Abnormal Expression And Molecular Mechanism Of LINC00994 In Pancreatic Cancer

Pancreatic cancer is a highly malignant tumor of digestive system,with the morbidity and mortality increasing year by year.According to global statistics in 2018,the incidence of pancreatic cancer is 2.5%,and the mortality rate is 4.5%,ranking 14 th and 7th in the world respectively.The incidence of pancreatic cancer is concealed and progresses rapidly.It is not sensitive to radiotherapy and chemotherapy.The diagnosis is mostly in the middle and late stage.The surgical resection rate is only 20%,and the recurrence rate and metastasis rate are extremely high.The 5-year survival rate is only about 8%.Therefore,it is particularly important to explore the molecular mechanism of pancreatic cancer development and to find effective therapeutic targets now.In April 2003,the sequencing of the Human Genome Project(HGP)was completed,revealing that only 1%-1.5% of human genomic DNA could encode proteins,and more than 98% of the sequences were non-protein coding RNAs(ncRNAs),so-called "junk DNA".Following HGP,in September of the same year,the US National Human Genome Research Institute(NHGRI)launched another transnational genomics research project,the encyclopedia of DNA Elements(ENCODE),in order to resolve all the functional elements in the human genome,and over a period of nine years,it revealed that at least80% of the ncRNA in the human genome were biologically active rather than the previously considered "junk product".Depending on the number of bases in the transcript,ncRNA could be divided into long non-coding RNA(lncRNA)and short non-coding RNA,such as microRNA(miRNA).LncRNAs are a class of RNA molecules that are greater than 200 nt in length and are transcribed by RNA polymerase II,lacking an open reading frame(ORF),and are not involved or rarely involved in encoding proteins.Numerous studies have found that lncRNA exerted molecular regulatory functions at all levels of gene expression,participated in the pathophysiological processes of tumors and was closely related to the prognosis of the disease.Therefore,in-depth study of differentially expressed lncRNA in pancreatic cancer and understanding its mechanism of action will be of great significance for improving the prognosis of patients.In 2001,journal of Science published three articles on miRNAs and presented the concept ofmiRNA for the first time.Most of the miRNA genes are transcribed by RNA polymerase II into primary miRNAs(pri-miRNAs)with a length of 300-1000 nt,and then pri-miRNAs are processed by Drosha enzyme to form a 70-90 nt long precursor miRNA(pre-miRNA)with a stem-and-loop structure,pre-miRNA enters the cytoplasm and then cleaved into a 21-25 nt double-stranded miRNA by Dicer enzyme and then untwisted into a mature single-stranded miRNA.The mature miRNA can bind to the Ago protein to form an RNA induced silencing complex(RISC),which recognizes and acts on the 3' untranslated region(3 ' UTR)of the target gene by base complementary pairing,which degrades mRNA or inhibits its translation into proteins,thereby reducing the protein expression of target genes.One of the important molecular regulation modes of lncRNA is competitive endogenous RNA(ceRNA).LncRNA and the 3' UTR of the mRNA are enriched with miRNA recognition elements(MRE).By competitively binding miRNA,lncRNA partially relieve the inhibition of miRNA on its target gene,which leads to the up-regulation of target gene expression.In this study,high-throughput microarray technology was used to screen out differentially expressed lncRNAs in pancreatic cancer tissues.It was found that long non-coding RNA LINC00994 was highly expressed in pancreatic cancer,and there had been no relevant report before,so there might be extensive research value.We used real-time qPCR to verify the expression level in 10 pairs of clinical tissue samples.The results was consistent with the chip detection data,which laid the foundation for the subsequent experiments.Next,we selected two pancreatic cancer cells Panc-1 and AsPC-1 with high basal expression level of LINC00994,knocked down the expression of endogenous LINC00994 