| PART Ⅰ Detection and verification of lncRNAs differentially expressed in pancreatic ductal adenocarcinomaBACKROUND Pancreatic ductal adenocarcinoma was a type of refractory malignancies. Specific symptoms and signs were absent in early stage. Once signs and symptoms appear, the majority of patients have progressed to advanced stages. What’s more, as its complex anatomical features and unique characteristics of nerve invasion, surgical resection is difficult, which results in a low removal rate. Even if the surgery is available,5-year survival reported was less than 5% and the overall survival was 10-20 months. Pancreatic cancer was not sensitive to radiotherapy and chemotherapy. Effective treatment was lacking after recurrence. It was reported patients who did not receive any treatment would only survive 3-5 months on average. Therefore, in order to improve the prognosis of pancreatic cancer, development of molecular markers and therapy targets for early diagnosis and effective treatment of pancreatic cancer was urgently. Though studies of molecular genetic on pancreatic cancer have made great progress over the past 20 years, effective diagnostic markers and therapeutic targets were still not available. It was demonstrated by recent studies that non-coding RNA, closely related to cancers, was widely involved in the regulation of normal physiological and pathophysiological processes.The long non-coding RNA (lncRNA) has got many encouraging breakthroughs concerning the malignant tumors, which meanwhile have greatly enriched the content of epigenetic research. The study of lncRNAs related to pancreatic cancer has just begun. Therefore,the further study of the role lncRNAs plays on pancreatic neoplasia which will help further understanding of its mechanism of oncogenesis and open up new ideas to improve the level of its diagnosis and treatment.OBJECTIVES To study profiles of differencially expressed lncRNAs in patients with pancreatic cancer and to predict the impact of differencially expressed lncRNAs on aberrantly expressed coding gene in pancreatic cancer.METHODS In order to screen differential expression of lncRNAs and mRNAs in pancreatic cancer, Human LncRNA microarray v2.0 (8×60 K, Arraystar) chips were used in six pairs of pancreatic cancer and adjacent tissues. Then qPCR was performed to confirm the differential expression of five lncRNAs(AC09011013.1、CXCR2P1、 PPY2、HOXA11-86, DKFZp434J0226) in 61 cases of PDAC,20 cases of pancreatic serous cystadenoma(SCA),14 cases of solid pseudopapillary tumor(SPT),10 cases of neuroendocrine tumors(NET) and 12 cases of duodenal papillary adenocarcinoma cancer(DPAC). Then bioinformatics methods was used to predict functions of differentially expressed mRNAs and the role of specific lncRNAs on regulations of the abnormal expressed genes in pancreatic cancerRESULTS The data of microarray was analyzed, and the results showed 291 differentially expressed lncRNAs between tumor and normal tissues (absolute changed fold> 2, P≤0.05), among them,53 up-regulated,and 76 down-regulated. Clustering analysis showed 291 differentially expressed lncRNAs can distinct normal tissue from the aberrant. Among them,91 lncRNAs was consistently changed in all six pairs of samples (34 up-regulated,76 down-regulated), which indicated their high specificity. For verification of microarray results, qPCR was performed to detect the differential expression of five lncRNAs, namely AC09011013.1, CXCR2P1, PPY2, HOXA11-86 and DKFZp434J0226, in paired tumor-nontumorous specimens in 61 PDAC patients.The findings showed results from microarray was reliable. Meanwhile qPCR revealed that differences in expression of AC09011013.1, and DKFZp434J0226 had no statistically significance in SPT, SCA, NET and DPAC. Except in SPT, the expression of HOXA11-86 increased significantly in the other 3 kinds of tumor tissues. PPY2 was upregulated in NET tissue and CXCR2P1 was downregulated in SCA tissues.Results of microarray also revealed 222 differently expressed protein-coding genes between pancreatic cancer and adjacent tissue,66 upregulated,156 downregulated. GO analysis showed that 66 upregulate genes mainly involved in "response to