| Part Ⅰ Lnc RNA PVT1 upregulate proliferation,metastasis and glycolysis in pancreatic cancerObjective: To see how lnc RNA PVT affects pancreatic cancer cell proliferation,metastasis,and glycolytic effects,the correlation between the expression of lncRNA PVT1 in pancreatic cancer tissues and its clinicopathological characteristics and survival prognosis was analyzed by lncRNA PVT1 expression in pancreatic cancer tissues.Methods: Thirty patients with pathologically confirmed pancreatic ductal adenocarcinoma from December 2017 to December 2018 were collected at Hubei Cancer Hospital.q RT-PCR was used to analyze the correlation of PVT1 expression in pancreatic adenocarcinoma tissues using pancreatic adenocarcinoma tissues and paracancerous tissue specimens;the correlation between the above molecular indicators and the clinicopathological characteristics and prognosis of pancreatic adenocarcinoma was statistically analyzed in combination with clinical follow-up.PVT1 expression in human pancreatic cancer cell lines HPAC,PANC1,ASPC-1,DANG,BXPC3,and human normal pancreatic ductal epithelial cells HPDE6-C7(H6C7)was investigated using q RT-PCR.The knockdown efficiency of each interfering RNA-PVT1 was analyzed by q RT-PCR,and the group with the best knockdown efficiency was selected.The MTT assay,clone formation assay,scratch assay,and transwell assay have been used to examine changes in tumorigenesis and migratory ability;down-regulation of lncRNA PVT1 inhibited glycolysis and reduced the expression of HIF-1 and key glycolysis enzymes.After knockdown of lncRNA PVT1,the glucose and lactate contents in the culture medium before and after cell treatment were measured by absorbance value,and the glucose uptake,lactate secretion and intracellular ATP contents were calculated.WB detected the expression levels of HIF-1α and key enzymes of glycolysis(LDHA,GLUT1,HK2).Results: The findings revealed that the lncRNA PVT1 was more abundant in carcinoma tissues than in paraneoplastic tissues,high expression was associated with a poor prognosis and lymphovascular invasion.Lnc RNA PVT1 was highly expressed in pancreatic cancer cell lines compared to human average pancreatic ductal epithelial cells,lncRNA PVT1 was highly expressed in pancreatic cancer cell lines,with the highest expression of HPAC.q RT-PCR assay verified the best transfection efficiency of si RNA PVT1.Using the MTT assay and the clone formation assay,the cell proliferation potential was reduced after down-regulating PVT1.The cell metastasis ability was decreased after transfection using scratch assay and transwell assay.The glucose uptake,lactate secretion and intracellular ATP content were decreased by absorbance value assay,and the expression levels of HIF-1α and key enzymes of glycolysis(LDHA,GLUT1,HK2)were decreased by WB assay.Conclusion: Lnc RNA PVT1 is highly expressed in pancreatic cancer tissues,and its high expression is positively correlated with poor prognosis and lymph node metastasis in pancreatic cancer patients.Lnc RNA PVT1 could promote proliferation,metastasis and glycolysis levels of pancreatic cancer cells.Part Ⅱ Molecular mechanism of lncRNA PVT1 as ce RNA recruitment to downregulate miR-519d-3p to regulate HIF-1αObjective: Exploring the molecular mechanism of lncRNA PVT1 recruitment downregulation of miR-519d-3p and miR-519d-3p downregulation of HIF-1α m RNA at post-transcriptional level and it can verify the molecular mechanism of lncRNA PVT1 regulation of HIF-1α through miR-519d-3p.This could explain the biological significance of PVT1 in terms of theoretical mechanisms.Methods: First,bioinformatics analysis(microrna.org)was performed to predict the binding sites of lncRNA PVT1 and miR-519d-3p;overexpression of PVT1 or knockdown of miR-519d-3p,q RT-PCR to detect the expression levels of miR-519d-3p and PVT1 to demonstrate a negative correlation between the two expressions;pmir GLO-PVT1-wt plasmid and pmir GLO-PVT1-mut plasmid were constructed.Dual