The Mechanism Study On MiR-29b-2-5p/Cbl-b Or YAP Axis In Proliferation And Metastasis Of Pancreatic Ductal Adenocarcinoma

Objective:Among all tumors,pancreatic cancer has the lowest 5-year survival rate(9%).Among all types of pancreatic cancers,pancreatic ductal adenocarcinoma(PDAC)accounts for more than 90%of all pancreatic tumors,and has a poor prognosis.One-year survival rate is about 18%.Postoperative effective adjuvant chemotherapy regimen and its limited and unsatisfactory effect.At the same time,in clinical work,we found that the prognosis of patients with the same chemotherapy bill and the same stage of cancer is different.Therefore,in order to improve the prognosis of resectable pancreatic cancer patients,it is very important to select individualized adjuvant therapy for patients to accurately evaluate the efficacy.One of the reasons for the poor prognosis of pancreatic cancer is that pancreatic cancer cells have a strong ability to proliferate.A wide range of prognostic factors are related to proliferation.Previous studies on proliferation of pancreatic cancer mainly focus on protein level,but lack of research on gene level.MicroRNA(miRNA)is a non-coding small RNA with a length of 20-23nucleotides.By specifically binding to the target gene's 3'-untranslated region(3'-UTR),target gene degradation or translation inhibition degradation can affect cell proliferation.In more than 1000 identified miRNAs,one miRNA can target hundreds of different mRNAs,and a single RNA can be co-inhibited by several different miRNAs.Therefore,the biogenesis and regulation of miRNAs play an important role in gene regulatory networks.Because of its high stability,small size,strong tissue specificity and simple isolation,miRNAs are more suitable as biomarkers for predicting prognosis than RNA and protein.Cbl-b is one of the members of the CBL family with E3 ubiquitin ligase and connective protein functions encoded by CBL(Casitas B-lineage lymphoma)gene.In the immune system,Cbl-b inhibits the response of effector CD8~+T cells in a cellular way.In solid tumors,the function of Cbl-b is controversial.Previous studies on Cbl-b in tumors have focused on gastric cancer,breast cancer and non-small cell lung cancer.The function of these solid tumors is to inhibit proliferation.However,the relationship between Cbl-b and PDAC has been rarely reported.Yes-associated protein(YAP)is the core component of Hippo signaling pathway,which participates in cell proliferation and tissue regeneration.In other malignant tumors such as hepatocellular carcinoma,ovarian cancer and non-small cell lung cancer,the expression of YAP increased in succession.It has been reported that YAP complexes with other transcription factors such as TEAD1-4 or SMAD protein and mediates the transcription of genes,including connective tissue growth factor(CTGF)and cysteine-rich angiogenic inducer 61(61CYR61).At the same time,YAP can contribute to malignant transformation of tumors by inducing epithelial-mesenchymal transition,independent of adherent growth and loss of contact inhibition.Activation of YAP can also enhance the migration of cancer cells and inhibit the sensitivity of treatment.Recent studies by us and other researchers have shown that YAP activation plays an important role in malignant transformation of pancreas.However,how YAP affects PDAC proliferation needs further study.In this study,we randomly selected some cases(10 cases each)with similar clinical and pathological factors and different prognosis,and divided them into two groups:good prognosis and poor prognosis.We detected the expression profile of microRNAs in the two groups,compared the differentially expressed miRNAs in the two groups,and screened meaningful miRNAs.To verify the prognostic significance of differentially expressed miRNAs in tissue samples of independent large sample size validation patients.The results showed that mir-29b-2-5p was a good independent prognostic factor in resectable pancreatic cancer.In addition,miR-29b-2-5p negatively regulates Cbl-b to reduce Cbl-b-mediated ubiquitination and p53 expression,inhibiting the proliferation of PDAC cells.We also report that ECT2expression is regulated by YAP.We further demonstrate that ECT2 is required for pancreatic cancer cell proliferation and migration in vitro.Finally,using a syngeneic orthotopic xenograft mouse model for pancreatic cancer,we found that ablation of ECT2 expression reduces pancreatic cancer growth and dissemination to the liver.These findings highlight a critical role of ECT2 in promoting pancreatic cancer growth and metastasis and provides insights into development of novel methods for early detection and treatment.Methods:1.A total of 120 patients with primary PDAC who underwent surgical treatment from January 2009 to February 2011 in Shengjing Hospital affiliated to China Medical University were selected according to the criteria of inclusion and exclusion.2.The screening case group(10 cases with good prognosis