IQGAP1Plays An Important Role In The Tumorigensis And Invasion Of Papillary Thyroid Carcinoma

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid malignancies, accounting for more than80%of all thyroid cancer. Moreover, the incidence of PTC has increased dramatically in the past several decades. Several genetic factors are involved in the development of PTC, including multiple regulatory RNA genes, the activation of the MAPK signaling pathway as a result of point mutation within BRAF, RAS or RET/PTC rearrangement. Treatment for PTC by surgery and radio-iodine therapy is highly effective. Several certain clinicopathologic features have been associated with poor PTC prognosis, particularly, extrathyroidal invasion has been demonstrated as a significant independent prognostic factor for survival.The189-kDa mammalian IQ-domain GTPase-activating protein1IQGAP1protein which has ubiquitous tissue expression was the best characterized member of IQGAP family. The function of IQGAP1protein which contains several protein-interacting domains includes cytoskeletal regulation, coordinating cadeherin mediated cell-cell adhesion, cell polarization and actin reorganization to promote cell migration. IQGAP1binding partners involve in several cell signal pathways including mitogen-activated protein kinase (MAPK) cascade, Wnt pathway, phosphoinositide3-kinase (PI3K)/Akt signaling and cell processes affect cell proliferation, differentiation and apoptosis. Accumulating evidence strongly supports a role for IQGAP1in tumorigenesis, tumors invasion/metastasis and its binding partners coupled with the existing evidence for its role in neoplasia strongly suggests that IQGAP1is an oncogene.It has been approved that IQGAP1protein plays a role in thyroid tumorigenesis. It is not well known whether IQGAP1protein can promote papillary thyroid carcinoma invasion and metastasis. Therefore, our objective was to identify the different IQGAP1expressions in primary tumor, para-tumor and normal tissues and the metastatic nodules. We also investigated its relationship with tumor proliferation, invasion and metastasis.MethodsThe tumor samples were collected included primary tumor, para-tumor and lymph nodules metastatic samples from PTC patients who underwent surgeries from Mar to Jul2013in the first affiliated Xiangya Hospital of central south university. These samples were divided into two groups, with metastatic lymph nodules group and without metastatic lymph nodules group.13normal tissues (from patients who underwent benign nodules dissection) were used as control group. Fresh samples were detected for expression levels of IQGAP1by RT-PCR, western blotting, immunochemistry assays. Genetic knockdown with siRNA was used to study IQGAP1protein function. We examined in vitro cell growth by MTT assay and soft-agar colony formation, cell proliferative index (PI) and apoptosis by Flow cytometry assay. Cell migration/invasion ability was investigated by transwell assay and Scratch assay.Results1. IQGAP1expression is elevated in papillary thyroid cancer tissues and cellsWe detected the expression of IQGAP1mRNA and protein in papillary thyroid cancer tissues and cells by RT-PCR, Western blotting and immunohistochemical staining. The levels of IQGAP1mRNA and Protein in cancer tissues were significantly higher than para-tumor tissues and normal thyroid tissues. No significant difference in IQGAP1mRNA and protein expressions was observed between normal thyroid tissues and para-tumor tissues. In addition, we also observed the highest expression level of IQGAP1in metastatic lymph nodules, whereas there is no difference between primary tumors from each group. PTC cell lines K1, BCPAP and TPC-1had higher IQGAP1mRNA and protein expression levels than normal cell line, and K1, TPC-1cells had higher IQGAP1expression levels than BCPAP cells.2. RNA interference down-regulates IQGAP1expression in PTC cellsTwenty four hours after transfection, the efficiency was over80%, and the results by Western blotting assay demonstrated that siIQ2had the strongest inhibitory effects for both K1and TPC-1at the dose of2μmol/L. Compared to blank control cells, siIQNC cells showed no reduction of IQGAP1level, while siIQ2cells exhibited more than70%reduction of IQGAP1level.3. Gene silencing of IQGAP1inhibits proliferation of PTC cellsCell proliferation was monitored for4d after K1, TPC-1cells transfected with siIQNC and siIQ2. During these four days, the growth rate of K1and TPC-1cells was reduced markedly (p<0.05). In addition, no difference was found between the blank and negative control cells over the entire experiment period. Flow cytometry assay was used to analyze the cell cycle distribution profile. The results showed that siIQ2cloud significantly decreased the proliferation of K1and TPC-1cells according to the percentage of cells in S phase and PI (p<0.05). Down-regulation of IQGAP1expression caused G2cell cycle arrest, decreased the proliferation rate and increased apoptosis rate in K1and TPC-1cells. Down-regulation of IQGAP1suppressed the colony-forming capacity of K1and TPC-1cells.4. Down regulation of IQGAP1protein inhabit PTC cellular invasion and migration in vitroIn the invasion assay, the date showed that the K1and TPC-1cells had more numbers of the migrated cells in the blank control and siIQNC groups competed with siIQ2groups (p<0.05). These results indicated that reduced IQGAP1expression attenuated the invasive potential of PTC cells in vitro. Cells infected with siIQ2migrated more tardily and filled in the would more slowly than siIQNC group and blank control group.ConclusionIQGAP1is an integral component of cell motility and participates in cell migration and invasion of PTC. IQGAP1over-expression is correlated with tumor progression. IQGAP1may serve as a biomarker for diagnosis of malignant PTC.