CircRNA RNA NOL10 Act As Mir-874-3p Sponge To Promote Pancreatic Ductal Adenocarcinoma Proliferation

BackgroundMost patients with pancreatic ductal adenocarcinoma(PDAC)remain asymptomatic in early stage,patients always reaches an advanced stage when they were diagnosed.PDAC has been the most lethal disease,for which mortality closely parallels incidence.A growing amount of research suggests that the aberrant expression of circRNAs play important roles in the progression of malignant tumors.Unlike linear RNAs that are terminated with 5’caps and 3’tails,circRNAs is characterized unique closed loop structures without 5’caps and 3’ tails nor polyadenylated tails.Attributing to the covalently closed loop structure,circRNAs are more stable than liner RNA and insusceptible to degradation by RNA exonuclease or RNase R,so circRNAs are abundant,conserved and stable in mammalian cells.When it was first discovered,circRNAs were commonly considered as molecular flukes or products of aberrant RNA splicing and its biological function also has not been paid enough attention.However,with the development of high throughput sequencing technology and bioinformatics,researchers gradually confirm that circRNAs have abundant expression in mammalian cells and play important biological roles.One of the important biological roles of circRNAs is acting as miRNA sponge and then regulating the expression of target gene.Previous studies have confirmed that circRNAs play important roles in the progression of various malignant tumors but the role in PDAC is still unclear and remains to be investigated.Methods1.Through high throughtput sequencing technology,we found that circRNAs was dysregulated in PDAC;2.To further verify the authenticity of the sequencing results,we obtained eighty-four fresh-frozen pancreatic cancer samples(22 with adjacent non-cancerous tissues)from the Institute of Hepatopancreatobiliary Surgery,Southwest Hospital,Army Medical University.After RNA extraction,we investigated the expression level of 5 most upregulated circRNAs in PDAC tissues and adjacent non-cancer tissues and finaly confirmed that Circular RNA NOL10 was upregulated in PDAC tissues compared to adjacent non-cancerous tissues;3.In this study,we explored the ceRNAs mechnasims of circRNAs,to investigate the biological mechanism of Circular RNA NOL10,bioinformatics was used to predict the downstream miRNAs and the target gene of the miRNA and finally select miR-874-3p and PLK1 as potential target of Circular RNA NOL10;4.To investigate the biological roles of Circular RNA NOL10,siRNA was transfected into PDAC cell lines to inhibit the expression of Circular RNA NOL10.After siRNA transfection in PDAC cells,EdU assay,CCK-8 assay,colony formation assay and flow cytometry was used to determine the changes of proliferation capacity,clone formation ability and cell cycle respectively.5.Dual luciferase assay was adopted to investigated whether there is a direct binding relationship between Circular RNA NOL10 and miR-874-3p,miR-874-3p and PLK1.FISH assay was adopted to determine the sublocalization of circular RNA NOL10 and miR-874-3p in PDAC cells.6.In order to explore the biological correlation between Circular RNA NOL10 and miR-874-3p,miR-874-3p mimics and inhibitor were constructed and transfected to PDAC cells.Through rescue assay,we detemmined whether Circular RNANOL10 regulate PLK1 expression via miR-874-3p and then regulate the proliferation ability and cell cycle of PDAC.7.Immunohistochemistry(IHC)and Western blot was used to explore the expression level of PLK1 in PDAC tissues and adjacent non-cancerous tissues and the relationship between PLK1 expression level and clinical significance.8.Circular RNANOL10 was knocked down via a lentiviral infection system to establish two stable low Circular RNANOL10 expression pancreatic cancer cell lines.Subcutaneously implanted tumor model in nude mouse was constructed to investigate the effect of Circular RNANOL10 in tumor proliferation in vivo.Results1.The results of qRT-PCR revealed that Circular RNANOL10 is significantly upregulated in PDAC tissues compared with adjacent noncancerous tissues.Survival analysis revealed that patients with high Circular RNANOL10 expression have lower overall survival rate compared with those with low Circular RNANOL10 expression.2.CCK-8,colony formation and flow cytometry assay revealed that knockdown of Circular RNA NOL10 in PDAC cells inhibited proliferation,colony formation capacity and induced cell cycle arrest.3.Dual luciferase assay confirmed that Circular RNA NOL10 could directly bind miR-874-3p as miR-874-3p sponge and miR-874-3p could directly bind to PLK1 3’ UTR then induced reduced luciferase activity.FISH assay confirmed that Circular RNA NOL10 and miR-874-3p have common sublocalization in the cytoplasm of pancreatic cancer cells which reveal that Circular RNA NOL10 and miR-874-3p may paly important roles in post-transcriptional regulation.4.Transfection of miR-874-3p mimics recapitulated the biological function of Circular RNA NOL10 siRNA and cotransfection of miR-874-3p inhibitor and Circular RNA NOL10 siRNA abrogated the inhibitory effect induced by Circular RNANOL10 siRNA.5.The results of IHC and WB confirmed that PLK1 is overexpressed in PDAC tissues and PLK1 expression is closely associated with with the clinical stage,the pathologic tumor status and prognosis.6.Subcutaneously implanted tumor model in nude mouse confirmed that knockdown Circular RNA NOL10 inhibited pancreatic cancer progression in vivo.Western Blot assay further revealed that the Circular RNANOL10 knockdown group showed lower PLK1 expression.ConclusionCircular RNA NOL10 is aberrantly unregulated in PDAC tissues and high Circular RNA NOL10 is closely related to lower overall survival rate.Functional studies confirms that Circular RNANOL10 promotes PDAC proliferation in vitro and in vivo.Mechanistic investigations demonstrate that confirmed Circular RNANOLIO promotes PDAC proliferation by acting as the sponge of miR-874-3p to promote PLK1 expression.Taken together,Circular RNA NOL10 may serve as potential therapeutic target and prognosis predictor.