dietary excess", "nuclesome" and "UDP-galactosyltansferase activity"; as for 156 down-regulated genes, their functions mainly related to "immune system process", "extracellular region" and "chemokine receptor binding". Pathway analysis revealed that genes differentially expressed mainly distribute in the glycosylation-related complex metabolic pathways (glycosaminoglycan biosynthesis-keratan sulfate) and Staphylococcus aureus signal pathway. Coding and non-coding RNA co-expression (CNC) network analysis showed that more genes were related with AC091013.1, CXCR2P1 and DKFZp434J0226; less with HOXA11-86 and PPY2.CONCLUSIONS Differently expression of lncRNAs and mRNAs exist in pancreatic cancer and adjacent tissues. Differentially expressed protein-coding genes mainly exist in cell metabolism and immune biological processes. They were closely related to the nucleosome and extracellular regions, involving molecular function like chemokine binding and UDP-galactose transferase. CNC analysis shows AC091013.1, CXCR2P1 and DKFZp434J0226 were related with more genes and may have more important pathophysiological functions.PART II The clinical significance of differentially expressed lncRNAs in pancreatic ductal adenocacinomarBACKGROUND Cancer transcriptome was far more complex than we currently understood. In addition to protein-coding genes and microRNA, the abnormal expression of lncRNAs were prevalent in human malignancies. LncRNAs were still rarely known, but previous studies showed that lncRNAs were playing an important role on the development of tumors. They had potential to be novel biomarkers for malignant diagnosis and prognosis and new molecular targets to improve the therapeutic effect of cancer. In some early stage of tumorigenesis, expression of cancer-related proteins can be regulated by lncRNAs. And at this point, lncRNA was of greater value as an early diagnosis tool when the tumor was too small to be recognized by imaging. Disease free survival and overall survival in breast cance patients with high expression of HOTAIR were significantly short than that with the low expression. Multivariate analysis further indicates that it was an independent risk factor concerning breast cancer metastasis and poor prognosis. Hence HOTAIR was considered to be an important marker for the diagnosis and prognosis for breast cancer. The invasion capability of breast cancer cells, especially in PRC2 overactivated cancer, can be inhibited with downregulated HOTAIR, which indicated that HOTAIR may be the molecular target of breast cancer therapy. Therefore, when the tumor cells epigenetic regulation error was occurring in the tumor cells, lncRNA may be utilized to correct such errors to kill the original tumor cell, so as to realize early cure of tumors. So for lncRNAs differentially expressed in pancreatic cancer, we may expand samples to determine its value of predictive clinical prognosis.OBJECTIVES To evaluate the relation between two lncRNAs (AC091013.1 and DKFZp434J0226) and clinicopathological parameters of pancreatic cancer and the value of clinical prognosis.METHODS One hundred and nine patients with pancreatic cancer were enrolled the study. The expression of AC091013.1 and DKFZp434J0226 were detected by qPCR in cancer. According to the level of lncRNAs, the patients were classified into high level group and low level group. Respectively, analysis of variance, chi-squared test and bilateral T test was applied for statistical analysis of the data; Logistic regression, Kaplan-Meier and log-rank analysis were applied for different purposes.RESULTS Of 109 cases with pancreatic cancer, the median DFS was 324 days, and median OS was 370 days. Log-rank test showed that TNM stage (x2= 9.486, P= 0.002), tumor size (χ2= 8.738, P= 0003) and lymph node metastasis(χ2= 7.152, P= 0.028) and age (χ2= 5.975, P= 0.015) affect the patient’s DFS; TNM stage (χ2= 6.741,P=0.009), lymph node metastasis (χ2= 5.322,P=0.21) and age (χ2 6.684, P=0.010) affect the patient’s OS. COX model reveals age and lymph node metastases were risk factors for OS (Age:OR=1.55, P=0.044; Age:OR=1.620, P =0.019).In 109 cases,53 were with high expression of AC091013.1, the rest 56 