Effect And Mechanism Of Circular RNA CiRS-7 Promotes The Proliferation And Metastasis Of Pancreatic Cancer By Regulating MiRNA-7 Mediated EGFR/STAT3 Signaling Pathway

Pancreatic cancer(Pancreatic ductal adenocarcinoma PDAC)is a kind of digestive system tumor with high degree of malignancy.Due to the development of living standard and poor diet and living habits,the incidence of Pancreatic cancer has increased about sixfold in the past 20 years.Surgical resection is still the preferred treatment for pancreatic cancer.Therefore,how to improve the preoperative diagnosis rate of pancreatic cancer,master the surgical indications,and improve the surgical resection rate is the key to ensure good efficacy of pancreatic cancer patients.The epidemiological studies of pancreatic cancer in recent years have found that the 5-year survival rate is still low,about 8.5%,the most important reason is the high malignant degree,strong invasion and may associate with lymph node metastasis along with nerve vascular infiltration in an early stage,therefore many patients have been in an advanced stage when diagnosed,and can soon appear local and distant metastasis.For lack of the early detection markers of pancreatic cancer,finding markers for early occurrence and development of pancreatic cancer will be a key factor in improving the prognosis of patients,In recent years,more and more scholars have noticed that the non-coding genes including micro RNA,circular RNA,Long non-coding RNA which were neglected before were closely correlated with occurrence and development of cancer,and part of them can even be applied to tumor diagnosis as early detection markers.MiRNA is a kind of non-coding single strand small RNA with the size of about 22-24 nucleotides,existing in eukaryotic cells and is involved in gene regulation after transcription.It can specifically identify and bind to the 3'-untranslated area(3'UTR)in Messenger ribonucleic acid(mRNA)of target gene,causing inhibition of target gene mRNA translation,thus implementing regulation on post transcriptional expression of target gene,and finally taking part in development of malignant tumor by affecting target gene expression.It is via schorlarly searching engines like Pubmed,Wan Fang Data and Google scholar can we find that the miRNA-7,a kind of non-coding anti-cancer gene widely exists in cells,palys an important role in the proliferation and metastasis of numerous tumors(including liver cancer,cervical cancer,lung cancer,etc.),and it is also the marker for early diagnosis and key factor affecting patients' prognosis,however the research of miRNA-7 on proliferation and metastasis of pancreatic cancer have been rarely reported until now.CicrRNA is a kind of non-coding RNA,with the function of gene expression regulation,can be produced by means of reverse stitching or lasso introns,having a series of characteristics including evalutionary conservative,structure stability and tissue expression specificity,which give different functions to circRNA,for instance,as the miRNA sponges,can compete with mRNA for binding to miRNA,can interfere the inhibition and regulation function of miRNA to target mRNA,thus affecting the expression and relevant protein synthesis of downstream gene.One of the representative studies found that CDR1as,also known as ciRS-7,contains more than 70 miRNA loci,which can competitively inhibit miRNA-7 activity,thereby improving the expression of miRNA-7 target genes and affecting biological functions.In recent studies of squamous cell carcinoma of the larynx and non-small cell lung cancer.It was confirmed that ciRS could regulate the expression of PIK3CD,EGFR,CCNE1,STAT3 by inhibiting miRNA-7,and affect the proliferation and invasion of tumors.Previous studies have confirmed that EGFR/STAT3 signal transduction pathway is abnormally expressed in many tumors,such as breast cancer,glioma and lung cancer,and affects the proliferation and metastasis of tumors.Based on this,we aim to explore the expression of ciRS-7 and miRNA in pancreatic cancer and the effect of which on biological characteristics of pancreatic cancer,to further identify the effect and mechanism of ciRS-7 affecting the activity of EGFR/STAT3 signaling pathway by regulating the expression of EGFR etc.,the target genes of miRNA-7 via competitively binds to miRNA-7,and explore the mechanism of ciRS-7 influencing the biological characteristics of pancreatic cancer.It was found that few relevant literatures were reported so far after reviewing literatures.This topic is mainly divided into three parts:Part I Expression of CiRS-7 and miRNA-7 expression in pancreatic cancer tissues and their clinical significanceResearch objectives:To study the expression of