Direct Interaction Between MiR-203 And ZEB2 Suppressed Epithelial-mesenchymal Transition (EMT) Signal Decreasing Chemotherapy Resistance In Lung Adenocarcinoma

Background and objectionLung cancer is one of the most important cancer contributed to deaths in the last 20 years all over the world. The incidence of lung cancer was also gradually increasing in cities and counties. Non-small cell lung cancer which accounts for 80%-85% lung cancer. Adenocarcinoma in some developed countries has become the most common lung cancer. The incidence increased year by year in our country, and has become the most common style than squamous carcinoma of non-small cell lung cancer. Tumors can occur in the bronchial levels, but the small bronchi as much as possible, and therefore the common peripheral mass. Non-small cell lung cancer patients seek treatment because of clinical symptoms at the time of diagnosis with 20%-30% of stage Ⅰ and stage Ⅱ,40%-50% of stage Ⅲ and 30% stage Ⅳ. Therefore, chemotherapy is the cornerstone of treatment of lung cancer. The two drugs combined with platinum-containing chemotherapy is the current standard regimen, wherein the cisplatin is one of the most common chemotherapy drugs in the treatment prone to relapse or resistance to chemotherapy.There is the main modes of failure because of chemotherapy resistance.At present, some molecular and cellular mechanisms of resistance to chemotherapy drugs known mainly includes changes and enhances the expression of drug targets to prevent drug into the target cells, drug metabolism and excretion of inactivation, DNA mismatch repair damage and enhance, reduce apoptosis, drugs drug-induced metabolic changes and karyotype changes. Studies have shown that multi-drug resistant MDR tumor cells and tumor-related genes and their abnormal signal transduction pathways involved in the formation mechanisms of resistance. miRNA plays an important role in the regulation of tumor metabolism of the drug, and thus can regulate the expression of miRNA thereby intervention mechanisms of chemotherapy resistance.MicroRNA (miRNA) is single-stranded noncoding RNA of 18-25 bases length, and its main function is widely involved in the pathological and physiological adjustment process of tissues and organs of the body. miR-203 as a member of miRNA family, is a miRNA epithelial tissue-specific expression factor. Some studies have found that miR-203 plays an important role in the development of skin-related diseases, epithelial tissue tumors and other pathological and physiological processes. According to current research reports, miR-203 is considered as a tumor suppressor widely involved in various tumors including gastric cancer, colorectal cancer, breast cancer, melanoma, and hepatocellular carcinoma pathogenesis. In lung cancer, miR-203 also acts as a tumor suppressor, can inhibit cancer cell proliferation and metastasis, which is mainly play a role by directly targeting the Bmil and PKCa in lung cancer.There are 108 cases with bladder cancer using cisplatin,the expression levels of miR-203 bladder cancer celll was detected in vitro, the expression level of miR-203 in patients with advanced bladder cancer is significantly lower than no-progress patients and could predict and distinguish whether tumor progress or not. The prognosis of patients with low expression of miR-203 is even worse, such as shorter PFS and OS, and could be used as an independent prognostic factor. Bladder cancer cells in vitro studies, the expression of miR-203 cells were more sensitive to cisplatin and promote cell necrosis and apoptosis. In the study of colorectal cancer using oxaliplatin chemotherapy drug showed that the higher expression of miR-203 may protect colorectal cancer cells by reducing the toxicity of oxaliplatin and an increment of resistance which may reverse the phenomenon by anti-miR-203 expression, thereby reducing the generation of platinum-based chemotherapy drug oxaliplatin. This study showed that miR-203 may play different roles and functions in different tumors.ZEB2 also called ZFHX1B, SIP-1, encoded by gene on chromosome 2q22 ZFHX1B on with 8 introns and 9 exons, and its CDS region was 3572 base pairs, act as a zinc finger transcription factors of the members of the E-box binding zinc finger protein (zinc finger E-box-binding protein, ZEB) family. ZEB2 contains two zinc finger clusters (zinc-finger cluster) and the two variable sequences of zinc finger clusters, where N and C-termini of the zinc finger could independently binding region of the target gene regulated by the 5’-CACCT (G) nucleotide sequence. The current study reveals the expression and function of ZEB2 was in the early embryonic development, but initial reports on ZEB2 was found in Xenopus