MicroRNA-494 Regulates Gli3 Expression And Inhibits Growth And Migration In Pancreatic Carcinoma

Pancreatic cancer(PC)is a highly malignant and highly invasive digestive system tumor.Currently,the treatment effect is not good,and the 5-year survival rate is still very low,most reports are between 5%and 10%.About 80%of patients have distant metastasis at the time of diagnosis,only less than 20%of the patients have the opportunity to undergo radical resection.Therefore,it is the focus of current research to search for effective diagnosis and therapeutic targets in the development of pancreatic cancer.Pancreatic carcinogenesis is a multistep process involving multiple genetic and epigenetic alterations.Hence,a better understanding of the molecular mechanisms involved in pancreatic carcinogenesis will be helpful for identification of new and effective prognostic biomarkers.MicroRNAs(miRNAs)are small non-coding regulatory RNAs that suppress gene expression through partial complementary elements in the 3'-untranslated regions of their target messenger RNAs(mRNAs).miRNAs have been shown to be involved in various critically important physiological processes such as cell proliferation,cell division,cell differentiation,cell apoptosis,tumorigenesis,hematopoiesis,and the nervous system patterning.A particular type of miRNA-miR-494-is downregulated in different types of cancer tissues.Moreover,it is a tumor suppressor and induces cell cycle arrest,cell senescence,and apoptosis,but suppresses cell proliferation.We have indentified the clinical significance of miR-494 in pancreatic cancer,but the function of miR-494 in PC remains unknown.The hedgehog(Hh)signaling pathway,which consists of three distinct homologues(sonic,indian,and desert Hh)is constitutively activated in a variety of human tumor entities.The zinc-finger protein Gli3 is a mediator of Sonic hedgehog signaling.Bioinformatics has shown that the 3-UTR of Gli3 contains a putative binding site for miR-494.Several studies have shown that GIi3 has an important role in cancer progression by stimulating cancer cell proliferation.However,the regulation of miR-494 in PC or it association with Gli3 have not been reported.In this study,we focused on the expression and function of miR-494 in PC,and the potential mechanism of miR-494 in PC was investigated,which provides a potential novel therapeutic agent for the treatment of pancreatic cancer.Purpose1.To investigate the expression level of mir-494 mRNA in pancreatic cancer tissues and adjacent normal tissues of pancreatic cancer patients,and to analyze the correlation with clinicopathological factors.2.To investigate the expression level of Gli3 mRNA in patients with pancreatic cancer,and to analyze the correlation with clinicopathological factors and miR-494.3.To verify the targeted regulation of mir-494 on Gli3,and to explore the regulation mechanism of mir-494 and Gli3 in the development and invasion of pancreatic cancer.Methods1.The study protocol was approved by the Ethics Committee of Shandong Provincial Hospital affiliated to ShanDong University and Weifang People's Hospital,and all patients provided written informed consent for the use of their tissues.A total of 99 pairs of human pancreatic cancer tissues and matched normal adjacent pancreatic tissues were obtained during surgery between July 2006 and August 2012 at the Department of Hepatobiliary Surgery,Shandong Provincial Hospital affiliated to ShanDong University and Department of Hepatobiliary Surgery,Weifang People's Hospital.The level of miR-494 expression was determined in 99 pairs of primary pancreatic cancer and their corresponding adjacent non-tumor tissues by using quantitative reverse transcriptase polymerase chain reaction.The associations between miR-494 expression and clinicopathological features were analyzed,and the survival correlations were analyzed by using the Kaplan-Meier method and Cox proportional risk model.2.The level of Gli3 expression was determined in 99 pairs of primary pancreatic cancer and their corresponding adjacent non-tumor tissues by using quantitative reverse transcriptase polymerase chain reaction and Western-blot.The associations between Gli3 expression and clinicopathological features were analyzed.In addition,association between Gli3 and miR-494 were also discussed by pearson correlation analysis.3.The effect of miR-494 in regulating PC cells biological properties were analyzed with miR-494 mimics/inhibitor-transfected cells.CCK-8 and Transwell method were used to detect the proliferation and migration activity of transfected cells,and the dual-luciferase report gene experiment were used to verify the targeted regulatory effect of miR-494 on Gli3.Results1.The level of miR-494 expression was significantly downregulated in pancreatic cancer tissues(mean relative expression level ±SD,0.48±0.11)when compared with matched adjacent normal tissues(1.80 ±0.28,P<0.05).The relative miR-494 expression level was classified as high or low in relation