ciRS-7 and miRNA-7 in pancreatic cancer tissues,and to explore the relationship between ciRS-7 expression in tissues and the biological characteristics of pancreatic cancer,such as the relationship with tumor development,invasion and metastasis,and analyze the significance of ciRS-7 in the prediction of clinical prognosis of pancreatic cancer.Research methods:This study was approved by the Ethics Committee of Anhui Provincial Hospital.A total of 41 PDAC tumors and their paracancerous tissues were obtained from General Surgery Department of Anhui Provincial Hospitall.All patients had signed the informed consent before the follow-up study.1.Quantitative real-time PCR(QRT-PCR)was used to detect the expression of ciRS-7 and miRNA-7 in 41 cases of PDAC tumors and paracancerous fresh frozen tissues.Correlation analysis was conducted on the expression of ciRS-7 and miRNA-7 to explore the relationship between the expression of ciRS-7 and miRNA-7 along with the occurrence and development of tumor.2.The expression of ciRS-7 in pancreatic cancer tissues and the clinical data were statistically analyzed to explore whether the expression of ciRS-7 was correlated with location,size of tumor,lymph node metastasis,local nerve infiltration and other factors.3.Expression of EGFR and STAT3 in pancreatic cancer and paracancerous tissues detected and analyzed by Immunohistochemistry(MHI)along with the clinical data were statistically analyzed.4.Postoperative follow-up was conducted,analyzing the significance of high ciRS-7 expression on clinical prognostic prediction of pancreatic cancer.Results:1.CiRS-7 expression in pancreatic cancer tissues was higher than that in adjacent tissues,and the difference was statistically significant(P<0.01),while miRNA-7 expression in pancreatic cancer tissues was lower than that in adjacent tissues,and the difference was statistically significant(P<0.05).Spearman correlation analysis revealed a negative correlation between ciRS-7 and miRNA-7 expression in pancreatic cancer(r=-0.035,P<0.05).2.According to statistical analysis of clinical data,expression level of ciRS-7 in pancreatic cancer was associated with lymph node metastasis(P=0.016)and nerve invasion(P=0.028)of tumor,but not gander and age of patients,with diabetes or not,location,size of tumor(P>0.05).3.After detected by IHC,the expression level of EGFR,STAT3 in pancreatic cancer tissue was higher than adjacent tissue,and was correlated to lymph node metastasis of tumor(P<0.05),but not gender and age of patients,location of tumor(P>0.05).4.After 1 year of follow-up,it was found that the tumor-free survival rate of pancreatic cancer patients with up-regulated ciRS-7 expression was lower than those with down-regulated ciRS-7 expression,and the difference was statistically significant(P<0.05).Conclusion:CiRS-7 expression in pancreatic cancer is higher than that in adjacent tissues,while miRNA-7 expression in pancreatic cancer is lower than that in adjacent tissues,and the expressions of the two are negatively correlated.CiRS-7 expression is correlated with lymph node metastasis and nerve invasion.Expression of EGFR and STAT3 could be found in pancreatic cancer tissue and was correlated with biological characteristics like proliferation and invasion etc.of tumor.Follow-up survey showed that higher ciRS-7 expression may be an indicator of prognosis of pancreatic cancer.Histological studies have shown that ciRS-7 and miRNA-7 expression may be correlated with tumor development,and provide a basis for the diagnosis and prognosis of pancreatic cancer.Part II To investigate the effect and mechanism of circular RNA ciRS-7 promoting proliferation and metastasis of pancreatic cancer by regulating miRNA-7 mediated EGFR/STAT3 signaling pathway in vitroResearch objectives:The role and mechanism of ciRS-7 and miRNA-7 in the proliferation and metastasis of pancreatic cancer were investigated in vitroResearch methods:1.The expression of ciRS-7,miRNA-7 in pancreatic cancer cell lines(BXPC-3,PANC-1)and normal pancreatic cells HPC-Y5 was examined by method of QRT-PCR.Silenced the expression of ciRS-7 and miRNA-7 in pancreatic cancer cells BXPC-3,PANC-1 by small interfering RNA(siRNA),and screened the ciRS-7-1 that stably low expressing ciRS-7 along with siciRS-7-1+simiRNA-7 that silenced expression of miRNA-7.2.Detecting expression of miRNA-7 in siciRS-7 group,siciRS-7&simiRNA-7 group,control group and negative control group(NC)by QRT-PCR.3.Testing the proliferation of cells in every group through MTT cell proliferation test.4.Cell invasiveness in every group was tested by Transwel Small chamber.5.Expression of EGFR/STAT3 in every group were examined by QRT-PCR and Western blotting.6.Change of cell