Silencing Of CCDC65 Affects The Proliferation Ability On HBV-Related Hepatocarcinoma Cells

BackgroundHepatocarcinoma is one of the common malignant tumors in China.It has the clinical characteristics of occult onset,rapid progress,and poor prognosis.According to the 2015 epidemiological survey of malignant tumors in China,the incidence of liver cancer ranks fourth among all malignant tumors in China,and the mortality rate ranks second,which threatens people’s life and health seriously.The main risk factors for liver cancer include hepatitis B virus(HBV),hepatitis C virus(HCV),alcoholic fatty liver,long-term intake of aflatoxin,etc.Among them,HBV infection is the most common factor for liver cancer patients in China.Surgical resection is still the main treatment for liver cancer at present,but the high recurrence and metastasis rate after surgery seriously affects the survival prognosis.Numerous research reports indicate that HBV is not only closely related to the incidence of liver cancer,but also very important to the recurrence and metastasis.Therefore,it is of great significance to actively explore the HBV-related pathogenic factors in liver cancer.CCDC65 is a member of the coiled-coil protein family.It encodes sperm tail protein and is an important component of the connexin-kinin regulatory complex,which is essential for ciliary movement.Mutations in the CCDC65 gene have been reported in the literature to cause primary ciliary dyskinesia.There are few related studies on the role of CCDC65 in tumors.This study intends to explore the differential expression of CCDC65 in Hepatocarcinoma and its relationship with survival prognosis through TCGA database and Hepatocarcinoma tissue microarry,and further analyze the role of CCDC65 in HBV-related Hepatocarcinoma cell and related molecular mechanisms.Results1.The mRNA expression levels of CCDC65 was explored in 371 Hepatocarcinoma samples and 50 normal samples in TCGA database through a public database platform.Results showed that CCDC65 were significantly increased in Hepatocarcinoma samples;further by detecting several Hepatocarcinoma cells and normal liver cell,It was found that CCDC65 expression level was significantly increased in HBV-positive Hepatocarcinoma cells;2.The effect of CCDC65 on the survival rate was explored by public database,results showed that HBV-related Hepatocarcinoma patients with high expression of CCDC65 had low long-term survival rate;The expression of CCDC65 in Hepatocarcinoma tissues and adjacent tissues was detected by IHC in Hepatocarcinoma microarray,results showed that CCDC65 expression was significantly increased in HBV-related Hepatocarcinoma tissues;3.The effects of CCDC65 on the proliferation of HBV-related Hepatocarcinoma cells were detected by MTT and EdU assays.The results showed that the proliferation of HBV-related Hepatocarcinoma cells was significantly inhibited after interfering with CCDC65;4.Bioinformatics was used to analyze and predict the physical and chemical properties,subcellular localization,and the structure of CCDC65 protein.RT-qPCR and WB assays showed that interfering CCDC65 inhibited the proliferation of Hepatocarcinoma cells by inhibiting the PI3K/AKT signaling pathway;COIP assays showed that CCDC65 interacted with PTEN and regulated the expression level of PTEN,which is the core regulatory protein;5.HBx expression levels was decreased after interfering with CCDC65 in HBV-positive Hepatocarcinoma cells by RT-qPCR and WB assays.ConclusionCCDC65 is significantly up-regulated in HBV-related Hepatocarcinoma and correlates with poor prognosis.Interference with CCDC65 inhibits the proliferation of Hepatocarcinoma cells by combining PTEN to inhibit the PI3K/AKT signaling pathway,and downregulates the expression of key oncoprotein HBx.