The Study On The Expression And Regulation Mechanism Of MiR-203 In Esophageal Squamous Cell Carcinoma

PartⅠIdentify the expression level and function of miR-203 in esophageal squamous cell carcinomaBackgroundEsophageal cancer is the carcinoma which arise in the esophageal epithelia and is one of the high frequency carcinoma which have lethal effects on human. The histologic classification of esophageal caner include adenocarcinoma,esophageal squamous cell carcinoma(ESCC) and undifferentiated carcinoma. ESCC always arise in Asian. The 5-year survival rate of ESCC is low,so the earlier diagnosis,protection and therapy are of essential important. Investigation to the molecule mechanism of the carcinogenesis of esophageal cancer, will provide clues to the early prevention.MicroRNAs (miRNAs) have been shown to be of great importance in a wide variety of biological processes including cell differentiation, proliferation and apoptosis. Previous studies on miRNAs showed that they involve in the carcinogenesis and development by targeting gene associated with cell proliferation and differentiation.miR-203 is one of non-coding miRNAs which have close relation with differentiation of the stratified epithelial tissues: Sonkoly et al reported that the expression of miR-203 presents skin-specific model in twenty-one human tissues, especially in keratinocytes, it has higher expression level in esophagus and cervix than that in other tissues.Yi et al found that miR-203 is induced in the skin concomitantly with stratification and differentiation, it promotes epidermal differentiation by restricting proliferative potential and inducing cell-cycle exit. miR-203 has one conserved target p63 across vertebrates, which is an essential regulator of stem-cell maintenance in stratified epithelial tissues.Feber et al detected the miRNAs exchange in 1 ESCC sample and 7 normal esophageal epithelia samples, and in 7 ESCC samples and 1 normal esophageal epithelia sample with chip, they found that miR-203 is 2-10 times down-regulated in ESCC comparing with normal esophageal epithelial. It indicated that miR-203 might involve in the carcinogenesis of ESCC. The limited specimen couldn't completely reflect the expression level of miR-203 in ESCC, it is essential to identify the expression level of miR-203 in larger specimen.ObjectivesIdentify the expression level of miR-203 in ESCC specimen. Based on it, identify the effect of miR-203 on ESCC cell proliferation and the protein level of target gene△NP63 in ESCC.Methods and results1). In 93 cases of ESCC and 38 cases of non-tumor tissue, we performed the detection of mature miR-203 with Taqman probe by real-time quantity PCR. The expression level of miR-203 in 93 cases of ESCC are higher than that in 38 cases of non-tumor tissue(independent samples t-test, p=0.006). In 38 pairs of specimen, 55.3%(21/38) carcinoma have higher expression level of miR-203, 28.9%(11/38) non-tumor tissue have higher expression level of miR-203, 15.8%(6/38) pairs of specimen don't have significant difference in expression level of miR-203. The paired t-test showed that there is significantly difference in carcinoma and non-tumor tissue(p=0.031)2). We over-expressed miR-203 in EC109 cell line and identified the relationship between miR-203 and the proliferation of ESCC cell line with CCK-8 detection kit. The result showed that miR-203 inhibits the proliferation of EC109 in 48h after transfection.3). In order to identify the association between expression level of miR-203 and the protein level of△NP63, which is a typical target of miR-203, we performed western blotting in 17 pairs of carcinoma and non-tumor tissue. The result showed that 64.7%(11/17) non-tumor tissue has higher△NP63 protein level, and 35.3%(6/17) carcinoma has higher△NP63 protein level. This result indicated that the high expression level of miR-203 in most of the ESCC might lead to the low expression of△NP63 in carcinoma.Conclusions1. miR-203 is upregulated in the carcinoma specimen of most of the ESCC.2. In ESCC cell line EC109, the overexpression of miR-203 will inhibit the cell proliferation.3.△NP63, which is on of the target of miR-203, is downregulated in most of the ESCC, it might be the upregulation of miR-203 in ESCC. PartⅡThe study on the involvement of TCF4 in the regulation of miR-203 expression in ESCCBackgroundIn normal physiological status, the basal cells of esophageal epithelial will differentiate and migrate to the surface and then turn into the stratified squamous epithelial cells, its renew rate and the lost of cells in surface maintain the balance. The renew model in esophageal squamous epithelial is similar with that in skin. miR-203 can inhibit the stemness of basal cells in skin, its dysregulation might lead to the lost of the balance in normal esophageal renew. Found out the mechanism involving in its dysregulation will help us to understand the