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Diagnostic Application and Mechanism of miR-17-5p/20a in Gastric Cancer

Author: WangMei
Tutor: XuWenRong
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: miR-17-5p miR-20a gastric cancer molecular mechanism circulating microRNA prognosis
CLC: R735.2
Type: PhD thesis
Year: 2012
Downloads: 256
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Abstract


Objective:MiR-17-5p and miR-20a (miR-17-5p/20a) have been demonstrated to be dysregulated in many kinds of tumor. However, their expression profiles, clinical significance、mechanisms and application in gastric cancer diagnosis、therapeutic effect monition and prognosis prediction are still unclear. In the present study, the objectives of the study are to uncover their clinical significance and their roles, and elucidate the underlying mechanisms and determine the diagnostic application of their circulating levels in gastric cancer.Methods:The miR-17-5p/20a expression levels in 157 gastric cancer tissues and corresponding adjacent cancer tissues were determined by quantitative RT-PCR.Their clinical significance were analyzed combined with the clinical pathological materials. The expression levels of miR-17-5p/20a were compared among gastric cancer cells (HGC-27, BGC-823, SGC-7901 and AGS) by quantitative RT-PCR and gastric mucosal epithelium cell line GES-1 was served as a control. The gastric cancer cell line, with the lowest expression level of miR-17-5p/20a, was selected to be transected with mina mimics and overexposing miR-17-5p/20a, while the gastric cancer cell line with the highest expression level of miR-17-5p/20a was selected to be transected with mina inhibitors and underexposing miR-17-5p/20a. Cell count, Plate clone forming assay, Flow cytometry assay, TUNEL assay, Western blot for the proliferation and apoptosis related proteins were performed to determine the function of miR-17-5p/20a in gastric cancer cells. Gastric cancer cell derived xenografts model was established subcutaneously in BALB/c nude mice in vivo. Antagomir-17-5p and antagomir-20a were administered to inject in and treated the tumor generated in the nude mice. The therapeutic effect of targeting inhibition of miR-17-5p/20a on gastric cancer were evaluated by tumor volume measure, tumor growth curve, quantitative RT-PCR for miR-17-5p/20a expression levels in tumor tissues. TUNEL assay for cell apoptosis in pathological section and Western blot for the proliferation and apoptosis related proteins. The two bioinformatic programs TargetScan and PicTar were used to predict the targets for miR-17-5p/20a. Luciferase reporter vectors containing wildtype and mutant type 3’UTR were constructed. Lucifease activity test, quantitative RT-PCR and western blot were used to validate and determine the targets of miR-17-5p/20a. The relation between targets protein levels and miR-17-5p/20a levels in gastric cancer tissues and adjacent cancer tissues were investigated by Western blot. SiRNA against MDM2 or its expression vector cotransfected with miRNA mimics were conducted to elucidate the underlying mechanisms of miR-17-5p/20a in gastric cancer. The plasma from unpaired 65 pre-operative,16 post-operative, and 6 relapse gastric cancer patients and paired pre-operative and post-operative plasma from 14 gastric cancer patients were recuited in this study.90 healthy volunteers’ plasma were set as control. The circulating levels of miR-17-5p/20a in all groups were tested by quantitative RT-PCR. Their clinical significance was analyzed combined with the clinical pathological materials of the pre-operative groups. The overall survival rate for the pre-operative patients was followed up and recorded. Kaplan-Meier curve and Cox regression models were performed to analyze whether the circulating levels of miR-17-5p/20a was valuable for prognosis prediction. The alteration of miR-17-5p/20a levels in serum were measured by quantitative RT-PCR before and after the mouse tumor model treated with antagomir-17-5p/20, which was used to evaluate the role of the circulating levels of miR-17-5p/20a in therapeutic effect monitoring.Results:Quantitative RT-PCR method for miR-17-5p/20a were successfully established. The expression levels of miR-17-5p/20a were just associated with the volume of gastric cancer tissues. The expression levels of miR-17-5p/20a were lowest in AGS, but highest in SGC-7901. miR-17-5p/20a overexpression resulted in increasing cell number, promoting cell enter S phase in advance, reducing the number cell apoptosis, upregulating the levels of CDK2, p-ERK and BCL2. inhibiting Caspase3 activation. miR-17-5p/20a underexpression lead to inhibiting the cell clone forming, blocking cell in G1/G0 phase, inducing cell apoptosis, downregulating the levels of CDK2, p-ERK and BCL2, and activating Caspase3. The bioinformatic programs prediction combind with these validated experiments demonstrated that miR-17-5p/20a inhibit p21 and TP531NP1 expression at posttranscriptional levels through 3’untranslated regions (UTR) of mRNA. The inverse relation between miR-17-5p/20a and TP53INP1 exists in gastric cancer tissues. However, this kind of relation between miR-17-5p/20a and p21 was not seen. MDM2 protein level was found to be always negatively correlated with p21 in these tested tissues. MDM2 protein level could be increased when miR-17-5p/20a were upregulated and it was reduced when miR-17-5p/20a were downregulated. Knockdown of MDM2 reversed miR-17-5p/20a inhibition of p21 and abolished their roles in increasing cell number, promoting cell enter S phase in advance, reducing the number cell apoptosis, upregulating the levels of CDK2, p-ERK and BCL2, inhibiting Caspase3 activation. Restoration of MDM2 could particially recover miR-17-5p/20a inhibition of p21 and their function in promoting gastric cancer cell proliferation, which rescued the impair effect of MDM2 konckdown on miR-17-5p/20a. The concentrations of miR-17-5p/20a in the preoperative group were significantly associated with the differentiation status and TNM stages of gastric cancer. Compared to the circulating levels of miR-17-5p/20a in the preoperative group, their levels were obviously downregulated in the postoperative group and elevated in the relapse group which were similar to those in the preoperative group. Kaplan-Meier curve, logrank test and Univariate Cox regression analysis revealed that high expression levels of miR-17-5p/20a were significantly correlated with poor overall survival. Multivariate Cox regression analysis indicated that the level of plasma miR-20a was an independent risk predictor for prognosis. miR-17-5p/20a levels in serum from the mouse tumor model were notably and gradually reduced after treated with antagomir-17-5p/20a.Conclusion:miR-17-5p/2Oa promote cell cycle progression and inhibit cell apoptosis via post-transcriptional inhibition of p21 and TP53INP1, which lead to promote gastric cancer cell proliferation. MDM2 is an important mediator for miR-17-5p/20a negatively regulation of p21 and function, and synergy with miRNAs to act as oncogenes, which may provide novel evidence for the oncogenic roles of miR-17-5p/20a in gastric cancer. miR-17-5p/20a were detectable in plasma of gastric cancer patients by Quantitative RT-PCR. Their circulating levels could indicate the burden of gastric cancer and pathological progression, reflect therapeutic effect and predict prognosis. Our work will provide novel molecular mechanism evidence for the oncogenic roles of miR-17-5p/20a in gastric cancer, and effective therapeutic targets for gastric cancer therapy and novel noninvasive molecular marker for gastric cancer.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Gastric neoplasms
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