with low expression. Univariate chi-square test showed a significant difference between the two groups in gender (χ2=5.814, P=0.016) and vascular invasion (χ2=5.58, P= 0.018). Multivariate binary logistic regression also supported that gender and vascular invasion were the risk factors of expression level of AC091013.1 (gender:OR= 0.337, P=0.009; vascular invasion:OR= 0.290, P= 0.011). Log-rank test suggested there were no statistically differences in DFS as well as OS between AC091013.1 high and low groups.In 109 cases,54 were with high expression of AC091013.1, the rest 55 with low expression. Univariate chi-square test shows significant differences exist between the two groups in differentiation (χ2= 8.363,P= 0.015), nerve invasion (χ2=16.048, P=0.001) and TNM stage (χ=3.993, P=0.046). Multivariate binary logistic regression also showed that nerve invasion was related to the expression of DKFZp434J0226 and was independent risk factor for DKFZp434J0226 expression (OR=5.963, P?<0.001).Further log-rank test showed DKFZp434J0226 low expression group significantly longer than the high expression group in both DFS and OS (DFS:low expression group vs high expression group=388.000±15.889 vs 280±25.107,χ2= 6.701, P= 0.010; OS:low expression group vs high expression group=416.000±14.830 vs 320 ±12.247,χ2=8.734, P=0.003).COX model analysis showed DKFZp434J0226 was an independent predictor of pancreatic cancer recurrence and death (DFS:OR= 1.68, P= 0.037; OS:OR= 1.891, P=0.009), compared with the DKFZp434J0226 low expression group, the high group risk of recurrence was 1.68 times higher and risk of death was 1.891 times higher.CONCLUSIONS Expression of the lncRNA-AC091013.1 in pancreatic cancer were related with vascular invasion level and gender, but has no significant difference in DFS and OS. LncRNA-DKFZp434J0226 was significantly associated with nerve invasion. In either DFS or OS, DKFZp434J0226 low expression group significantly excel the high expression group. COX model analysis showed DKFZp434J0226 was an independent predictor for recurrence and death of pancreatic cancer. Compared with the DKFZp434J0226 low expression group, the risk of recurrence in high group was 1.68 times higher; risk of death was 1.891 times higher.PART III A study of biofunction and molecular mechanisms about DKFZp434J0226 in pancreatic cancer cell MIAPaCa-2BACKROUND The mechanisms of lncRNAs were very complex, and they could regulation genes expression in variable levels which included the transcription, alternative splicing of posttranscription, protein translation, post-translational modification, protein transport, positioning and ultimately activation, and so on.they can also could work as pre-miRNA, endogenous siRNA and scalfold in the regulation. They could affect cell proliferation, apoptosis, invasion and metastasis, drug resistance. However, because of their different to miRNAs in length and structure, to study the mechanism of them was quite complex.OBJECTIVES To study influences and molecular mechanisms of lncRNA-DKFZp434J0226 on invasion and metastasis in pancreatic cancer cell.METHODS Firstly real-time PCR was used to detect the expression of DKFZp434J0226 in pancreatic cancer cell line MIAPaCa-2, Capan-1 and PANC-1, and the one highest expressed was selected for the study object. DKFZp434J0226 knockout stably transfected cell lines was established. Then the wound healing assay and Transwell assay was to test changes about abilities of migration and invasive in cell line. Finally the real-time PCR and Western blot was used to detect the expression ofMMP-9andVEGF.RESULTS In three strains, expression of DKFZp434J0226 was the highest in MIAPaCa-2. In stably transfected cell lines, the level of DKFZp434J0226 was decreased over 90%. After knockout of DKFZp434J0226S, the wound healing assay and Transwell assay was showed the abilities of migration and invasion significantly impaired, and real time PCR as well as western blot showed expression of MMP-9 and VEGF was reduced.CONCLUSIONS Knockout DKFZp434J0226 can inhibit the invasion and migration in MIAPaCa-2 cells, which may be associated with reduced levels of MMP-9 and VEGF. |
PDF Full Text Request