Expression Of CCT3 In Papillary Thyroid Carcinoma And Its Effect On Tumor Cell Proliferation

Background InformationThyroid cancer is the most common endocrine malignancy,including well-differentiated papillary thyroid cancer(PTC),which accounts for 80%to 90%of the incidence of thyroid cancer.Patients with suspected thyroid nodules are usually examined by ultrasound,computed tomography(CT),etc.,followed by preoperative ultrasound-guided fine needle aspiration cytology(FNAC)and intraoperative pathological examination for the diagnosis of papillary thyroid cancer.FNAC is the most reliable clinical diagnosis method,but 20%of thyroid pathology is diagnosed by FNAC as atypical follicular lesion,follicular tumor or suspected follicularity tumor,or a suspected malignancy tumor.Limitation of accurate preoperative diagnosis increase the non-necessary surgical procedures.5 percentage of patients who underwent surgical resection of the tissue still required a second thyroidectomy.The incidence of recurrence rates detected during double surgery was significantly higher.Combination of molecular markers to diagnosis can improve the detection of tumors.CCT3 is involved in folding protein structure and cell proliferarion in tumor cells,and has important reference significance in the diagnosis and prognosis of liver cancer and lung cancer.The treatment of papillary thyroid carcinoma is mainly include total thyroidectomy(or subtotal resection),adenectomy of thyroid gland and isthmus,postoperative adjuvant treatment with radioactive iodine and endocrine suppression drugs.Although more than 90%of patients are effectively controlled by surgery and postoperative care,there are still 5-10%of patients with metastasis and recurrence.MAPK signaling pathway is the main pathway driving the pathogenesis and development of thyroid cancer.The activation of this pathway depends on the cell membrane surface receptors binding to ligands,such as VEGFR,FGFR and so on.The USA FDA-approved drugs for the treatment of relapsed metastatic refractory thyroid cancer are sorafenib and Lenvatinib,which can simultaneously target inhibit VEGFR,FGFR,PDGFR,c-Kit,and RET/PTCs.Multi-target inhibitors have also shown that the adverse effects of these two drugs are particularly strong,and the treatment of refractory thyroid papillary carcinomas still lacks effective and specific therapeutic targets and methods.Silence of CCT3 can inhibit the proliferation and development of hepatocellular carcinoma,but there are few reports on specific molecular targets in PTC.PurposesTo study the distribution and relationship between CCT3 and papillary thyroid carcinoma and adjacent tissues,and to analyze the possibility of CCT3 as a new diagnostic molecular marker for papillary thyroid carcinoma.To study the effect of CCT3 on the biological function of papillary thyroid carcinoma cells,and to evaluate the significance of CCT3 as a new therapeutic target.To study the qualitatively and quantitatively by observing the expression of CCT3 in clinical papillary thyroid carcinoma tissues.It was investigated whether CCT3 could be a new molecular marker for the diagnosis of papillary thyroid carcinoma.At the same time,the mechanism of CCT3 on the development of papillary thyroid carcinoma was studied,and whether CCT3 could become a new therapeutic target was explored.Methods(1)Diagnosis and identification of thyroid papillary carcinoma:The cancer tissues and paracancerous tissues of the patients with clinical papillary thyroid carcinoma were collected.The samples were confirmed by the pathologist to be diagnosed as papillary thyroid carcinoma by fixation,sectioning and HE staining.(2)Detection of CCT3 expression in papillary thyroid carcinoma:The confirmed postoperative tissue sections were analyzed by immunohistochemical staining.The expression of CCT3 in the samples was qualitatively and quantitatively analyzed by tissue fractionation,which were used to analyze the relationship of CCT3 and papillary thyroid carcinoma.(3)RNA interferes with CCT3 in PTC cells:shRNA lentiviral vectors that target CCT3 are constructed.The shRNA lentiviruses were used to infect the PTC cell with high expression of CCT3.Real-time quantitative PCR and Western Blot detected the silencing effects.(4)Detection of CCT3 expression in PTC cells:This study used real-time quantitative PCR to analyze the expression levels of CCT3 in two PTC cell lines.Selecting the cell line with higher CCT3 expression level as a research tool.Constructing RNAi target CCT3 vector knockdown the expression of the PTC cell,and study the effect of CCT3 on the cell biology of PTC cells.(5)Detection of cell proliferation ability:In this study,the cultured cells were counted by Celigo,and the cell viability was detected by MTT assay.The results were used evaluate the effect of silencing CCT3 on the proliferation and viability of PTC cells.(6)Cell cycle assay:Flow cytometry and nuclear staining were used to detect the cell ratio of CCT3 silenced PTC cells and control cells in each cell cycle to study the effect of CCT3 on cell cycle.(7)Cell apoptosis assay:In this study,apoptotic cells were stained with Annexin V,and the effect of silencing of CCT3 on PTC cells was detected by flow cytometry.Results:This study confirmed the clinical tissues of papillary thyroid carcinoma and detected the distribution of CCT3 in cancer tissues.Then,the effects of knockdown the CCT3 on proliferation,cell cycle and apoptosis on thyroid papillary carcinoma cells were studied.The results are as follows:(1)CCT3 is highly expressed in the clinical papillary thyroid carcinoma tissues,but the paracancerous tissues are almost not expressed,and the expression of CCT3 expressed in the nucleus and cytoplasm,and the relative nuclear distribution is higher.Quantitative analysis of tissue scoring showed that the expression level of CCT3 in cancer tissues was significantly higher than that in paracancerous tissues,and the difference was statistically significant.(2)The high expression of CCT3 in the two PTC cell lines and tissues indicated that CCT3 is important for the clinical diagnosis of papillary thyroid carcinoma.(3)shRNA lentiviral vector was used to knockdown the CCT3 in PTC cells.The expression of CCT3 mRNA and protein expression were detected by real-time quantitative PCR and Western Blot,respectively.The results showed that CCT3 was significantly silenced in PTC cells.(4)Silencing CCT3,which is highly expressed in PTC cells,significantly inhibited the proliferation and viability of PTC cells,indicating that CCT3 is associated with proliferation of papillary thyroid carcinoma cells.(5)Silencing CCT3 can reduce the ratio of cells in G1 and S phase of PTC cells,and arrest the cell cycle in G2/M phase,which indicated that CCT3 is involved in promoting the normal progression of the PTC cell cycle.(6)Inhibiting the expression of CCT3 can induce cell apoptosis of papillary thyroid carcinoma cells,suggesting that CCT3 plays a vital role in the survival of papillary thyroid carcinoma.Conclusion:Papillary thyroid carcinoma tissue expresses chaperone protein CCT3.CCT3 is distributed in the nucleus and cytoplasm,and the nuclear expression is relatively high.Clinically CCT3 could be a marker for the diagnosis of papillary thyroid carcinoma.CCT3 is commonly expressed in papillary thyroid carcinoma cell lines and promotes the regulation of proliferation of papillary thyroid carcinoma cells.Silencing CCT3 blocks the papillary thyroid carcinoma cell cycle in the G2/M phase,indicating that CCT3 is involved in the progression and regulation of the G2/M phase of the cell cycle.CCT3 has a protective effect on apoptosis of papillary thyroid carcinoma.CCT3 could be used as a new therapeutic target for clinical treatment of papillary thyroid carcinoma.