to the median value for clinical pathological correlation analysis.2.Significant correlations between miR-494 expression levels and the TNM stage(P=0.009),lymphatic invasion(P = 0.036),vascular invasion(P = 0.011),distant metastasis(P = 0.007)and tumor grade(P = 0.031)were found.However,we did not find a significant association of the miR-494 expression levels with sex(P=0.062),age(P = 0.21),tumor size(P = 0.11),and tumor location(P = 0.45).3.To evaluate the prognostic value of miR-494 expression in pancreatic cancer,survival curves were constructed by using the Kaplan-Meier method and compared by using the log-rank test.Pancreatic cancer patients with low miR-494 expression level had a shorter overall survival than those with high miR-494 expression level.The 5-year overall survival rate in the low-expression groups was 12.0%compared with 49.2%in the high-expression group(log-rank test,P = 0.036).4.To determine whether the expression of miR-494 was an independent risk factor for poor prognosis,both clinicopathological factors and the level of miR-494 expression were evaluated by performing multivariate Cox regression analysis.We found that the miR-494 expression level(hazard ratio[HR]= 2.85,95%confidence interval[CI]:1.54-8.45,P = 0.019)was an independent factor for predicting the overall survival of pancreatic cancer patients.5.The level of Gli3 mRNA expression was significantly downregulated in pancreatic cancer tissues(mean relative expression level ± SD,0.68±0.25)when compared with matched adjacent normal tissues(0.50±0.24,P<0.05).6.Western-blot method were used to evaluate Gli3-FL and Gli3R protein expression.The ratio between Gli3-FL and Gli3R were also discussed.Compared with matched adjacent normal tissues,the level of Gli3-FL expression was significantly increased in pancreatic cancer tissues,while no significant change was found in Gli3R protein expression.Moreover,an increase in Gli3-FL/Gli3R ratio was accompanied by a significant rise of Gli3-FL.7.We found significant correlations between Gli3 expression levels and the vascular invasion(P = 0.04),distant metastasis(P = 0.001),and tumor grade(P = 0.03).However,we did not find a significant association of the miR-494 expression levels with sex(P = 0.74),age(P = 0.19),tumor size(P = 0.84),tumor location(P = 0.71),TNM stage(P = 0.07),and lymphatic invasion(P = 0.09).The expression level,of GIi3 mRNA was significantly higher in patients with low differentiation degree than those in the middle and high differentiation group.8.Bioinformatics has shown that the 3'-UTR of Gli3 contains a putative binding site for miR-494.We therefore asked whether clinical PC samples having a low level of miR-494 overexpressed the Gli3.Here miR-494 mRNA level detected by qPCR was significantly decreased while upregulation of Gli3 were observed.These results reveal an An inverse relationship between miR-494 and Gli3 expression levels in PC tissues.9.Transdected with miR-494 mimics/inhibitor could intervene the miR-494 mRNA expression in PANC-1 cells presented as upregulation or downregulation of miR-494 mRNA expression.10.To further characterize the functional importance of miR-494 in PC tumorigenesis,we examined the effect of miR-494 on the proliferation of PC cells using CCK-8 assay.We observed that over-expression of miR-494 significantly restrained the proliferation of PANC-1 cells,whereas miR-494 inhibition promoted the proliferation of PANC-1 cells at 24,36,48 h after transfection,respectively(P<0.05).11.To evaluate the impact of miR-494 on cell migration,transwell assay were employed.We found that overexpression of miR-494 inhibited PANC-1 cell migration,whereas its knock-down induced PANC-1 cell migration.These observations suggested that miR-494 played an important role in migration supression of PC cells.12.Analysis by using publicly available programs,TargetScan and miRanda,indicates that Gli3 is theoretically the target gene of miR-494.We then performed a luciferase reporter assay to verify that miR-494 directly targets Gli3.We found that co-transfection of miR-494 mimics and pmiR-Gli3-wt significantly decreased the luciferase activity in PANC-1 cells as compared with the control.However,miR-494 mimics had no effect on the luciferase activity when co-transfected with pmiR-Gli3-mut.These data showed that Gli3 is one of direct targets of miR-494.Conclusions1.miR-494 expression was significantly downregulated in pancreatic cancer tissues.Reduction of miR-494 expression was associated with TNM stage,lymphatic invasion,vascular invasion,distant metastasis and tumor grade.2.miR-494 expression level was an independent factor for predicting the overall survival of pancreatic cancer patients.3.Gli3 expression was upregulated in examined PC sa:mples and inversely correlated with the expression of miR-494.4.Gli3 were identified as the functional downstream target of miR-494 by directly targeting the 3'-UTR.miR-494 was involved in tumorigenesis of PC at least in part by suppression of Gli3.