and 10 cases with poor prognosis)and the validation case group(n=100)were determined by clinical analysis.3.Total RNA including miRNAs was extracted from FFPE tissue samples by microNeasy FFPE Kit kit.4.Geno Explorer TM miRNA array was used to detect the expression of miRNA in selected cases.5.Cell culture.Human PDAC cell lines SW1990 and Apan-2were routinely subcultured in our laboratory.6.Cell transfection,over-expression of miR-29b-2-5p:miRNA mimic synthesized by Guangzhou Reebok Company:miR-29b-2-5p mimic and negative control microRNA NC mimic(NC).3XFLAG-CMV9(control group,NC)synthesized by Sigma Company and 3XFLAG-CMV9 Cbl-b(overexpression group,OE Cbl-b)were used.The siRNA and NC(negative control group)were transfected into PDAC cell lines SW1990 and CaPan-2 respectively by Lipofectamine 2000.Cbl-b and YAP siRNA synthesized by Shanghai GeneChem Company were transfected with siRNA and negative control NC.The siRNA(experimental group)and NC(negative control group)were transfected into PDAC cell line SW1990 by Lipofectamine 2000.7.Using Trizol to extract RNA from cells.8.Cell proliferation was measured by trypan blue staining counting.9.Western blot was used to detect the expression of Cbl-b,EGFR,BBRCA1,p53,phosphorylated p53,GAPDH,YAP and ECT2.10.RT-PCR was used to detect the expression of Cbl-b,YAP,ECT2,CTGF and CYR61.11.Double luciferase reporter gene assay was used to identify target genes and verify the binding between proteins.12.Immunohistochemical staining was used to detect the expression of Cbl-b,Ki-67 or ECT2 in PDAC tissues.13.Immunofluorescence technique was used to detect the expression of Cbl-b,p53,Ki67 and ECT2 in PDAC tissues.14.The expression of ECT2 was knocked out by CRSPR-Cas9technique in human and mouse PDAC cell lines.15.In vivo imaging of mice was used to detect the growth of ECT2 in pancreas and the metastasis of ECT2 in liver.16.Statistical analysis showed that chi-square test or Fisher's exact probability method were used to compare the expression of miRNAs with the clinicopathological characteristics of the patients.Kaplan-Meier method was used to analyze single factor survival,Kaplan-Meier method was used to draw survival curve,and Log-rank was used to test survival difference.Multivariate Cox proportional risk model(forward method)was used for multivariate survival analysis.The cell experiment part was repeated three times each time,and the data were expressed as mean.SPSS13.0 software was used for statistical analysis,with P<0.05 as the difference with statistical significance.Results:1.The characteristics of screened case group and verified case group:screened case group:10 cases in good prognosis group and 10 cases in poor prognosis group.The clinicopathological factors between the two groups were basically balanced(all P>0.05).Verified case group:100 cases of resectable PDAC patients with different stages had no significant difference in clinicopathological factors between screened case group and screened case group;2.MiRNAs expression chip was used to detect the expression of miRNAs in screened case group.Spectrum:There were significant differences in the expression of 30 miRNAs between the good prognosis group and the poor prognosis group(Bonferonni-adjusted t-test,P<0.05=.Compared with the poor prognosis group,the expression of 22 miRNAs in the good prognosis group was significantly up-regulated(miR-29b-2-5p,etc.)and 8 miRNAs were significantly down-regulated(Mi-152,etc.).3.To verify the prognostic significance of miR-29b-2-5p:screening in 20 cases and 40 cases of cancer and adjacent pancreatic cancer.The expression of miR-29b-2-5p in patients with good prognosis and adjacent tissues was higher than that in poor prognosis and cancer tissues,respectively.100 cases were divided into two groups:good prognosis and bad prognosis.It was found that the expression of miR-29b-2-5p was higher in good prognosis.It was preliminarily confirmed that miR-29b-2-5p was a prognostic marker for resectable PDAC patients.4.The relationship between miR-29b-2-5p and clinical and pathological characteristics of patients.Series:The expression of miR-29b-2-5p in resectable PDAC patients was correlated with gender,differentiation,surgical margin,pT stage,pN stage,vascular thrombus,vascular thrombus and CA19-9(all P<0.05=,but not with other clinical and pathological features(all P>0.05);5.Multivariate survival analysis confirmed the prognostic significance of miR-29b-2-5p:high expression of miR-29b-2-5p(HR,0.492;95%CI).0.300-0.807;P=0.005;P=0.005;P=0.005)was an independent predictor of overall survival in patients with resectable PDDAC;pT4(HR,0.612;95%CI,95%CI,1.004-1.646;P=0.046),CA19-9(>37 U/L(HR,3.74;95%CI,95%CI,1.484-8.112;P=0.004)and histologically poorly differentiated tumors(HR,1.472;95%CI,95%CI,95%CI,1.016-2.016-2.133;P=0.041;P=0.041;P=0.041;P=0 Poor independence Establishment of predictive factors;6.Cell experiments showed that miR-29b-2-5p inhibited the proliferation of pancreatic cancer cells;7.Nude mice experiments demonstrated that miR-29b-2-5p inhibited the proliferation of pancreatic cancer cells;8.miR-29b-2-5p could induce apoptosis and G1 cell cycle arrest.Flow cytometry detected cell cycle and apoptosis found that overexpression of miR-29b-2-5p could significantly arrest PDAC cells in G1 phase and promote the apoptosis of PDAC cells.