luciferase reporter system used to verify the PVT1 site bound to miR-519d-3p.RNA immunoprecipitation assay(RIP assay)was performed to verify that PVT1 and miR-519d-3p could bind.RNA pull down assay to detect that PVT1 can enrich miR-519d-3p.Targetscan analysis of HIF-1α and miR-519d-3p binding sequences.WB detection of HIF-1α protein expression and q RT-PCR detection of HIF-1α m RNA expression to verify the correlation between miR-519d-3p and HIF-1αexpression in pancreatic cancer cells.HIF-1α 3’-UTR-WT and HIF-1α 3’-UTR-mut plasmid were constructed.The fluorescence intensity of HIF-1α 3’-UTR was detected using a dual luciferase reporter system to verify that the miR-519d-3p target gene was HIF-1α.The correlation between miR-519d-3p and HIF-1α m RNA was analyzed by q RT-PCR for PVT1 and HIF-1α m RNA using pancreatic cancer specimens.Results: Bioinformatics analysis predicted the binding sites of lncRNA PVT1 and miR-519d-3p.q RT-PCR detected the negative correlation between lncRNA PVT1 and miR-519d-3p expression in pancreatic cancer cells,and the expression of both in pancreatic tissues was also negatively correlated.Dual luciferase reporter system,RIP assay and RNA pull down assay indicate that lncRNA PVT1 is negatively correlated with miR-519d-3p expression and can recruit and bind and downregulate miR-519d-3p level.Targetscan analysis of HIF-1α and miR-519d-3p binding sequences.Targetscan predicted HIF-1α as a target gene for miR-519d-3p binding.q RT-PCR and WB detected a negative correlation between miR-519d-3p and HIF-1α(m RNA and protein)expression,as well as a negative correlation between the expression of both in pancreatic tissues.The dual luciferase reporter system assay verified that miR-519d-3p could bind to the 3’-UTR of HIF-1α.Conclusion: Lnc RNA PVT1 is negatively correlated with miR-519d-3p expression and can recruit binding and downregulate miR-519d-3p;miR-519d-3p decreases HIF-1α m RNA expression by binding the 3’-UTR of HIF-1α and at the post-transcriptional level.Part Ⅲ The role of lncRNA PVT1-miR-519d-3p-HIF-1αpathway in the regulation of pancreatic cancer proliferation,metastasis and glycolysis levelsObjective: To investigate and elaborate the role of this lncRNA PVT1-miR-519d-3p-HIF-1α pathway in the regulation of proliferation,invasion and glycolysis levels in pancreatic cancer.Methods: Knockdown lncRNA PVT1 was cotransfected with knockdown miR-519d-3p,absorbance value method was used to detect changes in glucose,lactate and ATP contents after cotransfection,and to calculate glucose uptake,lactate secretion and intracellular ATP content.The changes of proliferation and migration ability of pancreatic cancer cells were analyzed by MTT assay,clone formation assay,scratch assay,and transwell assay.The expression levels of HIF-1α and key enzymes of glycolysis(LDHA,GLUT1,HK2)were detected by WB.The subcutaneous xenograft tumor model of HPAC cells with stable sh RNA-PVT1 and negative control was constructed to detect tumor size,tumor volume and tumor mass,and miR-519d-3p was detected by q RT-PCR;HIF-1α protein level was detected by IHC.Results: In pancreatic cancer cells,scratch assay and transwell assay verified metastatic ability,glucose uptake,lactate secretion,intracellular ATP content and glycolytic key enzyme protein expression levels verified glycolytic ability,MTT assay and clone formation assay verified proliferative ability,and knockdown of lncRNA PVT1 showed a significant decrease in proliferation,metastasis and glycolysis levels.The proliferation,metastasis and glycolytic capacity were enhanced after miR-519d-3p plasmid rescue.A stable transgenic cell line was constructed,and knockdown of PVT1 inhibited tumor growth in mice,increased the expression level of miR-519d-3p in mice,and decreased the protein expression level of HIF-1α in mice tumors.Conclusion: The lncRNA PVT1-miR-519d-3p-HIF-1α pathway regulates its proliferation,invasion and glycolysis levels in cell lines and in vivo. |
