Study Expression Of ARC In Nasopharyngeal Carcinoma And Effect On Radiotherapy And Chemotherapy Sensitivity In Nasopharyngeal Carcinoma Cells

Cancer is a complex process by the interaction of oncogenes activation and tumor suppressor gene and the mechanism of cell death inactivation. Since reduction in apoptosis closely relates to tumorigenesis, it suggests that during apoptosis the negative regulation of genes may be carcinogenic and the promotion of apoptosis-related genes may be tumor suppressor genes. Therefore, the apoptosis-related proteins has been a hot area of cancer research. This article will discuss a novel anti-apoptotic protein ARC, and study the relationship between ARC and nasopharyngeal carcinoma (NPC) development and the relationship between ARC and NPC radiotherapy and chemotherapy sensitivity. Object Detect the protein level of ARC in NPC tissues and inflammatory tissues of the nasopharynx, analyse the relationship between ARC and tumor clinical pathology parameters including T stage, clinical stage, and with or without lymph node metastasis. Detect the expression level of ARC in the nasopharyngeal carcinoma cell lines.Method Using immunohistochemistry to detect the expression level of ARC in82cases of NPC tissues and20cases of chronic inflammatory tissues of the nasopharynx. Analysis the relationship between its expression and clinical pathological parameters in NPC. Western blot detects ARC expression in NPC cell lines. SPSS17.0statistical software for statistical analysis of experimental data.Result The expression of ARC in NPC was significantly higher than the inflammatory tissue of the nasopharynx (P<0.001). The expression of ARC in NPC is dependent with clinical stage, pathological grading factors (P<0.001), while independent with patient age and lymph node metastasis (P>0.05). The expression of ARC in NPC cells showed significantly high expression, while weak expression in nasopharyngeal immortalized epithelial cells NP69. The descending order of the expression of ARC in cell lines is CNE-2>CNE-1>5-8F>6-10B> NP69.Conclusion These results suggest that ARC high expression relates to the occurrence and the development of NPC, it provides new clues for the study of NPC radiotherapy and chemotherapy sensitivity. Object RNA interference technology and gene cloning techniques were used to silent and overexpress ARC in NPC cell line, and filtered out the stably transfected cell lines.Method Build vector pcDNA6.2-GW/EmGFP/miRNA for ARC RNA interference plasmid and vector pEZ-M03for gene clone plasmid. ARC-miRNA and control no-targe-miRNA plasmid are stably transfected in CNE-2cells with high expression of ARC by lipofection. pEZ-M03-ARC and control pReceiver-M03CT plasmid are stably transfected6-10B cells with low expression of ARC. Fluorescence quantitative PCR and Western blot are used to detect the ARC expression, flow cytometry is used to detected the cell cycle.Result1) ARC-miRNA-3plasmid showed the strongest effect on RNA interference. Silencing efficiency of ARC mRNA level reached to77.03%and ARC protein level reached to72.6%in ARC-miRNA-3plasmid stably transfected CNE-2cells.2) ARC mRNA level was2.06-fold increased in pEZ-M03-ARC stably transfected6-10B cells compared with the control cells, ARC protein level was1.89-fold increased in pEZ-M03-ARC stably transfected6-10B cells compared with the control cells.3) The proportion of the cell cycle G0-G1, G2-M and S phase showed no significant differences between experimental group cellls and the control group cells.Conclusion It was proved that miRNA silencing and overexpression of ARC plasmids were successfully constructed, and these plasmids were stably transfected in NPC cell lines, which laid the foundation for the subsequent experiments. Objective To investigate the influence of silencing or overexpression of ARC on radiosensitivity of NPC cells.Method Silencing or overexpression of ARC NPC cells and control cells were treated with different doses of X-ray irradiation of the gradient, CCK-8method was detected cell survival, Annexin V was used to assay of apoptosis, caspase3/8activity detection kit box was detected Caspase3/8activity.Result1) After X-ray irradiation, the cell viability of ARC-miRNA-3transfected cells was significantly lower than control cells, the cell survival curve of the experimental cells was lower than the control cells (P<0.001). The apoptosis ratio of ARC-miRNA-3transfected cells was52.2±3.4%which was higher than control cells of36.1±2.2%by Annexin V assay (P<0.001). 2) After X-ray irradiation, survival rate of pEZ-M03-ARC transfected cells was significantly higher than control cells, the cell survival curve of the experimental cells was significantly higher (P<0.001). The apoptosis ratio of pEZ-M03-ARC transfected cells was21.2±2.4%which was lower than the control cells of33.7±2.5%by Annexin V assay (P <0.001).3) After X-ray irradiation, the activity of caspase-3of ARC-miRNA-3transfected CNE-2cells was2.33±0.26fold increased compared with control cells. The activity of caspase-8of ARC-miRNA-3transfected CNE-2cells was1.65±0.17fold increased compared with control cells (P<0.001).4) After X-ray irradiation, the activity of caspase-3of pEZ-M03-ARC transfected6-10B cells was0.62±0.14times decreased compared with control cells. the activity of caspase-8of pEZ-M03-ARC transfected6-10B cells was0.73±0.11times decreased compared with control cells (P<0.001).Conclusion ARC could through exogenous and endogenous apoptotic pathway to inhibit radiation-induced NPC cell apoptosis. Objective To investigate the influence of silencing or overexpression of ARC on chemosensitivity of NPC cells.Method Silencing or overexpression ARC cells and control cells were treated with different concentrations of paclitaxel or cisplatin for48hours. CCK-8assay was detected cell growth and proliferation. After IC30concentration of the drug-treated cells24hours, Annexin V was used to assay of apoptosis, caspase3/8activity detection kit box was detected Caspase3/8activity.Result1) With cisplatin treatment, the cell viability of ARC-miRNA-3transfected cells was significantly lower than control cells, the cell survival curve of the experimental cells was lower than the control cells (P<0.001). IC50of ARC-miRNA-3transfected cells was (1.995±0.327)×10-5which was lower than control cells (3.006±0.344)×10-5(P <0.001). The apoptosis ratio of ARC-miRNA-3transfected cells was56.8±4.4%which was higher than control cells33.02±3.7%by Annexin V assay (P<0.001).2) With cisplatin treatment, survival rate of pEZ-M03-ARC transfected cells was significantly higher than control cells, the cell survival curve of the experimental cells was significantly higher (P<0.001). IC50of pEZ-M03-ARC transfected cells was (1.000±.244)×10-4which was higher than control cells (6.397±0.276)×10-5(P<0.001). The apoptosis ratio of pEZ-M03-ARC transfected cells was24.8±2.8%which was lower than control cells35.1±2.6%by Annexin V assay (P<0.001).3) With cisplatin treatment, the activity of caspase-3of ARC-miRNA-3transfected CNE-2cells was3.49±0.31fold increased compared with control cells. The activity of caspase-8of ARC-miRNA-3transfected CNE-2cells was2.17±0.20fold increased compared with control cells (P<0.001).4) With cisplatin treatment, the activity of caspase-3of pEZ-M03-ARC transfected6-10B cells was0.44±0.07times decreased compared with control cells. the activity of caspase-8of pEZ-M03-ARC transfected6-10B cells was0.69±0.12times decreased compared with control cells (P<0.001).5) There was no significant difference between A ARC-miRNA-3transfected cells and control cells in cell survival after the paclitaxel treatment (P>0.05), it was the same between pEZ-M03-ARC transfected cells and control cells (P>0.05). Conclusion ARC could through exogenous and endogenous apoptotic pathway to inhibit cisplatin-induced NPC cell apoptosis.