Establishment And Significance Of Lentivirus Vector-mediated CCDC67 Trasnfected Human PTC Cell Line TPC-1 Mice Tumor Xenograft Model

Background and ObjectiveIn recent years,the incidence of thyroid carcinoma shows a trend of increase year by year,about 4% of all human malignant tumors,and is the highest rates in the endocrine system tumor,female incidence of this disease is significantly higher than male.Thyroid carcinoma according to the pathological classification can be divided into: thyroid papillary carcinoma,follicular thyroid carcinoma,undifferentiated thyroid cancer and medullary thyroid carcinoma.The thyroid papillary carcinoma and follicular thyroid carcinoma are collectively referred to as differentiated thyroid cancer,thyroid papillary carcinoma accounts for about 95% of thyroid cancer pathology classification.In recent years,the research and clinical experience have already shown that the majority of differentiated thyroid cancer after treatment with specification of prognosis is good,long-term survival rate is as high as 90%.But differentiated thyroid cancer still have relatively high transfer rate and recurrence rate.With the rapid development of precision medicine,molecular markers that thyroid cancer was found gradually.Besides the inactivation of tumor suppressor genes,the imbalance of apoptosis regulation of gene expression level is another important element in thyroid cancer occurrence and development process.The latest study confirms that coiled-coil domain containing 67 gene in molecular cytology level was preliminarily confirmed as a new tumor suppressor genes,and shows certain relations with the mutated BRAF gene mutation,the Bcl-2 / Bax expression level.Therefore,this study first used immunohistochemical method to test the CCDC67 and Bcl-2 protein expression of clinical surgical resection of thyroid carcinoma and adjacent tissues to analyzethe correlations of protein expression and clinical characteristics.A tumor-burdened nude mice model was built by lentivirus vector-mediated CCDC67 trasnfected human PTC cell line TPC-1.After the in-vivo imaging confirmed cell lines into tumor effect,nude mice was executed after the analysis of tumor weight,volume and tumor inhibitory rate.Then,mice tumor was paraffin embedded,HE staining was used to determine pathological types.Immunohistochemical SP method was used to detect CCDC67 protein the Bcl-2 protein,Bax protein,Caspase3 protein beta-catenin protein and the expression of Cox2 protein in mice tumor tissue section in order to preliminary explored the mechanism of difference between gene transfection and non-transfection group.Finally,TUNEL method was used to detect apoptosis in rat tumor tissue biopsies,and to analyze the apoptosis index of transfection group and the transfection.The experiment was designed to explore the biological effect of CCDC67 gene in nude mice in vivo,and preliminary analyses the molecular mechanisms of its role in the body.Material and Method1.Immunohistochemistry was used to detect the expression of CCDC67 and Bcl-2 gene in the surgical romoval papillary thyroid carcinoma tissue specimens and to analyze thecorrelation between CCDC67 and Bcl-2.2.Lentivirus vector carrying CCDC67 gene was used to construct cell line OE and empty vector was used to construct cell line NC.3.Cell line TPC-1 was preliminarily used to build 3 tumor mice model to confirm the tumor formation possibility and ability of TPC-1.Cell line OE,NC,CON were used to separately inoculate 30 BALB/c female nude mice.In vivo imaging system was used to detected the fluorescence peak intensity,and put mice to death on the 20 th day,measure and analyze the tumor volume,weight and tumor inhibit rate.4.RealTime-PCR was used to detect and comfirm the expression of CCDC67 in the mice tumor.Immunohistochemistry was used to detect the expression of CCDC67,Bcl-2,caspase-3,cox-2,β-catenin gene in the mice tumor papillary thyroid carcinoma tissue specimens and to analyze the result with ImageJ.5.SPSS software version 22.0 was used to analyze the statistical data,one-way ANOVA,chi-square test and t-test were used to analyze the significance.P<0.05 was deliberated statistically significant.Result1.CCDC67 protein expression rate is 43.86%(25/57)in the PTC tissue,which is correlated with tumor size,TNM stage,pathological grade,lymph node in patients。Bcl-2 protein expression rate is 57.89%(33/57)in the PTC tissue,which was correlated with TNM stage,pathological grade and lymph node in patients。The down-regulation and loss of CCDC67 and high-regulation of Bcl-2 present a positive correlation(r=0.400,P﹤0.05).2.Flow cytometry by Annexin V/PI shows higher apoptosis level in cell line OE than cell line CON and NC(P﹤0.05).3.Papillary thyorid carcinoma cell line TPC-1 has the capability of building BALB/c tumor-bearing mice.4.Tumor average size,average volume and the total amount of fluorescence of tumor area in OE group are lower than them of gourp CON and gourp NC(P﹤0.5).5.GourpOEnudemicetumorpresenthigh-regulated CCDC67 mRNAexpression,and gourp NC and CON shows an expression loss of CCDC67 mRNA(P﹤0.5).6.Immunohistochemical results show that CCDC67 gene in group OE present high expression in tissue section,the group NC and group CON expression level is lower than the OE(P ﹤ 0.05);Bax protein expression level in tumors of group OE is higher than that of group CON and NC and expression level difference between CON and NC group had no statistical significance(P ﹤ 0.05);the Bcl-2 protein expression level in group OE is lower than group CON and NC,and expression level difference between CON and NC group had no statistical significance(P ﹤ 0.05);β-catenin protein in group OE abnormal expression rate is lower than group CON and NC,and the expression level difference between CON and NC group has no statistical significance(P﹤0.05);Caspase-3 expression level in group OE is higher than group CON and NC(P﹤0.05);Cox-2in group OE is lower than group CON and NC(P﹤0.05).ConclusionCCDC67 gene expression in clinical specimens of patients after surgical resection of thyroid papillary carcinoma is missing,and down-regulation/missing of CCD67 gene expression has correlation with the patient’s tumor size,TNM stage of tumor and clinical pathological stage;The Bcl-2 gene expression is missing in PTC tissues,and it is correlated with the patient’s tumor TNM stage and clinical pathological stage;The expressions of CCDC67 and Bcl-2 proteins displayed a negative correlation.We use lentivirus to transfect CCDC67 gene to TPC-1 to construct a transferred cell line in order to build tumor-burdened nude mice model.In CCDC67 high expression group,tumors volume,weight and growth rate is inhibited,and according to the immunohistochemical detection results of Caspase-3 Bax/Bcl-2,β-catenin and cox-2 protein,it prompts CCDC67 may participate in the mechanism of regulation of apoptosis of tumor.Considering of the cytology and molecular biology experiment in vitro,we completely proved CCDC67 gene to be a new tumor suppressor genes.But the specific and exact signal transduction pathways involved by CCDC67 gene in the molecular mechanism remains unknown and needs further research in the future.