| Objective: Nasopharyngeal carcinoma(NPC)is a malignance arising in the nasopharyngeal mucosa.NPC has a high degree of concealment,malignancy and metastasis,which seriously threatens the patient's physical and mental health.Plenty of studies have found that overexpression of oncogenes is an important biological factor for the occurrence and development of nasopharyngeal carcinoma.In recent years,it has been found that Cell Devision Cycle Associated 2(CDCA2)is up-regulated in a variety of tumors.More importantly,studies had shown that up-regulation of CDCA2 was related to the occurrence,development and prognosis of some tumors.However,it has not been reported in NPC.This study aimed to investigate the expression of CDCA2 in NPC and its effect on the biological function of NPC cells.Methods: 1.Detection of the expression of CDCA2 mRNA and protein in nasopharyngeal carcinoma tissues and nasopharyngeal mucosa chronic inflammation tissues was performed by quantitative reverse transcription polymerase chain reaction(qRT-PCR)and immunohistochemistry.In addition,the relationship between CDCA2 expression and clinicopathological features of NPC was evaluated with the data from this two experiments.2.The basic expression of CDCA2 protein in NPC cell lines HK1,HONE1,5-8F,CNE1,CNE2 and CNE2 Z was detected by Western blot(WB)to determine the cell lines used in the subsequent experiments.3.Lentiviral vector-mediated RNA interference(RNAi)was used to knock down CDCA2 expression in HK1,HONE1 and 5-8F cell lines in vitro.Cells transfected with lentivirus containing CDCA2 silencing fragment were defined as experimental groups(HK1-shCDCA2,HONE1-shCDCA2,5-8F-shCDCA2).Cells treated with lentivirus containing empty control fragment were defined as negative empty groups(HK1-shCtrl,HONE1-shCtrl,5-8F-shCtrl),and cells untreated were considered as blank control groups(HK1,HONE1,5-8F).The efficiency of lentivirus infection was observed with a fluorescence microscope.The expression of CDCA2 mRNA and protein in each group was detected by qRT-PCR and WB,respectively,to determine the silencing effect of CDCA2.4.CCK-8 assay and cell colony formation test were used to evaluate the effect of knockdown of CDCA2 on the proliferation of HK1,HONE1 and 5-8F cells.5.Wound-healing assay,transwell migration and transwell invasion assay were used to detect the effect of CDCA2 expression on migration and invasion of HK1,HONE1 and 5-8F cells.6.The cell cycle distribution and apoptosis ratio of cells in each group was detected by flow cytometry.7.Using WB,we detected whether CDCA2 affects the expression of cell cycle-associated proteins in NPC cells.Results: 1.The relative expression levels of CDCA2 mRNA in 16 cases of NPC and 16 cases of nasopharyngeal mucosal chronic inflammation were 5.319±3.198 and 1.148±0.759,respectively.Compared with the nasopharyngeal mucosal chronic inflammation tissues,the relative expression of CDCA2 mRNA in NPC tissues was significantly increased(P<0.001).The positive rate of CDCA2 protein expression in NPC was 60.47%(52/86),which was significantly higher than that in the chronic inflammatory tissues of nasopharyngeal mucosa(11.76%,6/51).And the expression of CDCA2 protein was found to be associated with the locality infiltration(P=0.033)and lymph node metastasis(P=0.034)of the cancer.2.Compared with CNE1,CNE2 and CNE2 Z,CDCA2 was expressed relatively higher in NPC cell lines HK1,HONE1 and 5-8F.Therefore,HK1,HONE1 and 5-8F were used in follow-up vitro experiments.3.The lentiviral transfection efficiency of the CDCA2 knockdown groups and the negative empty groups was more than 90%.Compared with the negative empty groups and the blank control groups,the expression of CDCA2 mRNA and protein in CDCA2 knockdown groups was significantly decreased(P<0.001).This indicates that the stable nasopharyngeal carcinoma cell lines with CDCA2 knockdown was successfully constructed.4.The results of CCK-8 assay showed that the proliferation ability of HK1,HONE1 and 5-8F was significantly decreased after knockdown of CDCA2 expression.The cell colony formation assay showed that the clone formation rates of HK1,HK1-shCtrl and HK1-shCDCA2 were(63.733±3.859)%,(63.467±4.601)% and(41.133±2.344)%,respectively;the clone formation rates of HONE1,HONE1-shCtrl and HONE1-shCDCA2 were(44.200±3.143)%,(45.667±2.516)% and(35.200±2.553)%,respectively;the clone formation rates of 5-8F,5-8F-shCtrl and 5-8F-shCDCA2 were(64.667±3.113)%,(65.667±2.516)% and(53.267±2.610)%,respectively.After knockdown of CDCA2 expression,the clone formation ability of HK1,HONE1 and 5-8F was significantly decreased(P<0.05).5.After silencing CDCA2 expression,the migration and invasion ability of nasopharyngeal carcinoma cell lines HK1,HONE1 and 5-8F cells was not significantly changed(P>0.05).6.Flow cytometry analysis showed that the proportion of G0+G1 phase cells in HK1,HK1-shCtrl and HK1-shCDCA2 was(62.380±2.868)%,(62.723±1.806)% and(69.507±2.607)%;(33.697±1.825)%,(32.603±2.365)% and(45.320±1.360)% for HONE1,HONE1-shCtrl and HONE1-shCDCA2,respectively;(62.050±3.080)%,(62.810±2.440)% and(68.373±2.437)% for 5-8F,5-8F-shCtrl and 5-8F-shCDCA2.Therefore,the number of cells in the G0+G1 phase of CDCA2 knockdown groups were significantly higher than that of the empty groups and the blank control groups(P<0.05).The changes of the three cell lines in the G2+M phase and the S phase were different.The total apoptotic rate of HK1,HK1-shCtrl and HK1-shCDCA2 was(7.303±0.702)%,(7.170±0.310)% and(8.967±0.656)%,respectively;the total apoptotic rate of HONE1,HONE1-shCtrl and HONE1-shCDCA2 was(7.040±0.616)%,(6.907±0.708)% and(16.397±1.903)%,respectively,the total apoptotic rate of 5-8F,5-8F-shCtrl and 5-8F-shCDCA2 was(18.570±1.415)%,(17.883±1.734)% and(23.343±2.270)%.Taken together,the total apoptotic rate of NPC cells increased slightly after knocking down CDCA2(P<0.05).7.WB data showed that the expression of cell cycle-associated proteins CDK4 and CDK6 was significantly decreased after CDCA2 silencing,while the expression of p21 protein was significantly increased;and that the expression of Cyclin D1 was not significantly changed.Conclusions: 1.Compared with chronic inflammatory tissues of nasopharyngeal mucosa,the expression of CDCA2 mRNA and protein in NPC is up-regulated and the up-regulation of CDCA2 may be related to local infiltration and lymph node metastasis of the cancer.2.CDCA2 may participate in the regulation of cell cycle by affecting the expression of cyclin CDK4,CDK6 and p21 in NPC cells,promoting the proliferation of NPC cells and possibly affecting apoptosis. |
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