embryos.ZEB2 as a transcription factor, initial studies suggested that ZEB2 located in the nucleus, but with further research, ZEB2 not only found in the nucleus,but also in the cytoplasm and its nucleus, it involved cell inflammation and the formation of cell growth, differentiation, apoptosis, embryonic development, and other life activities regulation. The current study suggested that ZEB family plays a role in the regulation of the involvement in tumorigenesis and development. In non-small cell lung cancer studies had shown that the expression of ZEB2 contributed to tumor T stage, tumor diameter, clinical stage and correlated with survival of patients with a negative correlation; In Gastric Cancer, ZEB2 correlated with its intestinal type and had a higher lever associated with histological type; In breast cancer, ZEB2 expression indicated a shorter overall survival time and poor prognosis; In ovarian cancer showed that the expression of ZEB2 in cancerous exudated higher than the primary tumor; Expression of ZEB2 were higher among the different levels of glioma, further analysis showed that the expression of ZEB2 during the tumor invasion and metastasis were positively correlated. In pancreatic cancer, higher ZEB2 promote d disease progression; In colorectal cancer, ZEB2 in adjacent tissues compared with the central region of the tumor tissue, adjacent tissues was expressed at higher levels, could promote the progress of the disease, may be used as an effective biological markers or as a new target for treatment of colorectal cancer; In malignant melanoma, ZEB2 was expressed at the lower levels, lower expression of ZEB2 may promote its progress and cell transformation and colony formation. In hepatocellular carcinoma, carcinoma group compared with the edge organization, ZEB2 was reduced, had a good survival after surgery; In summary, in different tumor and tissues,the ZEB2 have different expression levels and different function.In this study, we respectively investigated the effect of miR-203 and ZEB2 on chemotherapy resistance of DDP to lung adenocarcinoma and their molecular mechanism.Objective1. To explore the chemotherapy sensitivity of over expression of miR-203 A549 and SPCA-1 cells to cisplatin.2. To explore the chemotherapy sensitivity of of miR-203 inhibitor A549 cells and SPCA-1 to cisplatin.4. To study the influence and its mechanism of ZEB2 expression level and EMT marker expression after miR-203 inhibitor.5 To explore the chemotherapy sensitivityto cisplatin after ZEB2 knocking down in A549 and SPCA-1 cells.6. To explore the effects of ZEB2 on miR-203 expression7. To explore the mechanismof ZEB2 on regulating miR-203.Contents and methods1 Construction of stably overexpression of miR-203 in lung adenocarcinoma cell1) Use miR-203 lentivirus infection A549 and SPCA-1 cells to construct stably overexpressing miR-203 in lung adenocarcinoma cell line2) Total RNA was extracted from stabilized expression of miR-203 in lung adenocarcinoma cells3) Clontech kit RNA into cDNA4) TaKaRa SYBR Premix Ex TaqTM fluorescence quantitative PCR detection kit infection miR-203 lentivirus human lung adenocarcinoma cell line A549 and SPCA-1, in order to identify stably overexpressing miR-203 cell line whether to build success.2 MTT detected miR-203 overexpression on A549 and SPCA-1 cell sensitivity to cisplatin.3 miR-203 inhibitor of miR-203 stabilized chemosensitivity to cisplatin of A549 and SPCA-1 cells.1) The miR-203 inhibitor instant has transferred stably overexpressing miR-203 in A549 and SPCA-1 cells;2) Transient transfected cells extracted total RNA before and miR-203 inhibitor;3) Clontech kit RNA into cDNA4) TaKaRa SYBR Premix Ex TaqTM fluorescence quantitative real-time PCR kit fluorescence quantitative PCR, to detect inhibition of miR-203 inhibitor efficiency of miR-203 expression;5) MTT method to detect the A549 and SPCA-1 cells chemosensitivity to cisplatin aftertransient transfected miR-203 overexpression stabilize.4. Effect of miR-203 on ZEB2 and EMT-related protein1) Extraction stably overexpressing miR-203 A549 and SPCA-1 cells before and afterand measuring the protein concentration of ZBE2, E-cadherin, N-cadherin expression levels2) Western blot detected the protein expression5.Impact of miR-203 inhibitor of miR-203 stabilized A549 and SPCA-1 cells on ZEB2 expression1) The miR-203 inhibitor instant transferred stably overexpressing miR-203 in A549 and SPCA-1 cells;2) Extract the instantaneous turn around miR-203 inhibitor cells and measuring the protein concentration;3) Western blot of transient transfected miR-203 inhibitor, stable overexpression of miR-203 in A549 SPCA-1 cells and ZEB2.6.MiR-203 gene target validation1) TargetScan and RNAhybrid online software predicted target