proliferation and invasiveness ability in every group was examined by MTT cell proliferation test and Transwell Small chamber after Gefitnib,the specific blocker of EGFR/STAT3 signaling pathway was added.Results:1.CiRS-7 expression in pancreatic cancer cells was higher than that in normal pancreatic cells(P<0.05),and low expression ciRS-7 group was selected for further study.2.QRT-PCR detection revealed that the expression of miRNA-7 in the siciRS-7-1 group was higher than that in the control group and the blank control group(P<0.05).3.MTT assay showed that the proliferation of cancer cells in the siciRS-7-1 group was significantly lower than that in the control group and the blank control group(P<0.05).while no significant difference of cells proliferation ability was found between siciRS-7-1+simiRNA-7 group and control group(P>0.05).Transwell chamber method also found that the invasion ability of cancer cells in the siciRS-7-1 group was significantly lower than that in the control group and the blank control group(P<0.05),while no significant difference of cells invasion ability was found between siciRS-7-1+simiRNA-7 group and control group(P>0.05).4.The expression of EGFR and STAT3 in pancreatic cancer cells of siciRS-7-1 group was lower than that in control group(P<0.05),while the expression of EGFR and STAT3 in siciRS-7-1+simiRNA-7 group was similar to that in control group(P>0.05).5.After Gefitnid,the blocker of EGFR/STAT3 signaling pathway was added,no influence was found on proliferation and invasion ability of cells in siciRS-7-1 group(P>0.05),while proliferation and invasion ability changed significantly in control group(P<0.05).Conclusion:It has been clarified by the vitro study that down regulating ciRS-7 expression could promote miRNA-7 expressing,and regulated the expression of miRNA-7 downstream signaling pathway,the EGFR/STAT3 signaling pathway,thus inhibiting the proliferation and metastasis of tumor.This study show that ciRS-7 promotes the proliferation and metastasis of pancreatic cancer.It provided a research basis for target treatment of tumor.Part III To investigate the effect and mechanism of circular RNA ciRS-7 promoting proliferation and metastasis of pancreatic cancer by regulating miRNA-7 mediated EGFR/STAT3 signaling pathway in vivoResearch objectives:To verify ciRS-7 can affect proliferation and metastasis of pancreatic cancer by regulating miRNA-7 mediated EGFR/STAT3 signaling pathway by vivo study.Research methods:1.Injected recombinant siciRS-7-1 and siciRS-7-1+simiRNA-7 pancreatic cancer cells PANC-1 in subcutis and abdominal cavity of nude mice respectively,and established the nude mice model of subcutaneous transplantation tumor and peritoneal metastatic tumor.2.All nude mice were executed after being raised for 4 weeks.Measured the weight and volume of tumors stripped from mice along with observed the liver metastasis.3.Measured the expression of miRNA-7 in tumor model tissue by QRT-PCR method4.Tested the expression of EGFR and STAT3 in tumor model tissue by IHC.Results:1.Subcutaneous transplantation and peritoneal metastasis model of pancreatic cancer in nude mice were established2.Volume and weight of subcutaneous transplantation tumor in siciRS-7-1 group was significantly lower than control group(P<0.05).Hepatopulmonary metastasis in siciRS-7-1 group was significantly less than that in control group in abdominal metastasis group(P<0.05).3.By QRT-PCR method,we could find that the expression of miRNA-7 in siciRS-7-1 group of both models were than that in control group(P<0.05).4.The IHC showed that protein expression of EGFR and STAT3 of siciRS-7-1 group was lower than control group in both models.Conclusion:The in vivo study has confirmed the results of the in vitro study that the down-regulation of ciRS-7 can promote the expression of miRNA-7,then up-regulated miRNA-7 can inhibit the proliferation and metastasis of tumor by down-regulating EGFR/STAT3 signaling pathway.Conclusion of the article:CiRS-7 expression in pancreatic cancer tissues is obviouse higher than in adjacent tissues,and its expression is correlated with lymph node metastasis and nerve infiltration,which is an important indicator for the prognosis of pancreatic cancer.The low expression of ciRS-7 can inhibit the proliferation and invasion of pancreatic cancer,by up regulating the expression of miRNA-7 and obstructing EGFR/STAT3 signaling pathway.Comprehensive studies show that ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miRNA-7 mediated EGFR/STAT3 signaling pathway.ciRS-7 is expected to be a new potential target for the early diagnosis and treatment of pancreatic cancer,and provides a research basis for the follow-up targeted therapy of tumor genes.