Role Of CCDC34 In Angiogenesis Of Pancreatic Cancer And Its Prognostic Significance

Pancreatic cancer is insidious and is progressing rapidly.Nowadays,the incidence and mortality of pancreatic cancer are increasing in parallel.It is a multifactorial disease,which is a complex process of interaction between cancer cells,microenvironment and organism(including heredity,metabolism,immunity,etc.).Although we have a deeper understanding of the epidemiology,pathogenesis and diagnosis and treatment of pancreatic cancer,the effects of various treatments are limited,mainly due to the particularity of the pancreas,the difficulties of early detection and surgical resection,and the poor effect of chemotherapy.In the past 30 years,the molecular mechanism of pancreatic cancer,biological targeting therapy,the improvement of drug transport in the body and the regulation of immune system have developed rapidly.However,the study of KRAS has not achieved significant results,and the recovery of TP53 and other suppressor genes is difficult to achieve.The therapeutic effect of targeted therapy is still unsatisfactory and the treatment of human pancreatic cancer is still a big challenge.Therefore,exploring the pathogenesis,early diagnosis,drug screening,treatment strategy and prediction of recurrence and metastasis of pancreatic cancer has become a hot research direction nowadays.Coiled-coil domain containing 34(CCDC34)is a gene that encodes protein and related to disease,also known as NY-REN-41,RAMA3,or L15.Recent studies have found that CCDC34 was highly expressed in several kinds of malignant tumors,including renal cell carcinoma,non-small-cell lung cancer(NSCLC)and bladder cancer,and was related to tumor proliferation,apoptosis and angiogenesis.Our study team recently used the bioinformatics method to predict the following results using Oncomine and TCGA data:compared with the normal control group,CCDC34 mRNA was highly expressed in pancreatic cancer,and high expression of CCDC34 mRNA was significantly related to the angiogenesis-related indicators(VEGF,CD31,etc.).So,in the present study,we focused on the verification and exploration of the role of CCDC34 in angiogenesis of pancreatic cancer and its clinical significance.The study will provide a theoretical basis for finding new targets and new strategies for the treatment of pancreatic cancer.Part 1:Expression of CCDC34 and its prognostic significance in pancreatic cancer Objective:CCDC34 promotes cell proliferation and invasive properties in human cancer.The aim of this study was to compare the expression of CCDC34 in pancreatic adenocarcinoma with normal pancreatic tissue,and to evaluate the prognostic significance of CCDC34 expression in patients with pancreatic adenocarcinoma,using bioinformatics.Methods:The expression and prognostic value of CCDC34 were initially predicted using Oncomine and The Cancer Genome Atlas(TCGA)databases.Pancreatic adenocarcinoma tissue samples(N=90)and matched normal pancreatic tissues(N =90)were studied using immunohistochemistry to measure CCDC34 protein expression levels.Univariate Kaplan-Meier,and multivariate Cox analysis were used to determine the prognostic role of CCDC34 expression.Results:Oncomine and TCGA databases predicted that CCDC34 mRNA expression levels were significantly increased in pancreatic adenocarcinoma compared with normal pancreatic tissues(P<0.05),and that patients with increased CCDC34 mRNA expression levels had significantly lower overall survival(OS)(P =0.031).Immunohistochemistry showed that expression levels of CCDC34 protein in pancreatic adenocarcinoma were significantly increased,compared with normal pancreas(P=0.000).Patients with pancreatic adenocarcinoma with increased expression of tissue CCDC34 had significantly reduced OS compared with patients with low expression(P=0.000).Univariate and multivariate survival analysis showed that increased expression of CCDC34 was an independent predictor of poor prognosis in patients with pancreatic adenocarcinoma(all P =0.000).Conclusions:Compared with normal pancreas,CCDC34 expression was significantly increased in pancreatic adenocarcinoma,and increased CCDC34 expression was an independent predictor of poor patient prognosis.Part 2:Relationship betw een CCDC34 and angiogenesis in pancreatic cancer and its potential mechanismObjective:Recent studies have found