genes of miR-203, ZEB2 as a candidate target genes;2) Construction of dual luciferase reporter gene vector psicheck-2/ZEB2 3’UTR and dual luciferase reporter gene mutation carrier psicheck-2/ZEB2 mt 3’UTR;3) Wild-type carrier, mutant plasmid vectors and psicheck-2, respectively, miR-203 mimics and inhibitor co-transfected 293T cells,48h after detection of firefly luciferase and Renilla luciferase activity, determined by the ratio of luciferase whether direct regulation of miR-203 ZEB2.7 ZEB2 regulation of A549 and SPCA-1 cells chemosensitivity to cisplatin1) ZEB2 three siRNA fragments and control fragments into instant A549 and SPCA-1 cells;2) Transfected cells after extraction of instantaneous total RNA and protein;3) TaKaRa DNA reverse transcription kit to be transcribed into RNA cDNA;4) The use of real-time quantitative PCR fragment three siRNA interference efficiency ZEB2 mRNA levels;5) Western blot and real-time quantitative PCR to identify the best clips interference interference effect on ZEB2 protein levels;6) MTT interference ZEB2 of A549 and SPCA-1 chemosensitivity to cisplatin;8. The impact of ZEB2 on the expression of miR-2031) The specificity of siRNA targeting ZEB2 fragments and control fragments into A549 and SPCA-1 cells, interfering ZEB2 expression;2) Total RNA was extracted after transient transfected cells;3) Clontech kit RNA was reverse transcribed to cDNA;4) Real-time PCR to detect interference ZEB2 lung adenocarcinoma cells and SPCA-1;9. The mechanism of ZEB2 on regulation of miR-203.1) JASPAR and ALGGEN database analysis and forecasting ZEB2 and miR-203 binding of the promoter region of the boot2) chromatin immunoprecipitation (ChIP) experiments ZEB2 and miR-203 binding of the promoter region of the boot.Results1 Construction of miR-203 in lung adenocarcinoma cell line stably overexpressingMiR-203 with lentivirus particles infected lung adenocarcinoma A549 cells and SPCA-1 cells to over-expression of miR-203, and identify its overexpression efficiency by fluorescence quantitative Q-PCR method. The results showed that in lung adenocarcinoma A549 cells and SPCA-1 cells compared to the negative control group transfected miR-203 lentivirus expression levels of miR-203 cells was significantly higher (P<0.05), described expression of miR-203 in lung adenocarcinoma cell line was successfully constructed, can be used for subsequent experiments.2 over-expression of miR-203 in A549 and SPCA-1 cell sensitivity to cisplatinDetected by MTT after overexpressing miR-203 A549 and SPCA-1 cells to chemotherapy drug cisplatin dose response, dose response curves plotted, statistical analysis showed that A549 and SPCA-1 cell lines, over-expression of miR-203, the cells are more sensitive to cisplatin, IC50 values were lower, there was a significant difference (P<0.001) and between two different treatment groups. Description overexpression of miR-203 can increase the sensitivity of lung adenocarcinoma cells to cisplatin chemotherapy, reduce lung adenocarcinoma cells to cisplatin resistance.3 miR-203 inhibitor can reverse stable over the A549 and SPCA-1 cells chemosensitivity to cisplatin after expression of miR-2031) To further investigate the influence of miR-203 human lung adenocarcinoma cells to chemotherapy, we are stably overexpressing miR-203 lung adenocarcinoma cells transfected with miR-203 inhibitor again and quantified by fluorescence detection of Q-PCR expression of miR-203 in lung adenocarcinoma A549 cells and SPCA-1 cells with stable overexpression of miR-203 group compared to cells, miR-203 inhibitor levels in these cells expressing miR-203 was significantly reduced (P<0.05), indicating that miR-203 inhibitor can inhibit the expression of miR-203 can be used for subsequent experiments.2) To study the influence of miR-203 human lung adenocarcinoma cells to chemotherapy drugs from the other direction, we stably overexpressing miR-203 in A549 cells with miR-203 inhibitor inhibited the expression of miR-203, and through MTT method to detect the sensitivity of A549 cells to cisplatin, drawn A549 cisplatin dose response curve was found later transferred to miR-203 inhibitor, stable overexpression of miR-203 sensitive A549 and SPCA-1 cells to DDP reduced the, IC50 values increased, indicating that miR-203 inhibitor reversed the A549 and SPCA-1 cells chemosensitivity to cisplatin after stable overexpression of miR-203, miR-203 down-regulated expression can be increased in lung adenocarcinoma cells cisplatin resistance.4 Effection of tmiR-203 on ZEB2 and EMT related proteinsPrevious studies have shown that, ZEB2 is an important factor in the regulation of inter-cell epithelial-mesenchymal transition, and EMT-associated protein in tumor resistance also plays an important role.I n