The Functional Role And Underlying Mechanisms Of MicroRNA-19b In Necrotizing Pancreatitis And Pancreatic Ductal Adenocarcinoma

The incidence of acute pancreatitis(AP)is increasing in recent years,and AP is divided into interstitial edematous pancreatitis and necrotizing pancreatitis according to the revised Atlantic guidelines.Pancreatic parenchyma necrosis and/or peripancreatic necrosis may occur in necrotizing pancreatitis.Pancreatic parenchymal necrosis alone is seen in only 5%of patients and peripancreatic necrosis alone is seen in only 20%of patients,the most common type of necrotizing pancreatitis is combined pancreatic parenchymal necrosis with peripancreatic necrosis.All 3 types necrotizing pancreatitis can be sterile or infected.Necrosis complicates as many as 10%to 20%of all cases of AP,and these cases of necrotizing pancreatitis are associated with a markedly increased rate of complicating with infection and multiorgan dysfunction.Therefore,the morbidity and mortality rates of the necrotizing pancreatitis patients are relatively high and the prognosis of these patients is not satisfied at present.The natural process of necrotizing pancreatitis includes the acute phase characterized by inflammation and the delayed phase featured with sepsis.The mortality rate of necrotizing patients combined with infection increases significantly.So,it is essential to assess the prognosis of AP patients in acute phase by evaluating laboratory examinations and clinial parameters and positive treatment strategies should be adopted for patients with a high risk of developing necrotizing pancreatitis to minize the acinar cell necrosis,which is valuable for improving prognosis.MicroRNAs(miRNA)is a kind of non-coding RNA with a single strand,18-25 nucleotides.It can inhibit the transcription of the targeted genes by combining with the 3'-untranslated region(3'-UTR)of the mRNAs and suppress the expression of corresponding proteins.Although miRNAs accounts for only 1 percent of human genome,about 60 percent of human protein-coding genes have been under selective pressure to maintain pairing to miRNA.It has been demonstrated that miRNA can modulate various cellular processes,including proliferation,differentiation and apoptosis and so on.Moreover,it plays an essential role in inflammation and cancer.However,to our known,there is no literature about miRNA in necrotizing pancreatitis at present.In the present study,rat necrotizing pancreatitis models were established in vivo and miRNA chip assay was performed to select the miRNA correlated with pancreatic acinar cell necrosis.Furthermore,the functional role of the selected miRNA in acinar cel necrosis was confirmed in vitro,and the potential mechanisms were also explored,which may make a foundation for applying miRNA in the early diagnosis and treatment of necrotizing pancreatitis.[Objective]To select the miRNA associated with necrotizing pancreatitis,and demonstrate its role in necrotizing pancreatitis for applying miRNA in the early diagnosis and treatment of necrotizing pancreatitis.[Materials and Methods]1.The necrotizing pancreatitis SD rat models were established through injecting L-arginine intraperitoneally,and HE staininng was performed to confirm whether the models were successfully established.miRNA chip assay was carried out to select the miRNA in relationship to necrotizing pancreatitis.2.The AR42J cells were treated with taurolithocholic acid 3-sulfate disodium salt(TLC-S)to establish the necrotizing pancreatitis cellular model in vitro,the amylase level was detected to verify if the models were successfully established.3.mimic miR-19b,miRNA antisense oligonucleotide(miR-19b ASO)and negative control(NC)were transfected into the AR42J cells and qRT-PCR was used to measure miR-19b levels.The necrotizing rate of AR42J cells was examined after transfection.4.The levels of miR-19b in clinical serum samples,including interstitial edematous pancreatitis,necrotizing pancreatitis and healthy control,were detected by qRT-PCR for investigating its relationship with necrotizing pancreatitis.5.The mRNA levels of IL-1?,IL-6 and TNFa in AR42J cells were evaluated on qRT-PCR assay followed by transfection with mimic miR-19b,miR-19b ASO and NC.