Research On Expression Of CD1d In Human Papillary Thyroid Carcinoma Cells And Its Mechanism Of Clinical Significance

The whole study was divided into three parts:Part I:Expression and significance of CD1d in human papillary thyroid carcinoma tissuesObjective:The aim of this study was to observe the expression of CD1d in human papillary thyroid carcinoma tissues and normal thyroid tissues,and to preliminarily investigate the relationship between CD1d and clinical features of human papillary thyroid carcinoma and its clinical significance in the diagnosis and treatment of papillary thyroid carcinoma.Methods:1.Tissue samples of 34 cases of papillary thyroid carcinoma and 30 cases of paraneoplastic tissues were collected from patients with primary papillary thyroid carcinoma treated 2.Immunohistochemical staining was used to detect the expression of CD1d in papillary thyroid carcinoma tissues and paraneoplastic tissues.3.The relationship between CD1d expression and patients’age,gender,cell subtype,centrality of lesions,size of cancer foci,presence of local invasion and lymph node metastasis was analyzed byx~2 test.4.Kaplan-Meier survival curves were used to analyze the survival differences between patients with papillary thyroid carcinoma in the high and low CD1d expression groups.Results:1.CD1d expression was significantly higher in papillary thyroid carcinoma tissues compared to paraneoplastic tissues,with its OD mean value of 0.22±0.01 in papillary thyroid carcinoma and 0.19±0.01 in paraneoplastic tissues,with statistically significant differences(p<0.05).2.The expression level of CD1d correlated with age,whether the lymph nodes metastasized and the difference was statistically significant(p<0.05),but CD1d did not correlate with gender,pathological subtype,centrality of the cancer foci,size of the cancer foci,clinical stage,or whether it was extravasated.3.High CD1d expression was associated with poor prognosis in papillary thyroid carcinoma with a statistically significant difference(p<0.05).Conclusions:1.CD1d expression was elevated in human papillary thyroid carcinoma tissues;2.CD1d expression was closely correlated with age,lymph node metastasis,CD1d did not correlate with gender,pathological subtype,centrality of the cancer foci,size of the cancer foci,clinical stage,or whether it was extravasated;3.Overexpressed CD1d suggested that patients had poor prognosis and high recurrence rate,suggesting that CD1d may play an important role in the metastatic invasion of papillary thyroid carcinoma.Part II:Effect of CD1d in the malignant biological behavior of human papillary thyroid carcinoma cellsObjective:To detect the expression of CD1d in human papillary thyroid carcinoma cell lines by quantitative real-time polymerase chain reactions(q RT-PCR)and Weston Blot in an in vitro assay.CD1d knockdown cell lines were constructed in human papillary thyroid carcinoma cell lines with lentivirus to detect the proliferation,apoptosis,migration and invasion ability of papillary thyroid carcinoma cells by altering CD1d expression in papillary thyroid carcinoma cell lines,and to preliminarily investigate the biological functions of CD1d in human papillary thyroid carcinoma cell lines and its possible role in tumor development.Methods:1.Western blotting was used to detect the expression of CD1d in human papillary thyroid carcinoma cell line TPC-3,and Western blot technique was used to detect CD1d protein expression.2.CD1d-targeted silencing of TPC-3 cells by interfering RNA(si RNA), construction of knockdown CD1d cell lines using lentiviral transfection,and detection of the effect of CD1d on cell proliferation ability using CCK-8 and cell cycle analysis assays.The effect of CD1d on the migration and invasion ability of thyroid cancer cells was detected using cell scratch assay technique;the effect of CD1d on apoptosis was detected by flow cytometric apoptosis.Results:1.CD1d expression was significantly higher in the papillary thyroid carcinoma cell line TPC-3 than in normal thyroid follicular cells Nthyori3-1.2.CCK8 assay revealed that interference with CD1d expression significantly inhibited the proliferative capacity of TPC-3.3.CD1d affects the cell cycle of papillary thyroid carcinoma and promotes cell transformation to G2/M phase;4.Scratch test showed that the invasion and migration ability of papillary thyroid carcinoma cell lines were significantly reduced after interfering with CD1d.5.Flow cytometry detection of apoptosis revealed increased apoptosis in papillary thyroid carcinoma cell lines after interference with CD1d expression.Conclusions:1.CD1d is highly expressed in papillary thyroid cancer cells and low expressed in normal thyroid cells.2.Downregulation of CD1d can significantly inhibit the proliferation of thyroid cancer cells,and the expression of proliferation-related proteins ALMS1,CDKN3,CDK6 and Ki-67 is decreased.3.Downregulation of CD1d can significantly reduce the proportion of cells entering the phase of cell division,and also reduce the migration ability of thyroid cancer cells.4.Downregulation of CD1d can significantly increased the apoptosis rate and decreased the expression of apoptosis-related protein Bcl2,and increased the expression of Bax and Caspase 3.Part III:Exploration of CD1d-related molecular biological mechanismsObjective:To explore the possible mechanism of CD1d in promoting the invasive proliferative capacity of human papillary thyroid carcinoma cells,and to clarify the molecular mechanism of CD1d regulation:κ-light chain enhancement of MAPK(mitogen-activated protein kinase)/NF-κB(nuclear factor-activated B cells).To elucidate the process mechanism of CD1d in the progression of human thyroid cancer cells.To investigate the molecular signaling pathway of the effect of CD1d on the malignant biological behavior of papillary thyroid carcinoma and provide new ideas for finding therapeutic targets for thyroid cancer.Methods:1.Differential gene expression mi RNAs were sequenced,and the sequencing results were enriched and analyzed using Kyoto Genome(GO)and Encyclopedia of Genes(KEGG).2.Combined with KEGG Mapper analysis to screen the molecular signaling pathway of CD1d affecting the development of human thyroid cancer,and further quantitative verification of molecular signaling pathway protein expression by Western blot.3.Western blot was used to detect changes in EMT(Epithelial-mesenchymal transition)-related indicators.Results:1.Analysis of RNA-Seq results yielded a total of 2541 differential genes,including 1260up-regulated genes and 1281 down-regulated genes.2.By analysis of GO gene enrichment,KEGG pathway enrichment functional annotation analysis,selected biological processes with p<0.05,the main enrichment of differentially expressed m RNAs was found in the tumor pathway,MAPK/NF-κB.3.After inhibition of CD1d expression in papillary thyroid carcinoma cells,E-Cadherin expression minus increased and N-Cadherin,Sani12and Vimentin expression decreased in the cells;4.PCR,Western Blot assay showed a statistically significant increase in total p-NF-κB(p-P65),NF-κB(P65),HRAS,p-P38 MAPK,and P38 MAPK expression.Conclusions:1.Downregulation of CD1d expression in papillary thyroid cancer cells suggested that CD1d promoted EMT function of thyroid cancer cells and induced cell migration and invasion.2.CD1d gene may affect the malignant biological behavior of papillary thyroid carcinoma cells through activation of MAPK/NF-κB signaling pathway.3.CD1d gene may provide a theoretical basis for gene targeted therapy of papillary thyroid carcinoma through activation of MAPK/NF-κB signaling pathway.