The Expression Of MiR-124and The Mechanism Of MiR-124in Suppressing Tumor Proliferation, Invasion And Metastasis By Targeting IQGAP1in Thyroid Papillary Cancer

BackgroundThyroid cancer is one of the most common endocrine malignant tumor worldwide and account for94.5%of all endocrine tumor and2.6%of all malignant tumor. The incidence of thyroid cancer is ranked first place in head and neck cancer. Since the1980s, the incidence of thyroid cancer has been increasing. Among which the incidence of thyroid papillary cancer(PTC) account for85%-90%.The occurrence and developing of thyroid cancer consider to be a complex process, including several oncogenes, suppressor genes, tumor-related protein aberrant expression, As a result, inducing the abnormal proliferation and mutation, eventually the formation of tumor. Therefore, making clear the molecular events underlied the pathogenesis of PTC, searching the biomarker for early diagnosis, metastasis prediction, and treatment, are important for further increase of cure rate, survival rate.In recent years, MicroRNA (miRNA) have been recognized as one of the many types of noncoding small RNAs families. Mature miRNA can target specific mRNA by binding its3’UTR with incomplete complementary, resulting in the degradation or translation suppression of targeted mRNA. Previous studies have suggested that the deregulation of miRNAs may contribute to the development of many of human cancer including liver cancer, gastric cancer, gallbladder carcinoma, breast cancer and thyroid cancer. Thus further research in miRNAs expression and fiction will contribute to understand the pathogenesis of tumor and may profit in the early diagnosis treatment and prognosis of cancer.IQGAP1(IQ motif-containing ATPase-activation protein1) is a protein family found recent year, the up-regulated expression of which are discovered in many cancers and cancer cell lines. As an oncogene, IQGAP1can bind to several specific proteins belonging to signal pathway. Several studies have validated that IQGAP1involves in invasion and metastasis of thyroid cancer. However, the molecular mechanism underlied of differential expression of IQGAP1is still unclear.This study aims to investigated the expression of miR-124in human PTC and explore the function and mechanism of miR-124in tumor invasion and metastasis of PTC. To validate IQGAP1was the target gene of miR-124.Methods1. Investigation the expression of miR-124by Real-time PCR in15without metastasis PTC tumors,8with lymph node metastasis primary PTC tumors, matched8lymph node metastasis tumor tissues and6in adjacent thyroid tissues from thyroid adenomas. These tissues had been collected from thyroid resection and were diagnosed by Xiangya Hospital in Central South University during2013.3-2013.10.Three human PTC cell lines (K1, BCPAP, TPC-1) and a normal thyroid cell line (Nthy-ori3-1) were detected the difference of miR-124expression.2. Transfecting Has-miR-124mimics into Kl cell line by use of LipofectamineTM2000.Cells transfected with no significant homology in negative group and blank group were treated in parallel. After successful transfection, Real-time PCR were performed to detect miR-124expression. Real-time PCR and western blot were using to detect the expression of IQGAP1; In vitro assay, the proliferation of cells was evaluated and graphed by MTT assay. The migration was evaluated by Wound Healing assay. The migration and invasion ability were detected by using the Transwell assay. Cell proliferative index (PI) and apoptosis by Flow cytometry assay3. Utilizing three bioinformatics tools miRanda、TargetScan、 miRbase to predict IQGAP1maybe the downstream target gene of miR-124and validated by Real time PCR,Western bolt and Luciferase Reporter assay.Results1. Real-time PCR analysis showed the levels of miR-124 expression is significantly downregulated in PTC tissues and PTC cell lines compared with adjacent thyroid tissues from thyroid adenomas and normal cell line(P<0.01).2. Real-time PCR analysis showed that miR-124expression of Kl cells was increased by having been transfected with the miR-124mimics (P<0.05). MiR-124mimics reduced cell proliferation after transfection24-72h compared with the negative control and blank control (P<0.05). Flow cytometry showed that the up-regulation of miR-124could significantly decreased the proliferation of Kl and cells according to the percentage of cells in S phase and PI (P<0.05). Up-regulation of miR-124expression caused G2cell cycle arrest, decreased the proliferation rate and increased apoptosis rate in Kl cells. Enhanced miR-124reduced the migratory ability of Kl cells (P<0.05) The invasive capacity was also suppressed in the enhanced miR-124transfectants compared with the NC cells(P<0.01).3. IQGAPl is a target gene of miR-124in Kl cells through bioinformatics and dual-luciferase reporter assay system. Both Real-time PCR and Western blot analysis showed that the expression levels of IQGAP1was suppressed after being transfection with miR-124compared with in the NC.Conclusions 1. The levels of miR-124expression in the PTC tumors tissues especially in lymph node metastasis tumor tissues and cell lines were lower than in the normal tissues and cell lines.2. As a suppression gene, the expression of miR-124can reduce the cell proliferation, migratory and invasive capacity,suggesting its potential therapeutic target role.3. Up-regulation of miR-124can suppress the expression of IQGAPl.Both of them maybe involve in the regulation of PTC signaling pathways.