that CCDC34 was highly expressed in several kinds of malignant tumors.The purpose of this study was to explore and validate the relationship between CCDC34 and angiogenesis in pancreatic cancer and its potential mechanisms.Methods:Initially,pancreatic cancer data in TCGA database was downloaded to analyze the mRNA expression levels of CCDC34 and key molecules in PI3K/AKT pathway(PIK3CA,AKT1,mTOR,HIF1A,VEGFA and PECAM1)and its relationship.Besides,immunohistochemical method was used to verify the protein expression levels of above indexes and to analyze the correlation between the expression of CCDC34 and the above indexes.Finally,quantitative real-time PCR(qRT-PCR)and western blot were performed to detect the mRNA and protein expression levels of key molecules in PI3K/AKT signaling pathway.Results:Bioinformatics results showed that compared with the normal control group,the mRNA expression levels of CCDC34 gene and PI3K/AKT pathway key molecules(PIK3CA.AKT1,mTOR,HIF1A.VEGFA and PECAM1)increased significantly(All P<0.05).Subsequent immunochemical data were consistent with the prediction results.Moreover,bioinformatics results revealed that CCDC34 and PI3K/AKT pathway key molecules(PIK3CA,AKT1,mTOR,HIF1A,VEGFA and PECAM1)showed a significant positive correlation,with the correlation coefficient listed as follows:CCDC34 and PIK3CA(R =0.24,P =0.0012),CCDC34 and AKT1(R =0.29,P=6.6e-05),CCDC34 and mTOR(R =0.24,P =0.0015),CCDC34 and HIF1A(R =0.15,P=0.041),CCDC34 and VEGFA(R =0.19,P =0.011)and CCDC34 and PECAM1(R=0.17,P =0.026).Then,immunohistochemical results revealed that CCDC34 and VEGF and MVD counts showed a significant positive correlation,the correlation coefficient was 0.464 and 0.378 respectively(Both P =0.000).MVD in CCDC34 high expression group was significantly higher than that in CCDC34 low expression group(P<0.001).Additionally,qRT-PCR and western blot validated that high expression of CCDC34 might contribute to the angiogenesis of pancreatic cancer by activating the PI3K/AKT pathway.Conclusions:CCDC34 high expression were positively correlated to the key molecules expression(PIK3CA,AKTI,mTOR,HIF1A.VEGFA and PECAM1)in PI3K/AKT pathway in pancreatic cancer,suggesting that high expression of CCDC34 may promote the angiogenesis of pancreatic cancer through activation of the PI3K/AKT pathway.Part 3:Effects of CCDC34 on the cell function of pancreatic cancer and its molecular mechanismObjective:To further investigate the effects of CCDC34 on the cell function of pancreatic cancer and its molecular mechanism based on the first two parts.Methods:We selected 3 kinds of pancreatic cancer cell lines(BxPC-3,PANC-1 and SW1990)and 1 kind of normal pancreatic ductal cell line HPDE6-C7 to detect the basal expression of CCDC34.Then,PANC-1(the highest expression levels of CCDC34)was used as a tool cell line to silence CCDC34 by RNA interference.The assays of cell proliferation,cell cycle,apoptosis,colony formation and angiogenesis were performed.Moreover,screening by whole genome expression profiling was done to find the differentially expressed genes after CCDC34 silence.Additionally,the KEGG pathway enrichment was analyzed by DAVID website.The key molecules of the pathway were verified by western blot.Results:Compared with the control group,the proliferation and clonal formation of pancreatic cancer cells in the CCDC34-silencing group were significantly reduced,the cell cycle was arrested in the G2/M phase,the number of apoptotic cells was significantly increased,and the angiogenic ability was significantly reduced(All P<0.001).Besides,the whole gene expression profiling showed that a total of 410 genes showed significant expression changes after CCDC34 silence(P<0.05),of which 218 genes were up-regulated and 192 genes were down-regulated.Subsequently,David online tool showed that PI3K/AKT signaling pathway enrichment was the most significant among the top 10 enrichment pathways.Finally,western blot analysis confirmed that the levels of PI3K and AKT were not significantly changed after silencing CCDC34,while the levels of p-PI3K and p-AKT were significantly decreased.Conclusions:Silencing CCDC34 can significantly inhibit the cell proliferation,clonal formation and angiogenesis of pancreatic cancer and promote its apoptosis.The mechanism may be related to the regulation of PI3K/AKT signaling pathway by CCDC34.