The Expression Level Of LncRNA RP11-263F15.1 In Pancreatic Cancer And Its Effect On Biological Behavior Of Pancreatic Cancer Cell

Backgroud Pancreatic cancer is one of digestive system malignances.Pancreatic cancer,characterized by rapid development and high mortality rate(5-year survival rate is less than 5%),is a common malignance threatening the human life and health.In China,the morbidity of pancreatic cancer is about 300,000-400,000 and the trend is increasing.It is reported that the common risk factors of pancreatic cancer are smoking,obesity,diabetes and chronic pancreatitis.Currently,the diagnosis method of pancreatic cancer is limited and CA19-9 is the main biomarker to detect pancreatic cancer.However,due to low sensitivity and specificity,CA19-9 is not an ideal biomarker for diagnosis in pancreatic cancer because it will express abnormally in the case of chronic hepatitis and chronic pancreatitis.Patient is usually at the advanced stage of pancreatic cancer with nerve invasion and distant metastasis when diagnosed,losing the best time to treat it.Besides,although there are many kinds of therapies including surgery,chemotherapy and radiotherapy treating pancreatic cancer,the effectiveness is unsatisfied.The molecular mechanism underlying pancreatic cancer is in the exploring process,which should be further investigated.It is generally acknowledged that the pathogenic factors of pancreatic cancer can be roughly divided into two categories: external environment factors and inherent genetic factors.Gene mutations including but not limited to KRAS,SMAD4,TP53 and CDKN2 A are common pathogenic factors of pancreatic cancer.The mutant KRAS,which can be found in 80%-90% pancreatic cancer cases,is the most common factor among them.Secondly,mutant TP53 exists in about 50% pancreatic cancer cases.Additionally,mutant CDKN2 A and SMAD4 can be found in about 20% pancreatic cancer cases.To some extent,the aforesaid common gene mutations can illuminate the biological mechanism of pancreatic cancer.However,gene mutation alone insufficiently fully accounts for the biological mechanism of pancreatic cancer,for the reason that gene expression in the human body can be regulated and controlled by the process of gene transcription and translation.Consequently,seeking a new point to break through in the exploration process of pancreatic cancer mechanism is needed so as to have a more comprehensive view and to design effective drugs aiming to curb the development of pancreatic cancer.Long noncoding RNA,whose transcript sequences are more than 200 bp,is unable to code protein.In the past ten years,a mass of Lnc RNAs were discovered by methods.Nevertheless,their functions in biological mechanism remain to be illuminated.Presently,lnc RNA has been proved to be involved in multiple biological processes in the human body.Moreover,some lnc RNAs play a role in key process.More importantly,lnc RNA,different from m RNA and its protein products characterized by wide expression,most of which only express in specific biological stages of corresponding organs.This provides a new strategy for clinical diagnosis of diseases including tumor.In the last decade,a large number of studies have found that lnc RNA is related to the cause and development of multiple diseases.Researches show that there is a correlation between lnc RNA and the morbidity of malignance in many parts of body.lnc RNA MALAT-1 has been proved to be relevant to the early stage of non-small cell lung cancer.Studies indicate that the expression levels of lnc RNA PAC3 in the cases of prostate cancer increase significantly.lnc RNA H19 has also been proved to be related to the incidence of gastric cancer.In the cases of pancreatic cancer,researches about lnc RNA indicate that a number of lnc RNA and relevant m RNA expression change during the progression of this disease and the specific functions of only a few lnc RNA can be explored.For instance,during the progression of pancreatic cancer,the control and regulation imposed by lnc RNA HOTAIR located near HOX gene towards HOX expression is different from that in normal physiological conditions,which changes the tumor cells' ability of proliferation and invasion and encourages the cancer incidence.In consideration of aforesaid research background and present situation,we make the following assumption: with the method of sequencing to detect a number of lnc RNAs expressing abnormally in pancreatic cancer,then relevant lnc RNAs would be selected to serve as the clinical markers exploring the clinical significance of lnc RNA in diagnoses and disease prediction so as to provide new insights into the diagnosis and therapy of pancreatic cancer.Aims