by transfecting shRNA interference plasmid,analyzed the effect of sh-LINC00994 on the biological behavior of pancreatic cancer cells from the perspective of lack of function.The results showed that silencing the expression of LINC00994 inhibits the proliferation,migration and invasion of pancreatic cancer cells and promotes apoptosis.Therefore,we speculated that LINC00994 might play a role in cancer-promoting genes in pancreatic cancer.To explore the potential molecular mechanisms by which LINC00994 plays a role in cancer promotion,we used three previous tissue samples,using Agilent Human miRNA,Release 21.0(8*60K,Design ID:070156)gene chip to screen differentially expressed miRNA molecules in pancreaticcancer.Furthermore,the screening range was further narrowed by ?Fc ??10 and P value?0.01.Finally,miR-765-3p(Fc=23,p=0.00089)with the most significant difference was screened out.Based on the information of miRanda,TargetScan and RNAhybrid databases,we found that there are two potential site of action between miR-765-3p and LINC00994 and one potential binding site in the 3'UTR of RUNX2(Runt-related transcription factor-2),as a downstream target gene of miR-765-3p.The GEPIA database predicts that RUNX2 is highly expressed and significantly positively correlated with the expression of LINC00994 in pancreatic cancer.(spearman correlation analysis: p =0.0022,r = 0.23).Real-time qPCR and Western blot experiments confirmed that miR-765-3p was down-regulated in pancreatic cancer tissues and had a significant negative correlation with the expression of LINC00994,and the RUNX2 expression was significantly higher than the adjacent cancer control tissue.Overexpression of LINC00994 could significantly increase the mRNA and protein expression levels of RUNX2 in pancreatic cancer cells,while knocking down the expression of LINC00994 or RUNX2 could significantly reduce the expression level of each other,indicating that there was a positive regulation relationship between LINC00994 and RUNX2.Furthermore,through overexpression and silencing,we found that miR-765-3p is able to negatively regulate the expression of LINC00994 and RUNX2.Based on this,we hypothesized that LINC00994 would promote the development of pancreatic cancer by interfering with the targeted binding of miR-765-3p to RUNX2.To demonstrate the above scientific hypothesis,we constructed a pmirGLO dual luciferase reporter vector containing a LINC00994(1015 or 647 locus)fragment or a RUNX2 3'UTR,using dual luciferase reporter gene system to detect whether miR-765-3p directly targets LINC00994 or RUNX2 and to verify the binding site.The results showed that luciferase activity was significantly decreased in HEK293 cells co-transfected with miR-765-3p-mimic and pmirGLO-LINC00994 WT(1015 or 647)or pmirGLO-RUNX23'UTR WT vector,and the inhibitory effect of miR-765-3p on luciferase activity disappeared when co-transfected with miR-765-3p-mimic and pmirGLO-LINC00994MT(1015 or 647)or pmirGLO-RUNX2 3'UTR MT vector with mutation at the binding site.Similarly,we co-transfected miR-765-3p-mimic and pmirGLO-RUNX2 3'UTR WT vectors into sw1990 cells,and the luciferase activity was significantly reduced,whileoverexpression of LINC00994 reversed luciferase activity,indicating that LINC00994 can competitively bind to miR-765-3p to regulate the expression of RUNX2.Finally,in order to further completely explain the mechanism of the LINC00994/miR-765-3p/RUNX2 axis,we performed a rescue experiment and found that miR-765-3p could reverse the regulation of RUNX2 expression by LINC00994,and miR-765-3p-inhibitor could reverse the inhibitory effect of sh-LINC00994 on the proliferation and migration of pancreatic cancer cells,suggesting that LINC00994 could regulate RUNX2 through miR-765-3p,and influence the biological behavior of pancreatic cancer.In summary,this study found that LINC00994 was highly expressed in pancreatic cancer,knocking down the expression of LINC00994 could inhibit the proliferation,migration and invasion of pancreatic cancer cells,and LINC00994 was capable of playing the role of a cancer-promoting gene through the LINC00994/miR-765-3p/RUNX2 axis.