order to the mechanism of miR-203 chemotherapy sensitivity of lung adenocarcinoma cell, after the over-expression of miR-203, was detected by Western Blot.ZEB2 and EMT related protein were changed. The results showed that in lung adenocarcinoma A549 and SPCA-1 cells, miR-203 overexpression can inhibit the expression of the transcription factor ZEB2 and EMT mesenchymal markers N-cadherin and promoting expression of epithelial markers E-cadherin.Conversely, when the stable overexpression of miR-203 cells introduced into miR-203 inhibitor, the expression is elevated ZEB2. miR-203 is capable of mediating ZEB2 and regulating EMT associated proteins.5.miR-203 targeting the 3’UTR of ZEB21) We use the online software TargetScan predicted target of miR-203 gene, as found ZEB2 target candidate gene, then the UCSC Genome Bioinformatic online software to obtain the predicted target genes ZEB2 3’UTR, by miRBase find mature hsa-miR-203 in sequence, then RNAhybrid software hsa-miR-203 sequence and the target gene ZEB2 3’UTR were compared, the final predicted ZEB2 3’UTR and miR-203 binding sites.2) We successfully constructed psicheck-2/ZEB2 wt 3’UTR Plasmid and psicheck-2/ZEB2 mut 3’UTR plasmid, respectively, and miR-203 mimics and inhibitor co-transfected 293T cells 48h after transfection luciferase assay active. The results show, psicheck-2/ZEB2 wt 3’UTR and miR-203 mimics plasmid co-transfection significantly reduced luciferase activity, the difference was statistically significant (P<0.001), with the miR-203 inhibitor co-transfected can significantly enhance luciferase activity (P<0.001). After the mutant plasmids psicheck-2/ZEB2 mut 3’UTR respectively miR-203 mimics and inhibitor co-transfection, the luciferase activity did not change significantly (P> 0.05). These results indicate that, miR-203 is capable of specifically binding to the 3’UTR ZEB2 end.6 Effects of ZEB2 on chemosensitivity to cisplatin A549 and SPCA-1 cells1) In order to further investigate the effects of cisplatin chemosensitivity ZEB2 lung adenocarcinoma cells, we can use three specific targeting of siRNA and A549 ZEB2 ZEB2 expression SPCA-1 cells using real-time quantitative PCR identification of its interference efficiency, choose the best of siRNA interference 2, and was confirmed by Western blot to identify the siRNA enables ZEB2 protein downregulation, indicating that siRNA can use for subsequent experiments.2) interference with siRNA A549 and SPCA-1 cells ZEB2, the detection sensitivity of A549 and SPCA-1 cells to cisplatin by MTT method, draw the A549 and SPCA-1 drug cisplatin dose-response curve. Statistical analysis showed that after interference ZEB2 A549 and SPCA-1 cells increased sensitivity to DDP, IC50 values decreased (P<0.05), illustrate the sensitivity of interference ZEB2 increase lung adenocarcinoma cells to cisplatin.7 Knocking down of ZEB2 increased miR-203In A549 and SPCA-1 cells with siRNA interference targeting silence ZEB2 ZEB2 after we use the expression of real-time PCR detection of miR-203, and the results show that compared with undisturbed ZEB2 group, A549 cells and SPCA-1 expression in miR-203 levels were increased. Explained ZEB2 when miR-203 targeted regulation, ZEB2 feedback can also modulate the expression of miR-203.8.ZEB2 miR-203 binding to the promoter region of miR-2031) We first find the promoter region of miR-203 in the NCBI database, and then JASPAR ALGGEN database predict miR-203 may regulate transcription factor, we found ZEB2 in miR-203 promoter region of a combination of two relatively conservative site.2) We used chromatin immunoprecipitation (ChIP) were combined verification situation ZEB2 and miR-203 promoter region two binding sites, the binding was found compared with the negative control antibody IgG, ZEB2 and miR-203 promoter region more; but with siRNA interference ZEB2, we found that miR-203 in combination with ZEB2 promoter region is reduced. These results indicate that the expression of ZEB2 by miR-203 binding to the promoter region and thus the regulation of miR-203.Conclusion1.Overexpression of miR-203 increased the chemosensitivity of A549 cells and SPCA-1 cells to cisplatin.2.miR-203 inhibitor can reverse chemotherapy sensitivity in lung adenocarcinoma cell.3. Overexpression of miR-203 inhibits the expression of ZEB2 and EMT-associated protein N-cadherin, and promote the expression of E-cadherin.4.miR-203 inhibitor reversibly turned expressing ZEB2 increase.5.miR-203 directly targeted ZEB2 in ZEB2 the 3’UTR.6 Knocing down ZEB2 increased chemosensitivity to cisplatin in adenocarinoma cells7. Knocing down ZEB2 increased the expression of miR-203.8. ZEB2 binding to the promoter region of miR-203.miR-203 and ZEB2 may be treated as the target of non-small cell lung caner.