Expression And Regulation Of MicroRNA-133b In Esophageal Squmaous Cell Carcinoma

Esophageal cancer (EC) is one of the most common malignancy worldwide, and the incidence and mortality of EC rank the eighth and sixth among all malignancy respectively. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are two main forms of EC in pathology, and ESCC is the dominant subtype of EC. In china, the incidence and mortality of EC is the highest worldwide. EC. is usually diagnosed as locally advanced stages with/without lymph node matastasis. Despite treated aggressively, the overall five-year survival rate remains poor which is only10-20%after operation. Therefore, exploring the biological behavior and pathogenesis of EC is necessary for the development of better diagnostic methods, therapeutic strategies and survival.MicroRNAs (miRNAs) are non-coding single-stranded, endogenous RNA molecules, only about21-25ribonucleotides in length, highly conserved in evolution, which negatively regulate gene expression at the post-transcriptional and/or translational level. MiRNAs play important roles in diverse biological process including development, cell differentiation, proliferation, apoptosis, hormone secretion and oncogenesis. Recently, cumulative evidences have shown that the generation and dysfunction of miRNAs are associated with the occurrence and development of tumors and function as oncogenes or tumor suppressor genes.Recent evidence suggest that miRNAs involved in the pathological processes of EC, but the mechanism is still not explained clearly. Therefore, further study of miRNA expression spectrum and aberrant expression of miRNAs in EC would be helpful to reveal the mechanism for EC and provide new ideas for early diagnosis, prognosis and therapeutic strategies.In this study, we planed to screen miRNA expression spectrum and study the function of abnormally expressed miRNAs in the onset and development of ESCC. First of all, we campared the miRNAs expression spectrum between ESCC and adjacent tissues to detect the abnormally expressed miRNAs by Gene Biochip (miRNA microarray). Then, we used real-time quantitative PCR to further verificate the abnormally expressed miRNAs which are specific to ESCC. Thirdly, we searched for the downstream target genes of the abnormally expressed miRNAs using bioinformatics. Finally, we used gain/loss-function study to analyze the mechanism and therapeutic potential of ESCC-related miRNAs in ESCC. This research project revealed that abnormally expressed miRNA played important roles in the onset and development of ESCC and provide new insights into the early diagnosis, prediction of prognosis and targeted therapy of ESCC.Part I Identification of Specific miRNAs in Esophageal Squamous Cell CarcinomaPurpose:To analyze and compare miRNAs expression profile and screened out the specific miRNAs which are abnormally expressed in ESCC.Methods:We compared the miRNA expression profile in13pairs of samples collecting from ESCC and adjacent normal esophageal epithelium tissues using TaqMan MicroRNA Array. According to the microarray results and literature reports, we selected10out of31candidate miRNAs to perform real-time quantitative PCR (qRT-PCR) in another20pairs of samples and found that three miRNA(miR-21, miR-135b, miR-133b) are abnormally expressed. Then, we verificated the three miRNAs and confirmed their specificity in ESCC from another50pairs of samples.Results:1, We screend out31candidate miRNAs by TaqMan MicroRNA Array.2, We verified three specific miRNAs which are related to ESCC(miR-21, miR-133b and miR-135b) in large ESCC samples by qRT-PCR.Conclusion:miR-21, miR-135b and miR-133b are specific miRNAs related to ESCC. Part II Prediction and Validation of the target genes of MiRNA-133bPurpose:To predict and validate the target genes of miR-133b.Methods:We predicted the downstream target genes of the abnormally expressed miRNAs from three databases (miRBase, TargetScan and PicTar) and literature reports, and screened out the candidate target genes of miR-133b in ESCC. Then, we detected the expression levels of these candidate genes by qRT-PCR, Western blot and Immunohistochemistry (IHC) and detected the expression level of miR-133b by qRT-PCR in40pairs of ESCC and adjacent normal esophageal epithelium tissue samples.Results:1, LASP1, CDKN2AIP and BCL2L2might be target genes of miR-133b by bioinformatics study.2, We found that the expression levels of miR-133b and LASP1were negatively related, while the expression levels of CDKN2AIP and BCL2L2were not correlated with miR-133b; the result of immunohistochemistry revealed that the expression of LASP1was located in cytoplasm and nucleus of ESCC tissue.Conclusion:LASP1is a target gene of miR-133b. Part Ⅲ The mechanism of MiRNA-133b and LASP1in ESCCPurpose:To clarify the pathogenesis mechanism of MiRNA-133b and LASP1in ESCC.Methods:We cultured the esophageal squamous cell carcinoma cell lines ECA109and KYSE510in vitro. We instantaneously transfected both cell lines with miR-133b-mimic and siRNA-LASP1and detected the expression level of miR-133b and LASP1and the impact of proliferation, apoptosis, invasion and migration by MTT assay, Annexin V-FITC assay, Millcell assays respectively.Results:1,72hours after the transfection of miR-133b-mimic, the expression level of miR-133b were obviously increased in ECA109(p=0.000) and in KYSE510(p=0.024) compared with the negative control; However, the expression level of LASP1were obviously downregulated by in ECA109(p=0.017) and in KYSE510(p=0.006) compared with the negative control.2, The expression level of miR-133b were not changed72hours after the transfection of siR-LASP1, but the expression level of LASP1on mRNA and protein level were obviously repressed by in ECA109(p=0.006,0.003) and in KYSE510(p=0.039,0.001) campared with control.3,The upregulation of miR-133b or silencing of LASP1by transfection with miR-133b-mimic or siR-LASP1at24,48and72hours in ECA109and KYSE510inhibited the proliferation using the MTT assay. Fouthermore, the decreasing effect on cell proliferation was enhanced after co-transfected with miR-133b-mimicand siR-LASP1compared with negative-control-siR and non-transfected in ECA109and KYSE510(p=0.000, p=0.002; p=0.007, p=0.027, respectively).4, The invasion ability were inhibited significantly after transfected with miR-133b-mimic (p=0.001, p=0.000) or siR-LASP1(p=0.001, p=0.000)at18hours in ECA109and KYSE510.5,The migration ability were inhibited significantly in ECA109and KYSE510cells after the transfection of miR-133b-mimic(p=0.01, p=0.005) or siR-LASP1(p=0.02, p=0.005) at8hours.Conclusion:1. LASP1is the target genes of MiR-133b, and MiR-133b could inhibit the expression of LASP1.2. LASP1could enhance the proliferation, invasion and magrition ability of ESCC which would be reversed by MiRNA-133b.