The Study On Regulatory Functions Of TUG1-miR-299-3p Axis In Pancreatic Cancer And Its Mechanism

Pancreatic cancer is one of the most malignant diseases worldwide.In the early stages of pancreatic cancer,most patients have no obvious symptoms or signs until the tumor cells metastasize to other organs and develop into a later stage.Therefore,the search for new biological indicators related to the development,early diagnosis and prognosis of pancreatic cancer is of great significance for the early detection of pancreatic cancer and the improvement of the prognosis of pancreatic cancer patients.In large numbers of non protein-encoded transcripts,long-chain non-coding RNA(lncRNA)is a new class of non-protein-encoded transcripts,as many new non protein-encoded transcripts have been identified with advances in genomic and transcriptome sequencing techniques.LncRNAs are defined as endogenous cellular RNAs with more than 200 nucleotides in length,and they play crucial roles in the regulation of various pathophysiological processes.Taurine upregulated 1(TUG1),a novel lncRNA with 7.1-kb nucleotides and located at chr22q12.2,which was initially characterized by a genomic screening study in mouse retinal cells.With the increase of the researches on TUG1,TUG1 is found in avariety of tumors.However,researches of TUG1 are still in initial stage in pancreatic cancer.The mechanism of TUG1 in the pathogenesis of pancreatic cancer remains unclear.miR-299-3p is a type of miRNAs,which has been rarely reported in domestic and foreign literatures.Studies have shown that miR-299-3p is down-regulated in osteosarcoma tissues,which is related to tumor cell proliferation.apoptosis and chemotherapy.Bioinformatics analysis showed that TUG1 can sponge and regulate the expression of miR-299-3p,but the specific mechanism of miR-299-3p in pancreatic cancer remains unclear.This study first detected the expression of TUG1 and miR-299-3p in human normal pancreas tissue,adjacent tissue of pancreatic cancer,pancreatic cancer tissue and pancreatic cancer cell lines,and analyzed the relationship between TUG1 expression and TNM stages of pancreatic cancer,and the relationship between TUG1 and miR-299-3p was predicted by Starbase2.0 online.Secondly,the dual luciferase reporter gene assay verified that TUG1 could bind to miR-299-3p,and the effects of TUG1-miR-299-3p on the proliferation,invasion,migration,apoptosis,EMT.the Notchl signaling pathway in PANC-1 and BXPC-3 cells were detected by MTT,scratch transwell,western blot,flow cytometry and ELISA assays.a series of molecular biological methods were used to further explore the molecular mechanism of TUG1-miR-299-3p in the development and progression of pancreatic cancer.Finally,the mice bearing pancreatic cancer transplanted tumor was established,and the effect and mechanism of TUG1-miR-299-3p on pancreatic cancer xenografts were further studied.In this study,TUG1-miR-299-3p was determined to regulate the proliferation,invasion,migration and apoptosis of pancreatic cancer cells by regulating the expression of miR-299-3p,and then could regulate the growth of pancreatic cancer,which provided a new idea for the treatment of pancreatic cancer.This study is divided into three parts:Part ?:The study of expression and clinical value of TUG1 and miR-299-3p in pancreatic cancerMethods1.qRT-PCR was used to detect the expression of TUG1 in 31 cases of pancreatic cancer tissue and adjacent tissue of pancreatic cancer tissues samples and 8 cases of normal tissue samples2.Analysis of the relationship between the expression of TUG1 in pancreatic cancer and the TNM stages of clinical characteristics in 31 cases of pancreatic cancer.3.qRT-PCR was used to detect the level of TUG1 in normal pancreatic and pancreatic cancer cell lines(PANC-1,PSN-1,BXPC-3,HPAC).4.Spearman's correlation analysis was conducted on the relationship between TUG1 and miR-299-3p expression in pancreatic cancer tissues.5.Starbase2.0 was used to predict the binding sequence of TUG 1 and miR-299-3p online.Results1.The result of qRT-PCR showed that TUG1 was significantly higher in pancreatic cancer tissues than that in adjacent tissue of pancreatic cancer tissues and normal pancreatic tissues and miR-299-3p expression was significantly reduced.2.Through analysing the clinical data of pancreatic cancer patients with 31 cases of pancreatic cancer and TUG1 expression in pancreatic cancer tisssues,the data showed that the expression of TUG1 in tumor tissues of pancreatic cancer patients with ?+? stage were significantly higher than that in pancreatic cancer patients with ?