The Expression Of CCT3 And IQGAP3 Related With Poor Prognosis In Hepatocellular Carcinoma And Its Mechanism

Hepatocellular carcinoma(HCC) is the third leading cause of cancer-related mortality worldwild, causing 696, 000 deaths each year. Although the prognosis of late-stage HCC has improved during the past two decades, its 5-year survival rate is very low.At present, the commonly used method of treatment for HCC is liver resection,but tumors at later stages may be inoperable. Traditional detection methods such as ultrasound, magnetic resonance imaging and computed tomography are helpful and efficient in detecting tumor location and metastasis, but cannot reliably detect early-stage HCC, and fail to diagnose HCC when they are used alone. To date,α-fetoprotein(AFP) is the most important serological marker recommended for screening patients at high-risk for HCC. However, nearly 40% of patients with HCC,including some with small tumors, have normal serological AFP levels. Therefore, it is important to develop reliable biomarkers for early detection and diagnosis of HCC for clinical treatment and prognosis.The chaperonin containing TCP1 complex(CCT), also called TRi C or c-cpn,mediates protein folding in the cytosol. Chaperonins are ATP-dependent protein-folding machines that are present in all kingdoms of life. They consist of two back-to-back stacked oligomeric rings with a cavity at each end, where protein substrate binding and folding take place. The chaperonin family includes white karor, mitochondrial heat shock protein 60, chloroplast Rubisco small subunit binding protein and molecular chaperone II archaea group. CCT3(60 k Da) is a critical subunit in CCT/TRi C complexes, which plays a significant role in specifically binding these factors during protein folding or refolding. Several studieshave shown by quantitative RT-PCR and western blotting that CCT3 is overexpressed in patients with HCC. CCT3 can affect the progression of HCC, by having an impact on the transport of phosphorylated signal transducer and activator of transcription(STAT)3/STAT3 into the nuclei of HCC cells. CCT3 may play a role in regulating microtubular structure and function(capture of kinetochores) and affect the cellular sensitivity to these microtubule-targeting agents.The IQ-motif-containing GTPase-activating protein(IQGAP) family comprises three members: IQGAP1, IQGAP2 and IQGAP3. IQGAP3 is the latest addition to this family. In the current study, it was involved in the proliferation of epithelial cells, however, its role in tumorigenesis remains to be determined. IQGAP3 promotes cell proliferation through the Ras/extracellular signal-regulated kinase(ERK) pathway. In the present study, expression of IQGAP3 was increased during proliferation at sites of cell–cell contact in hepatocytes. Therefore, one of the most important future issues is to identify the cell-density-sensing units at cell–cell contacts in association with IQGAP3. The IQGAP3-related signaling pathway may be one of the complex signaling routes involved in liver regeneration. The contribution of Ras-, Rac-, and Cdc42-binding IQGAP3 to liver regeneration suggests that IQGAP3 might lead to effective coordination of these signaling processes for cell proliferation and tissue remodeling.In this study, using the technology of gene transfection and RNA interference to further investigate the role of CCT3 and IQGAP3 in the development and invasion of liver cancer process. With different metastatic potential of hepatocellular carcinoma cell lines and non-tumor liver cell lines transfected with vector applications designed and synthesized for CCT3, IQGAP3 gene-specific small interfering RNA(small interfering RNA, si RNA). Using transiently transfected manner, si RNA transfected human hepatoma cell line and disturbed the expression of CCT3 and IQGAP3 gene. To observe the influnce on invasion of hepatocellular carcinoma cell lines after the knowdown the CCT3 and IQGAP3 gene. Meanwhile,to invastagate the expression of some signaling pathways using the Western-blot assay before and after cells to be transfected. It is purpose to explore that the possibility of CCT3 and IQGAP3 as tumor markers and therapeutic targets in HCC,and to improve the theoretical foundation of early diagnosis, prognosis and treatment of HCC.Innovations:1. In this study, we investigated CCT3 and IQGAP3 plasma levels in patients with hepatocellular carcinoma(HCC) or cirrhosis and in healthy individuals, and evaluated their application in detecting small and AFP-negative tumors in patients with HCC. Our findings indicate that CCT3 and IQGAP3 are novel biomarkers complementary to AFP in HCC diagnosis, whose expression is probably independent of AFP. This is especially valuable when AFP is negative and HCC is at an early stage. Thus, determination of CCT3 and IQGAP3 in combination with AFP increases the diagnostic sensitivity and specificity of HCC.2. To explore the mechanism of CCT3 promote occurrence, development and invasion of HCC. IT is indicated that there are close contact between the role CCT3 promoting tumor growth and JAK / STATs signaling pathway. The mechanisms of CCT3 promote invasion of HCC may be related to JAK / STATs pathway activation.3. TO learn the mechanism of IQGAP3 promote proliferation and invasion of HCC. There are closely linked between the role of IQGAP3 promoting tumor growth and Ras / ERK signaling pathway. Ras family proteins, the