[Results]1.Numerous pancreatic acinar cell necrosis of the L-arginine treated SD rats were detected by HE assay,which confirmed the successful establishment of the models.miRNA chip assay found the expression of miR-19b was correlated with necrotizing pancreatitis.2.The amylase level of the AR42J cells treated with TLC-S was increased obviously,which confirmed the successful establishment of the necrotizing pancreatitis cellular models in vitro.3.The level of miR-19b in the AR42J cells of the mimic miR-19b group was significantly increased relative to the NC group on qRT-PCR assay,and the miR-19b ASO group displayed obviously suppression of miR-19b expression compared with the NC group.Moreover,the cell necrosis rate of the mimic miR-19b group of the in vitro models was much higher than the NC group,on the contraty,the necrosis rate of miR-19b ASO group was statistically decreased compared with the NC group.4.The level of miR-19b in interstitial edematous pancreatitis patients was higher than that of healthy persons,while the level of miR-19b in necrotizing pancreatitis patients was significantly increased in comparison with those of the interstitial edematous pancreatitis patients and healthy persons.5.The IL-?,IL-6 and TNFa mRNA levels of the mimic miR-19b group were obviously higher than those of the NC group,while the IL-?,IL-6 and TNFa mRNA levels of the miR-19b ASO group were significantly decreased relative to those of the NC group.[Conclusions]1.miR-19b could promote the necrosis of acinar cells in necrotizing pancreatitits.2.miR-19b could modulate the inflammation of necrotizing pancreatitis via regulating the mRNA levels of IL-1?,IL-6 and TNF?.Pancreatic ductal adenocarcinoma(PDAC)is one of the most common reasons leading to death in spite of the advances in diagnosis and treatment.More than 80%patients are diagnosed with advanced stage and have no access to surgical resection because of PDAC insidious development process and highly invasive characteristic.Besides,the recurrence rate of resected patients is high,which contributes to a poor prognosis.The overall 5-year survival rate is less than 6%,and the median survival averages 24 months even for patients undergoing both surgery and adjuvant therapy.The adjuvant therapy for resected PDAC patients is gemcitabine based chemotherapy.In recent years,combined chemotherapy regimens have been suggested for improving the prognosis of metastatic patients.The overall response rate is 23-31%,the disease-free survival time is only 5.5-6.6 months,and the overall survival time is 8.5-11 months.So,the prognosis of PDAC patients is still poor.In addition,combination therapy obvously increases side effects.Early diagnosis and resection is the most efficient way for improving prognosis at present.It has been demonstrated that the high heterogenicity of PDAC contributes to its insenstitive to chemotherapy and poor prognosis.Thus,further investigation of PDAC development should be carried out to detect early diagnotic marker and more effective therapeutic targets.MicroRNAs(miRNAs)are small,non-coding RNA,which can target mRNAs mechanistically and induce the translational repression or degradation of mRNAs.The role of miRNAs in control of proliferation,differentiation,and apoptosis have been well elucidated and their aberrant expression in many tumors indicated that they can function as tumor suppressors and oncogenes.Furthermore,selected miRNAs may influence cancer response to chemotherapy.Moreover,miRNAs targeted therapy is illuminated as a promising regimen in cancer therapy.miR-19b is an oncogenic member of the miR-17-92 cluster.Prevous studies have showed that miR-19b could promote caner development by targetting TP53 and PTEN in breast carcinoma,lung carcer and gastric cancer.Besides,the structure of miRNAs is relatively stable and their levels could be examined in blood samples,which makes them an ideal marker for early dignosis and prognogis evaluation.However,the role of miR-19b in PDAC has not been verified at present.The present study explored the functional role of miR-19b in prolifetation,metastasis and stemenss of PDAC cell,and the potential mechanisms were also revealed.The results not only further elucidated the development mechanisms of PDAC,but also provided new ways for PDAC diagnosis and treatment.Part ? The role of miR-19b in PDAC proliferation and metastasis[Objective]To examine the expression of miR-19b in PDAC cell lines and patients' blood samples,and the functional role of miR-19b in PDAC cells line proliferation,migration and invasion will also be explored.