+? stage.3.The result of qRT-PCR showed that the expression level of TUG1 in PANC-1,PSN-1,BXPC-3 and HPAC cells was significantly increased compared with HPDE cells,and the expression of TUG1 is the mostly highest in PANC-1 and BXPC-3 cells.Compared with normal pancreatic cells,miR-299-3p levels were obviously decreased.4.Spearman's correlation analysis showed that the expression of TUG1 in pancreatic cancer tissues was negatively correlated with the expression of miR-299-3p.Starbase2.0 online software predicted that TUG1 had binding sequences with miR-299-3p.Part ?:The studies of the function of TUG1 and miR-299-3p in pancreatic cancer cellsMethods1.The dual luciferase reporter gene assay was used to verify whether TUG1 could sponge to miR-299-3p.2.Lentiviral vectors(interfering TUG1 and miR-299-3p)were constructed.After recombinant lentivirus were packaged and titrated,the virus particles infected pancreatic cancer cell lines(PANC-1 and BXPC-3).sh-NC,sh-TUG1.sh-TUG1+anti-NC,sh-TUG1+anti-miR-299-3p PANC-1 and BXPC-3 cells were gained.qRT-PCR was used to detect the effect of TUG1 knockdown on the expression of miR-299-3p.3.MTT assay,scratch assay.transwell assay,flow cytometry and western blot were used to detect the effects of TUG1-miR-299-3p axis on the proliferation,migration,invasion and apoptosis,EMT and Notchl signaling pathway in PANC-1 and BXPC-3 cells.Results1.The dual luciferase reporter gene assay verified that TUG1 could bind to miR-299-3p.2.TUG1 knockdown could promote miR-299-3p expression in PANC-1 and BXPC-3 cells.3.TUG1 knockdown inhibited cell proliferation,migration,invasion,EMT and activation of the Notchl pathway,as well as promoted apoptosis.Furthermore,miR-299-3p knockdown could inverse the effect of TUG1 knockdown on cell proliferation,migration,invasion,EMT,and apoptosis by promoting the activaty of Notch1 pathway.Part ?:Studies on the effect of TUG1 and miR-299-3p on tumor formation of pancreatic cancer cells in nude mice1.PANC-1 xenograft mouse models were constructed by left axillary intraperitoneal injection of sh-NC,sh-TUG1,sh-TUG1+anti-NC.sh-TUG1+anti-miR-299-3p PANC-1 cells.After the models were successfully established,the volume was measured every five days.After 30 days,the mice were sacrificed by cervical dislocation method.And then the weight of xenografts was determined.2.qRT-PCR was performed to detect the expression of miR-299-3p in xenograft tissues.3.Immunohistochemical staining was used to confirm the effect of TUG1-miR-299-3p on Ki67expression in pancreatic cancer xenograft tissues.4.Western blot was used to detect the influence of TUG1-miR-299-3p on the expression of EMT-related proteins(E-cadherin,N-cadherin and Snail)as well as the Notchl pathway-related proteins(Notchl,Survivin and CyclinD1).Results1.Compared with the control group,TUG1 knockdown significantly inhibited growth of transplanted tumor,and miR-299-3p knockdown reversed the inhibitory effect.2.TUG1 knockdown significantly promoted the expression of miR-299-3p in pancreatic cancer xenograft tissues.3.TUG1 downregulation markedly constrained Ki67 expression in pancreatic cancer xenograft tissues,and knockdown of miR-299-3p significantly promoted the Ki67 expression compared with TUG1 knockdown group.4.TUG1 knockdown significantly enhanced E-cadherin expression and inhibited N-cadherin as well as Snail expression in pancreatic cancer xenograft tissues.Moreover,knockdown of miR-299-3p significantly reversed the effect on E-cadherin,N-cadherin and Snail expression in pancreatic cancer xenograft tissues.5.Compared with control group,the Notchl,Survivin and CyclinD1 expression was significantly suppressed by TUG1 knockdown in pancreatic cancer xenograft tissues,whereas knockdown of miR-299-3p could reverse the inhibitory effect of TUG1 knockdownConclusion1.TUG1 expression was significantly increased in pancreatic cancer tissues and cells,while miR-299-3p expression was significantly decreased,and they showed a negative correlation.The expression of TUG1 was closely related to TNM stage.2.TUG1 could sponge miR-299-3p and inhibit the expression of miR-299-3p.3.TUG1-miR-299-3p axis could affect the proliferation,invasion,migration,epithelial mesenchymal transformation(EMT)and apoptosis of pancreatic cancer cells by regulating the activity of the Notch1 pathway.4.An animal model of pancreatic cancer xenograft was successfully constructed.TUG1-miR-299-3p axis could promote the growth of xenografts by promoting the activity of the Notch 1 pathway.5.TUG1 played a role of primoting cancer and miR-299-3p played a role of inhibiting cancer,which may be potential molecular therapeutic targets.