and transfected into H69AR cells by using lipofectamin2000. Compared with the other groups, the mRNA and protein expression level of ZEB2-642group reduced significantly (P<0.001). Therefore, ZEB2-642was chosen to be used in following experiments.(2) CCK-8method analysis showed that,after treatment of ADM、DDP andVP-16, survival and IC50values of H69AR cell transfected with ZEB2-642reduced significantly compared with that of negative control (NC) or mock control.(3) Apoptosis of H69AR cell transfected with ZEB2-642increased significantly compared with H69AR cell transfencted with that of negative control (NC) or mock control, while cell cycle changed.2、ZEB2is the target genes of miR-200b(1)According to miRNA target gene prediction software (PicTar, TarScan and miRBase), there are complementary binding sites between miR-200b and ZEB23’-UTR.(2) Regulation of ZEB2protein by miR-200b.After transfection of miR-200b mimic into H69AR cell, miR-200b expression increased (P<0.001). ZEB2expression did not change in mRNA level (P-0.472), but decreased in protein levels (P<0.001).(3) ZEB2is target of miR-200b①Successfully.../constructed psiCHECK-2-ZEB2-3’UTR-R vectorThrough the PCR amplification ZEB23’UTR, product length for1512bp, the transformation, the screening, pick resistance steps take monoclonal after extraction kit plasmid, Not I and Xho I double enzyme cut, product size is about6273bp/1512bp (figure8). Enzyme cut psiCHECK identification-2-ZEB2-3’UTR cloning1-4and psiCHECK-2-ZEB2-3’UTR-R cloning3correctly, start psiCHECK-2-ZEB2-3’UTR-1cloning and psiCHECK-2-ZEB2-3’UTR-R-no.3on cloning sequencing. Sequencing result with the theoretical sequence is consistent, kit extraction plasmid follow-up experiment.②ZEB2fluorescent element enzyme activity by miR-200b mimic and inhibitor regulationpsiCHECK-2-ZEB2-3’UTR-R or psiCHECK-2-ZEB2-3’UTR-mut were transfected into the H69AR cells, with miR-200b mimic or inhibitor co-transfected, and fluorescent element enzyme activity of ZEB2showed that, compared with the miR-NC group, miR-200b mimic significantly inhibited ZEB23’UTR (P<0.001) miR-200b inhibitor can significantly increase the ZEB2(P<0.001).As compared with ZEB23’UTR-mut,miR-200b mimic and miR-200b inhibitor had no effect on the enzyme activity of ZEB2.3、Up-regulation of miR-200b in H69AR cell resulted in reduced of resistance to ADM, DDP and VP-16After treatment of ADM, DDP and VP-16, survival and IC50values of H69AR cell transfected with miR-200b mimic reduced significantly compared with that of negative control (miR-NC) or mock control(P<0.001).4、Relationship between ZEB2expression and clinical pathological features in SCLC tissues.Positive ZEB2expression in SCLC tissues was located in the nucleus. Its positive rate was23.5%(16/68). ZEB2positive expression rate showed no significant differences in gender, age, and survival state (P=0.492,0.776,0.098), but in tumor spread range was statistically significant (P<0.001).5、Survival analysis of SCLC patientsKaplan-Meier method was taken to estimate patient survival, and found that there was no statistical difference between gender and age (P=0.152,0.097). The survival rate in LD patients was higher than ED patients indicating statistics difference(P<0.001). The survival rate in ZEB2negative patients was higher than positive patients, indicating a statistics difference (P<0.001).Conclusions1、ZEB2expression in SCLC multi-drug resistant cell line H69AR was higher than that in H69cell. Elevation or inhibition of ZEB2resulted in change of chmoresistance of cells. These results indicated that ZEB2be involved in regulation of chemoresistance of SCLC.2、miR-200b expression in SCLC multi-drug resistant cell line H69AR was lower than that in H69cell. Elevation of miR-200b resulted in change of chemosenstivity of cells. ZEB2is a direct gene of miR-200b. Effect of ZEB2on chemoresistance of SCLC is under the regulation of miR-200b.3、ZEB2expression in SCLC tissues correlated with stage and survival of patients. ZEB2may be a prognostic factor of SCLC patients.