The main aim of this work is to explore the assumption: with the method of sequencing to detect a number of lnc RNAs expressing abnormally in the cases of pancreatic cancer,then lnc RNAs with abnormal expression would be selected to validate the results of sequencing with a lot of clinical tissue cases.Meanwhile,with the method of cell experiment,we investigate the role of the specific lnc RNAs in the pathogenesis of pancreatic cancer so as to reveal its mechanism in the development of pancreatic cancer.Methods 1.Use microarray to detect the expression levels of 10(5 pairs)clinical lnc RNAs and relevant m RNAs in pancreatic cancer and adjacent tissues.2.Make Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis on the basis of the results of Microarray.3.3.Measure the expression levels of lnc RNAs characterized by abnormal expression using q RT-PCR technology then compare the results to the expression levels of lnc RNA s in pancreatic cancer tissues and their adjacent tissues.Make an analysis of how the results could be licked to the clinicopathologic features of patients with pancreatic cancer.4.Select cell lines including As PC-1,PANC-1,MIA Pa Ca-2,CFPAC-1 and Bx PC-3 and determine the expression of specific lnc RNAs in these cell lines.5.Select PANC-1 and As PC-1 in vitro experiment.Develop a model in vitro with the method of overexpression construction,interference recombinant plasmid.Then determine the cell proliferation with CCK-8 experiment.Subsequently apply the method of Transwell to determine cell invasion and migration.6.Use flow cytometry to test the over-expressed or sequence-interfering PANC-1and As PC-1 in specific lnc RNA cell lines transfected by plasmid or infected.Then make an analysis of whether the cell cycle changes.Results 1.According to the result of Microarray based on 33045 lnc RNAs and 30215 relevant m RNAs in 5 pairs of pancreatic cancers and their adjacent tissues,expression change were found in 4364 lnc RNAs(multiple of change?2.0,P< 0.05),including the expression of 2365 lnc RNAs increased and that of 1005 relevant m RNAs decreased.2.GO analysis revealed that up-regulated m RNA had the closest relationship with the protein function(e.g.protein location and the intracellular locations of proteasome complexes and large molecules).Down-regulated m RNA was related to material transport in cell,cytochrome c's release from mitochondria and the relationship with cell receptor.KEGG analysis demonstrated that 42 signal pathways in all were related to up-regulated m RNA and 8 signal pathways were related to down-regulated m RNA.3.The expression changes of lnc RNA CTD-2244C20.1,RP11-263F15.1 and LOC541471 in pancreatic cancer tissues showed obvious statistically difference(P< 0.05),compared with those in pancreatic cancer adjacent tissues.4.Lnc RNA RP11-263F15.1,related to the differentiated degree of pancreatic cancer (P< 0.05),showed diagnostic value of 0.843(95% confidence interval=0.774-0.911,P<0.001)in pancreatic cancer.Additionally,patients with high expression level of lnc RNA RP11-263F15.1 had relatively poorer prognosis.The total survival time of these patient was 12.0 months,which was significantly shorter than that of patients with low expression level of lnc RNA RP11-263F15.1(25.1 months)(P<0.001).5.In vitro,the ability of over-expressed cell lines in lnc RNA RP11-263F15.1 to proliferate,invade and migrate improved evidently and the cell ratio of S stage increased significantly.However,the ability of the interfering cell lines in lnc RNA RP11-263F15.1 toproliferate,invade and migrate reduced significantly while the cell ratio of S stage decreased significantly.Conclusions The expression levels of a series of lnc RNAs and relevant m RNAs change in pancreatic cancer.Evident change was found in the expression levels of lnc RNA CTD-2244C20.1,RP11-263F15.1 and LOC541471 in pancreatic cancer.The expression level of Lnc RNA RP11-263F15.1 in pancreatic cancer increases significantly.The diagnostic value of Lnc RNA RP11-263F15.1 in cancer is 0.843(95% CI=0.774-0.911,P<0.001)and Lnc RNA RP11-263F15.1 is related to the differentiated degree of pancreatic cancer.Patients with high expression level of lnc RNA RP11-263F15.1 has relatively poorer prognosis.Over-expressed Lnc RNA RP11-263F15.1 can enhance pancreatic cancer cell lines' ability to proliferate,invade and migrate.Besides,lnc RNA RP11-263F15.1 can increase the cell ratio of S stage in pancreatic cancer cell line.Lnc RNA RP11-263F15.1,which is expected to act as a new tumor marker,provides a new approach and insight for pancreatic cancer research and diagnosis and it has potential significance for clinical diagnosis and prognosis prediction.