Rac/Cdc42 plays an important role in development of HCC and establish a theoretical foundation to develop gene targeted therapy of HCC.Part I Expression and diagnostic value of CCT3 and IQGAP3 in hepatocellular carcinomaAim:To evaluate plasma chaperonin containing TCP1 complex subunit 3(CCT)3 and IQ-motif-containing GTPase-activating protein(IQGAP)3 for hepatocellular carcinoma(HCC) diagnosis.Methods:Blood samples were collected from 126 patients with HCC, 88 patients with cirrhosis and 50 healthy volunteers to detect plasma α-fetoprotein(AFP),chaperonin containing TCP1 complex subunit 3(CCT3) and IQ-motifcontaining GTPase-activating protein-3(IQGAP3) levels. Plasma AFP, CCT3 and IQGAP3 protein levels were measured by ELISA.Results:In the plasma of HCC patients, both CCT3 and IQGAP3 were significantly higher than in patients with cirrhosis and in healthy controls(P < 0.01). CCT3 and IQGAP3 protein level correlated well with HCC etiology, tumor size, TNM stage, and Child–Pugh classification. CCT3 protein had better sensitivity in the diagnosis of HCC when compared with AFP(0.846 vs 0.667). In addition,CCT3 and IQGAP3 protein were able to complement AFP in detecting AFP-negative HCC patients with sensitivity and specificity of 92.1% and70.5%and 81.6% and 71.6%, respectively. In the small HCC group, CCT3 and IQGAP3 protein had sensitivity of 76.1% and 75.3%, respectively. The combination of AFP + CCT3 + IQGAP3(0.954) had significantly superior discriminative ability than AFP alone(0.815; P < 0.01).Conclusion:CCT3 and IQGAP3 are promising complementary biomarkers for HCC diagnosis, especially for AFP-negative and small tumors.Part II Effect and mechanism of CCT3 in the Invasion of HCC cell linesAim:To study the role of CCT3 genes in HCC cell lines. Detection the expression of genes CCT3 with different invasive capability HCC cell line and non-tumor liver cell line. Designed and synthesized the specific small interfering RNA for CCT3 gene(small interfering RNA, si RNA), To study the effect and mechanism of invasion of CCT3 in HCC MHCC97 H and Hepg2 cell lines.Methods:Western blot was used to detect the expression of CCT3 protein in the cell lines with different invasion. Designed and synthesized the specific small interfering RNA(small interfering RNA, si RNA) for CCT3 gene. Using Lipofectamine transfect MHCC97 H and Hepg2 cell lines. Real-time quantitative PCR and Western blot were used to detect the expression levels of CCT3 m RNA and protein in each group after transfection. The invasion of the treated Hep G2 and MHCC97 H, which were transfected by the CCT3-si RNA, using Transwell cell culture chambers to analyzed.Using Western blot to detect the expression of STAT3 and(p)STAT3 in each groups.Results:The expression of CCT3 protein in Hep G2 and MHCC97 H was higher than that of HL7702. Knocking down the expression of CCT3 by si RNA into Hep G2 and MHCC97 H cell, the expression levels of CCT3 protein and m RNA were distinctly downregulated by Western blot and Rt PCR. The expression of CCT3 can inhibit by CCT3-si RNA. The MHCC97 H and Hep G2 cells were transfected with CCT3-si RNA. Using the transwell cell culture chambers to test the invasion capability of treated cells. The results indicated that the number of CCT3 si RNA-transfected Hep G2 and MHCC97 H that tranfered through the Transwell was distinctly less than the control group. We found that the protein expression levels and proteolytic acticity of STAT3 and(p)STAT3 were decreased in CCT3-si RNA-transfected MHCC97 H and Hep G2 using Western blot.Conclusion:The process of the invasion and metastasis of HCC, CCT3 may play the important role. Si CCT3 can reduce the invasion and the expression and proteolytic acticity of STAT3 and(p)STAT3. It means that silencing the expression of CCT3 could decrease the invasion of HCC cells through to regulate STAT3 and(p)STAT3.Part III Effect and mechanism of IQGAP3 in the invasion hepatoma cell linesAim:To clarify the role and the mechanism of IQGAP3 in promoting HCC cell proliferation, invasion and metastasis.Methods:Analysis the downregulation of expression of IQGAP3 in HCC cell lines, the change of Ras and ERK1 / 2 by Western-Blot and q RT-PCR. Study cell migration and invasion by Transwell and cell proliferation by MTT assay.Results:The expression of IQGAP3 protein in Hep G2 and MHCC97 H was higher than that of HL-7702(P<0.01). The IQGAP3-si RNA was transfected into MHCC97 H and Hep G2, and the expression levels of IQGAP3 protein and m RNA were effectively downregulated, detected by Western blot and real time PCR. The IQGAP3-si RNA can effectively inhibit the expression of IQGAP3. The Hep G2 and MHCC97-H cells were transfected with IQGAP3-si RNA, and the invasion capability of treated cells was tested by the transwell cell culture chambers. The results showed that the number of IQGAP3 si RNA-transfected MHCC97 H and Hep G2 cell lines that migrated through the Transwell was significantly less than the number of the control groups.We found that the protein expression levels and proteolytic acticity of Ras 和 ERK1/2were decreased in IQGAP3-si RNA-transfected MHCC97 H and Hep G2 using Western blot.Conclusion:Preliminary study the expression of IQGAP3 effect on biological behavior of hepatocellular carcinoma cells in vitro. We found that IQGAP3 can promote HCC cell proliferation and invasion, which the molecular mechanism may be active Ras /ERK1 / 2 signaling pathway.