The Role Of CCDC65 Gene In Cell Proliferation And Invasion Of Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma（NPC）is a malignant tumor of the nasopharyngeal mucosa,whose appearance is influenced by environmental and genetic factors influence,mainly distributed in southern provinces of China and Hong Kong,Macao and Taiwan regions and some Southeast Asian countries and regions,there exists obvious regional and ethnic aggregation.Influenced by various environmental factors,EB virus infection,genetic susceptibility,epigenetic changes and other various reasons eventually led to some key oncogenes overexpress and tumor suppressor gene expression be downregulated or absent[1],which makes pathological changes to the patients with nasopharyngeal mucosa.With the exacerbation of the disease,precancerous lesions will develop into nasopharyngeal carcinoma（NPC）,,and ultimately the patient’s death is often due to tumor metastasis[2,3].Therefore,it is very important to explore the mechanism of its pathogenesis,which is very important for the prevention and treatment of nasopharyngeal carcinoma and improve the prognosis of nasopharyngeal carcinoma.In 1999,Dr.Li Zhongkui guided by Yao Kaitai academician found a tumor suppressor related gene whice can inhibit tumor cell proliferation,invasion and metastasis of nasopharyngeal carcinoma cells called NESGI,whose official name is CCDC19（NM₀12337.1）.They also amplificated and spliced out of a continuous full length about 1850bp of NESGI was gene cDNA fragment.Through gene chip analysis,we got a lot of cell signaling pathway mediated by NESGI gene,but it is necessary to identify the key target that can interact with it directly.Therefore our research group has carried out NESG1 protein interaction studies,and through the yeast two hybrid assay screened several proteins included VPS33B,ENKUR and CCDC65,which could interact with NESG1,but it was so regret that we verified only VPS33B interacted with NESGI finally.However,we were surprised to find that CCDC65 expression was down regulated in the Q-PCR of the collected samples of nasopharyngeal carcinoma patients,which made us pay attention to the CCDC65 gene again.CCDC65（coiled coil protein 65）was mainly located in normal nasopharyngeal ciliated epithelial cells and sperm tail[4,5,6],in nasopharyngeal epithelium it is a key structural element of a protein complexe N-DRC（connection protein dynein regulation complex）with 12 subunits[5,7,8];and in the male reproductive system it is located in the sperm tail of a testis development related protein NYD-SP28[6].Protein and protein complexes composed of CCDC65 are mainly responsible for ciliary movement,sperm tail activity and sperm obtained function,but the current research on it is not so much,mainly concentrated on N-DRC and NYD-SP28 structural abnormalities in the CCDC65 deletion mutation triggering PCD（primary fiber hair movement disorders）,which leads to chronic airway infection,abnormal development of male infertility and body diseases[4,5,6].CCDC65 in Chlamydomonas reinhardtii has homologous protein FAP250（flagellum associated proteins 250）,it is also the key structure element of protein complex DRC2 and constitute the N-DRC of Chlamydomonas reinhardtii to control its flagella motion[8].Therefore,Chlamydomonas reinhardtii in most human CCDC65 research as a model.In addition,there are specialized studies on Chlamydomonas reinhardtii flagella pointed out modification enzyme of Tektin,RSPs,CCDC40 and CCDC65 in flagellar resorption was phosphorylated to activate the methylation pathway of CCDC65 and CCDC40,which leads to the disintegration of basal filament structure[9].Although CCDC65 did not have direct interaction with NESGI,Dr.Wang Hao in our study group had detected CCDC65 in nasopharyngeal carcinoma cells showed low expression by Q-PCR analysis of the collected nasopharyngeal carcinoma patients.The malignant degree of nasopharyngeal carcinoma is high and it is easy to invade the adjacent tissue,so we choose CCDC65 as the research object,to explore its role in the proliferation and invasion of nasopharyngeal carcinoma cells.Objective1.Establishment of a stable overexpression and transient interference CCDC65 gene in nasopharyngeal carcinoma cell line and cell function test2.Research the molecular mechanism of CCDC65 inhibiting the proliferation and invasion of nasopharyngeal carcinoma cells3.Preliminary study on the interaction of CCDC65 proteinContents and methods1.The establishment of a stable overexpression and transient interferenceCCDC65 gene in nasopharyngeal carcinoma cell lines and cell function test①Using the lentivirus packaged by company to infect nasopharyngeal carcinoma cell lines 5-8F and HONE-1,we established the stable overexpression of the CCDC65 gene in nasopharyngeal carcinoma cell lines.Western Blot analysis and Q-PCR identified the expression efficiency and screened the nasopharyngeal carcinoma cell lines which overexpressed CCDC65.②After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,MTT test detected changes in experimental and control groups in cell proliferation;③The cell proliferation in the experimental group and the control group was detected by plate cloning test after the nasopharyngeal carcinoma cell lines had overexpressed CCDC65;④)After the stable overex.pression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,EDU cell proliferation assay detected changes in experimental and control groups in proliferation;⑤After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,flow cytometry assayed cell cycle,compared experimental and control groups in the proportion of cells in S phase change;⑥After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,tumorigeniced in nude mice to assay the changes of ability that growing in vitro.Removed the tumor,recorded weight and volume of tumors;⑦After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,scratch assayed changes in experimental and control groups of cell invasion.⑧After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,Boyden and Transwell detected changes in experimental and control groups of cell invasion.⑨Western Blot analysis was used to identify the interference efficiency of CCDC65 gene in nasopharyngeal carcinoma cell lines 5-8F and HONE-1 interfered by siRNA,which was synthesized by the company.