The Role Of NCAPH In Nasopharyngeal Carcinoma Progression

Objective:To study the relative expression of NCAPH in head and neck squamous cell carcinoma(HVSC)and normal tissues and the survival rate of patients.On this basis,NCAPH was studied at the cellular level in nasopharyngeal carcinoma cells and normal.Relative expression difference in nasopharyngeal epithelial cells;then establish a stable NCAPH low expression cell line to study the effect of NCAPH on proliferation,migration and cell cycle of nasopharyngeal carcinoma cells;establish a subcutaneous xenograft model of nasopharyngeal carcinoma in nude mice at animal level To study the effect of NCAPH on the tumorigenic ability of nude mice,and to explore the role of NCAPH in the development of nasopharyngeal carcinoma.Methods:1.Firstly,the bioinformatics method was used to analyze the cancer and tumor gene map(TCGA)data.44 normal samples and 519 HNSC patients were selected to study the relative expression of NCAPH in HNSC and normal tissues and survival.Rate relationship.The mRNA and protein expression levels of NCAPH were directly detected in nasopharyngeal carcinoma cells SUNE-1,CNE-2,5-8F and normal control human immortalized normal nasopharyngeal epithelial cells NP69 by qRT-PCR and western blot.To analyze whether the expression level of NCAPH gene in nasopharyngeal carcinoma cells is abnormal,and whether it is consistent with the results of bioinformatics methods.2.Two NCAPH low expression vectors and control vector Scramble shRNA were constructed,and HEK 293T cells were transfected.Lentivirus-infected wild-type nasopharyngeal carcinoma cell lines SUNE-1,CNE-2,and 5-8F were collected to establish a NCAPH low expression cell line.The knockdown efficiency was identified by qRT-PCR and western blot.The effects of NCAPH on cell proliferation,migration,etc.were observed by growth curve,Brdu incorporation assay,colony formation assay,scratch assay,and chamber migration assay using a stable cell line with low expression of NCAPH.Flow cytometry is used to determine the effect of NCAPH on the cell cycle and to detect changes in the expression levels of cell cycle related markers at protein levels(eg,CDK1,CyclinB1,etc.).3.NACEH stabilized low expression and normal expression of nasopharyngeal carcinoma CNE-2,5-8F cells,established a subcutaneous xenograft model of nasopharyngeal carcinoma in nude mice,and observed tumor formation,including tumor formation time,tumor weight and volume,etc..The expression levels of NCAPH protein,proliferation-related protein KI-67 and Cleaved Caspase 3 in nasopharyngeal carcinoma xenografts were studied by immunohistochemistry.The expression of NCAPH in animals was studied at the animal level.And its effect on the ability of nasopharyngeal carcinoma cells to form tumors.Results:1.Through bioinformatics analysis,NCAPH was found to be highly expressed in a variety of solid tumors,and the difference was significant in HNSC;analysis of cancer and tumor gene map(TCGA)data,found that NCAPH protein expression differences Significantly(P<0.05),tumor samples were significantly higher than normal samples;in addition,the relationship between NCAPH protein expression and overall survival in HNSC patients was analyzed,and patients with low NCAPH expression were found to have better survival than those with high expression.mRNA and total protein were extracted from immortalized normal human nasopharyngeal epithelial cells NP69,nasopharyngeal carcinoma cell lines SUNE-1,CNE-2,5-8F,and NCAPH was further confirmed in nasopharyngeal carcinoma cells from mRNA and protein levels.High expression2.By constructing a NCAPH low expression plasmid and a control plasmid,a NCAPH low expression cell line was established,and the knockdown effciency was detected from the relative mRNA and protein expression levels,and the knockdown efficiency was good.Using the established NCAPH knockdown cell line,through cell growth curve assay,Brdu incorporation assay,colony formation assay,cell scratch assay and chamber migration assay,it was found that nasopharyngeal carcinoma cells SUNE-1,CNE after knockdown of NCAPH In vitro proliferation and migration of-2,5-8F were all inhibited.Cell cycle was detected by flow cytometry.It was found that nasopharyngeal carcinoma cells were arrested in G2/M phase after knockdown of NCAPH,and the expression levels of G2/M phase related proteins CDK1 and CyclinB 1 were changed accordingly.3.Through the subcutaneous xenograft model of nasopharyngeal carcinoma in nude mice,it was found that the weight and volume of xenografts in nude mice decreased after knocking down NCAPH.Immunohistochemistry showed that NCAPH expression was down-regulated in nude mice after knockdown of NCAPH,and there was no significant difference in the expression of proliferation-related protein KI-67,and the expression of apoptosis-related protein Cleaved Caspase 3 was up-regulated.Conclusions:1.NCAPH is highly expressed in HNSC tissues,and patients with high expression have a worse survival rate;NCAPH is highly expressed in nasopharyngeal carcinoma cell lines.2.The proliferation and migration of nasopharyngeal carcinoma cells were inhibited after knocking down NCAPH,and the cell cycle arrest was in G2/M phase,which effectively reversed the malignant biological characteristics of nasopharyngeal carcinoma cells.3.Knockdown of NCAPH can inhibit the growth of xenografted tumors in nude mice,and the expression of NCAPH protein in tumor cells is significantly inhibited,and the expression of apoptosis-related proteins is increased.4.NCAPH is a carcinogen related to the development of nasopharyngeal carcinoma,and it is expected to become a new target for the diagnosis and treatment of nasopharyngeal carcinoma.