molecule mechanism in carcinogenesis of ESCC.Bioinformatics analysis showed that transcription factor TCF4 can combine with the promoter of miR-203, it maybe take part in the upstream transcription regultion of miR-203. TCF4 acts as one of the effectors of typical Wnt signal pathway.In 2009, Nguyen et al reported that they established roles for Tcf3 and Tcf4 in long-term aintenance and woundrepair of epidermis, suggesting that Tcf proteins have both Wnt-dependent and Wnt-independent roles in lineage determination.TCF4 has multiple alternative splicing isoforms, different isoforms may code proteins with different molecular mass, the major difference lies in the binding site for C-terminal binding protein, there are two such binding sites in the long isforms but not in the short isforms because of the earlier termination of tranlation. The reports about the effect on the transcription of target genes of long and short isforms was controversial. Andreas et al reported that the long isforms promote the transcription of target genes but not the short isforms. Tsedensodnom et al showed that the typical long isoforms inhibit the transcription of target genes, conversely, the typical short isoforms promote transcription.The former studies always focused on the activation and translocation to the nucleus ofβ-catenin, and little focus was put on the expression and function of different isforms of TCF4. There is no report on whether TCF4 involves in the regulation of miR-203 in ESCC.Objectives1. Identify whether TCF4 involve in the expression regulation of miR-203 in ESCC.2. Identify the protein levels of TCF4 in carcinoma and non-tumor tissue of ESCC.3. Identify whether the different isforms will have different effects on the expression level of miR-203.Methods and results1). Firstly, we detected the protein level of TCF4 in ESCC cell lines. Western blotting results showed that the long and short isforms all expressed on KYSE150,KYSE450,EC109,EC9706. In order to identify whether TCF4 can bind to the promoter of miR-203, we performed ChIP in EC109 cell, the result showed that TCF4 can combine with the upstream of miR-203. The luciferase reporter gene assay showed that the mutation of TCF4 binding site on miR-203 strengthens the transcription activity of luciferase. It inditated that TCF4 might inhibit the transcription of miR-203 in EC109.2). The western blotting on the TCF4 in 21 cases of ESCC and 21 cases of non-tumor tissue showed that the 58kD short isoforms of TCF4 arose in the tumor specimen and non-tumor tissues, but its 79kD long isforms were lost in 21(0/21) cases of ESCC and express on 85.7%(18/21) non-tumor tissues. This result showed that TCF4 has significantly difference in protein level between ESCC and non-tumor tissues.3). we constructed typical long isform plasmid and short isform plasmid, the tranfection experiment in EC109 cell showed that the long isform plasmid inhibits the expression level of miR-203, and the short isform plasmid promotes the expression level of miR-203. The lost of long isoforms in ESCC might make the lost in its inhibition ability, and the anxo-action of short isoforms were presented.Conclusions1. The short isform and long isform of TCF4 all express on ESCC cell line KYSE150,KYSE450,EC109,EC9706.2. In EC109 cell, TCF4 involves in the transcription regulation of miR-203. TCF4 inhibits the transcript of miR-203 in EC109.3. The long isforms of TCF4 don't express in carcinoma of ESCC and express in non-tumor tissue of ESCC, it indicated that the protein level of TCF4 has significant difference between carcinoma and non-tumor tissue of ESCC.4. The long isform inhibits the expression of miR-203, the short isform promotes the expression of miR-203, this indicated that different isforms of TCF4 have different effects on the expression level of miR-203. The upregulation of miR-203 in the carcinoma of most of the ESCC patients, might be caused by the non-expression of long isforms of TCF4 in ESCC.As mentioned above, we set miR-203 as our subject, identified its expression level in ESCC patients. The result showed that miR-203 upregulated in the carcinoma of most specimen of the ESCC. Then we studied the regulation mechanism of miR-203 from transcription level and proved that TCF4 involve in the regulation of miR-203. We found that TCF4 has different isform expression between carcinoma and non-tumor tissue of ESCC. The long isoform of TCF4 only express in non-tumor tissue, but don't express in carcinoma. The long isform of TCF4 inhibit the expression of miR-203 and the short isform promote its expression. This different isform expression of TCF4 might be the reason why miR-203 is upregulated, it leads to the balance established by esophageal squamous epithelial itself, then causes the carcinogenesis of ESCC.This study provide a new clue for understanding of the molecular mechanism of carcinogenesis of ESCC.