⑩After siRNA had interfered CCDC65 expression of nasopharyngeal carcinoma cell lines,proliferation and invasion related function experiments,confirmed that these changeg are caused by CCDC65.2.Research the effect and molecular mechanism of CCDC65 on proliferation and invasion ability of human nasopharyngeal carcinoma cell①After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,Western Blot analysis detected changes in cell cycle-related proteins to find out related cycle-regulated signaling pathways that associated with CCDC65;②After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,Western Blot analysis detected changes of protein that associated with EMT,and searched the related signaling pathways of invasion which regulated by CCDC65.3.The preliminary study on the interaction of CCDC65 protein①CCDC65-Flag fusion plasmid was introduced into nasopharyngeal carcinoma cell lines,and Wester Blot analysis was used to detect the expression.②The co-immunoprecipitation experiment was carried out on the nasopharyngeal carcinoma cell lines which had been introduced the fusion plasmid,and the mass spectra were analyzed by mass spectrometry.③ According to the results of mass spectrometry,the co-immunoprecipitation experiment was carried out on the nasopharyngeal carcinoma cell lines and nasopharyngeal carcinoma cell lines which had been introduced the fusion plasmid to verify the relationship between the MYH-9 and CCDC65.Statistical Analysis:Using SPSS 19 statistical software for data analysis.Measurement data results are expressed as x±s.Overexpression of CCDC65 in nasopharyngeal carcinoma cell lines control between the control group and experimental group ratio,wearing number of cell membrane,fluorescence quantitative polymerase chain reaction（PCR）2-△△Ct value of two independent samples t test was used to compare;the expression of each cell sample instantaneous interference CCDC65 nasopharyngeal carcinoma（NPC）cell lines in the control group and the experimental group ratio,wear membrane cell number was used single factor analysis of variance（one way ANOVA）to compare,if there is significant difference,multiple comparisons using Dunnett method;MTT and scratch assays results using two way ANOVA;to P<0.05,the difference has statistical significance.Results1.Established the stable overexpression and transient interference of the CCDC65 gene in nasopharyngeal carcinoma cells lines 5-8F and HONE-1.The successful gene manipulation,was proven by Western Blot analysis and Q-PCR.These cell lines were used for a series of cell proliferation,migration and invasion assays.①After the stable overexpression of CCDC65 gene in the nasopharyngeal carcinoma cell lines,MTT assay and clonogenic assay were detected changes in cell proliferation in experimental group and control group.The results showed that overexpression of CCDC65,through a series of intracellular activities,can be suppressed NPC proliferation.②EDU and cell cycle test showed that stable overexpression of CCDC65 gene,the proportion of cells in the G1 phase increased,the proportion of S phase decreased,the cells were blocked in the division interval,resulting in decreased proliferation capacity.③After the interference of CCDC65 gene in nasopharyngeal carcinoma cell lines,cell proliferation function experimental results show that interference of CCDC65 can enhance the proliferation of nasopharyngeal carcinoma cells④Nude mice tumor experiment results showed that the weight and volume of the tumor cells in the nude mice were decreased after the overexpression of CCDC65 gene.Compared with the blank control group,the experimental results of the two groups were statistically significant（P<0.05）.It was confirmed that the overexpression of CCDC65 inhibited the tumor growth of nasopharyngeal carcinoma cells in vivo.⑤Transwell and Boyden results showed that after the stable overexpression of CCDC65 gene decreased cell invasion and CCDC65 could inhibit cell invasion.⑥The scratch test results showed that CCDC65 decreased cell invasion,which showed that CCDC65 could inhibit cell invasion.⑦After the interference of CCDC65 gene in nasopharyngeal carcinoma cell lines,invasion function experiment results showed that the interference of CCDC65 can enhance the invasion of nasopharyngeal carcinoma cells.2.CCDC65 inhibits the proliferation and invasion of cancer cells and its molecular mechanism in nasopharyngeal carcinoma.①Western Blot assay was used to detect the stable overexpression CCDC65 cell lines.Cell cycle related genes PTEN,p21 and p27 expression was up-regulated,and CDK4,P-RB,CCND1,c-myc,p-Akt,p-PI3K,Akt,PI3K and c-jun expression was down-regulated,which suggested that CCDC65 may through PI3K/Akt and β-catenin signaling pathway to regulate the cell proliferation ability of NPC cells.②After the stable overexpression of CCDC65 gene in nasopharyngeal carcinoma cell lines,Western Blot analysis showed the expression of EMT related genes of N-cadherin,slug,beta-catenin was down-regulated,and the E-cadherin expression was up-regulated;and the expression of p-Akt,P-PI3K,Akt and PI3K protein in PI3K/Akt signaling pathway was down-regulated,and the expression of PTEN was up-regulated;which suggested that CCDC65 could inhibit the invasion process,through the regulation of PI3K/Akt and β-catenin signaling pathway.2.The preliminary study on the interaction of CCDC65 protein①After the introduction of CCDC65-Flag fusion plasmid,the score of the nasopharyngeal carcinoma related protein MYH-9 in the mass spectrometry results was high.②The interaction between CCDC65 and MYH-9 in the presence of protein was preliminarily verified by co-immunoprecipitationConclusion1.CCDC65 can inhibit the proliferation and invasion of nasopharyngeal carcinoma cell.2.CCDC65 can inhibit the proliferation and migration of nasopharyngeal carcinoma cell by regulating the PI3K/AKT and β-catenin signaling pathway.3.The protein interaction between CCDC65 and MYH-9 was preliminarilyconfirmed.The innovation of this research:1.It was the first time confirmed that CCDC65 could inhibit the proliferation and invasion of nasopharyngeal carcinoma cells,and the molecular mechanism was verified.2.The interaction between CCDC65 and MYH-9 in the presence of protein was preliminarily confirmed.