-After 29b-2-5p,the expression of Bcl-2,CyclinD1 decreased and Bax increased;9.Prediction and validation of the target gene of miR-29b-2-5p:miR-29b-2-5p could significantly inhibit the expression of Cbl-b and YAP,while the expression of BRCA1,EGFR and IGFR remained unchanged;In PDAC cells,the expression level of Cbl-b protein was significantly down-regulated by miR-29b-2-5p in a concentration-dependent manner,but the expression level of Cbl-b protein was not significantly affected by double fluorescence.Reporter kinase assay showed that the expression of Cbl-b and YAP could be directly inhibited by the binding site of miR-29b-2-5p.10.Overexpression of Cbl-b promotes the proliferation of PDAC cells,which depends on the expression of miR-29b-2-5p:knockout of Cbl-b can significantly inhibit the proliferation of PDAC cells.Cbl-b promotes the proliferation of PDAC cells through miR-29b-2-5p.11.miR-29b-2-5p promotes the expression of Cbl-b binding to p53 by inhibiting Cbl-b and co-localizes it in cells.Cbl-b can degrade p53 by ubiquitination:Cbl-b is silenced and Cbl-b is overexpressed in SW1990 cell line,respectively.We found that the expression of p53 and p-p53 is reversed.We found that after using PS341,the expression of Ub protein in untreated group is increased,suggesting that Cbl-b degrades p53 protein by ubiquitination.Immunofluorescence assay showed that Cbl-b and p53 were co-localized,and the fluorescence showed that miR-29b-2-5p promoted p53 expression by inhibiting the expression of Cbl-b;12.In the verification concentration,Cbl-b was negatively correlated with prognosis and the expression of miR-29b-2-5p:Spearman grade correlation analysis showed that the expression of Cbl-b was negatively correlated with the expression of miR-29b-2-5p,r=-0.33,p=0.001;13.YAP promoted the expression of ECT 2.Da:YAP knockout can significantly reduce the level of ECT2protein in Mia PaCa-2 or Aspc1 pancreatic cancer cell lines,and the YAP inhibitor Viteporfin can also reduce the level of ECT2 protein expression.We conducted chromatin immunoprecipitation(CHIP)experiments to determine whether the binding of YAP was found in the promoter region of ECT2;14.ECT2 up-regulation promotes the progress of pancreatic cancer:at a larger sample size,Oncomine analysis database,a publicly available microarray data set,was used to conduct oncological analysis.It was found that the level of ECT2 gene in PDAC was higher than that in normal pancreas.The up-regulation of ECT2,especially its presence in nucleus,was related to the progress and metastasis of human PDAC.Through immunohistochemistry,we found that nuclear ECT2 could be detected in a small part of PANIN cells of KC mice and pancreatic cancer cells of KPC mice.15.ECT2 promoted the proliferation of pancreatic cancer.Through immunofluorescence staining,we found that the location of ECT2 in the nucleus was often associated with Ki-67 positive cells.CRISPR-CAS9method was used to reduce the expression of ECT2 in pancreatic cancer cell lines,and the expression of ECT2 was depressed.The proliferation of cancer cells was inhibited.16.Changes of signal pathway after ECT2 knockout:the decrease of ECT2expression mainly affects PI-3 kinase and STAT3 signaling pathway,not MAP kinase pathway;17.YAP and ECT2 participate in the regulation of cytokines:In ECT2-KO cells,the levels of IL-11 and IL-6 mRNA are significantly reduced,similarly,Yap knockout also leads to the decrease of IL-11 and IL-6 levels,and after ECT2knockout only leads to a moderate decrease of YAP expression;8.ECT2 plays a key role in the migration of pancreatic cancer cells:Using Transwell analysis,we found that the migration of pancreatic cancer cells decreased after ECT2 gene knockout;19.Animal experiments confirmed that ECT2 was involved in the proliferation and metastasis of pancreatic cancer cells:4662 cells of mouse pancreatic cancer cell line isolated from KPC mice were transfected to stably express firefly luciferase,and knockout ECT2 inhibited PD.Proliferation and migration of AC cells.Conclusion:1.miR-29b-2-5p is an independent prognostic factor for resectable PDAC patients.2.miR-29b-2-5p inhibits the proliferation of PDAC cells by promoting G1 arrest and inducing apoptosis.3.miR-29b-2-5p inhibits the proliferation of PDAC cells by inhibiting the expression of Cbl-b and reducing the ubiquitination and degradation of p53 mediated by Cbl-b.4.miR-29b-2-5p inhibits YAP expression.5.YAP promotes proliferation and metastasis of pancreatic cancer by binding with ECT2 promoter region.6.ECT2 up-regulates YAP feedback and further promotes the progress of pancreatic cancer.