[Materials and Methods]1.The expression level of miR-19b in human pancreatic ductal epithelial cell line(HPDE),human PDAC cell line(PANC1,MiaPaCa2 and BxPC3)and huamn PDAC metastasis cell line COLO357 and its fast-growing variant FG were quatified by qRT-PCR assay for investigating the role of miR-19b in PDAC.2.To further varify the function of miR-19b in PDAC,the blood samples of 63 PDAC patients and 50 healthy persons treated at Qilu Hospital of Shandong University and People's Hospital of Henan Province were collected and qRT-PCR assay was performed to evaluate the expression of miR-19b.3.To elucidate the oncogenic role of miR-19b in PDAC,the expression of miR-19b was inhibited or overexpressed in PDAC cell lines at first.Then,Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b inhibited or overexpressed.4.To examine the effect of miR-19b on proliferative ability of PDAC cell lines,MTT and colony formation assay were performed on PDAC cell lines with miR-19b silenced or overexpressed.[Results]1.The results of qRT-PCR showed the expression level of miR-19b in human pancreatic ductal epithelial cell line(HPDE)was much higher than that in human PDAC cell lines(PANC1,MiaPaCa2 and BxPC3)and huamn PDAC metastasis cell line COLO357 and its fast-growing variant FG,which suggested miR-19b might function as an oncogenic role in PDAC cell lines.2.It was confimed that the level of miR-19b in PDAC patients' blood samples was much higher than in healthy persons on qRT-PCR assay,which demonstrated the oncogenic role of miR-19b in PDAC again.3.Loss of miR-19b markedly decreased the invasion and migration abilities of PDAC cell line(FG)on Transwell assay and Matrigel assay,while overexpression of miR-19b significantly increased the invasion and migraion of PDAC cell line(BxPC3).The results indicated that miR-19b could promote the migration and invasion of PDAC cell line.4.MTT and colony formation assay verified the proliferation of PDAC cell lines would be decreased with miR-19b inhibition,accompined with the upregulation of proliferation markers(p21 and p27);while the proliferation of PDAC cell line would be increased in miR-19b overexpressed PDAC cell line,and the expression of proliferation markers(p21 and p27)would also be reduced.It could be concluded that miR-19b could promote the proliferation of PDAC cells.[Conclusions]1.miR-19b could promote the migration and invasion of PDAC cells.2.miR-19b could increased the proliferation capability of PDAC cells.Part ? The mechanism of miR-19b promoting metastasis of PD AC cells[Objective]Our studies have confirmed the functional role of miR-19b in promoting PD AC metastasis,which is the leading reason contributing to the death of PDAC patients.The detailed mechanism of miR-19b promoting PD AC metastasis would be further elucidated in the following studies.[Materials and Methods]1.To detect whether miR-19b promoting PDAC metastasis through regulating sternness,cancer stem cells(CSCs)markers of PDAC,including CD133,c-Met and FOXMI were examined in miR-19b silenced and overexpressed PDAC cells on Western blotting assay.Besides,the self-renewal ability of miR-19b silenced and overexpressed PDAC cells was also eluminated on sphere formation assay.2.The mRNAs targets of miR-19b were predicted by microRNA.org and TargetScan.Potential gene targets were selected based on the predict results in combination with the well-known molecules and signaling pathways regulating PDAC CSCs reported in previous literatures,and the expression of selected targets were further verified on Western blotting assay in miR-19b overexpressed PDAC cells.3.To define the role of the selected gene,its cDNA sequence was transfected into the miR-19b overexpressed PDAC cells,and the expression of PDAC CSCs markers(CD133,c-Met and FOXM1)were detected on Western blotting assay.In addition,the self-renewal ability of the miR-19b overexpressed PDAC cells transfected with the cDNA of the selected gene was also confimed on sphere formation assay.4.To investigate the function of the selected gene on miR-19b mediated migration and invasion of PDAC cells.Transwell assay and Matrigel assay were performed with the miR-19b overexpressed PDAC cells transfected with the cDNA of the selected gene.[Results]1.The PDAC CSCs markers,including CD133,c-Met and FOXM1 were downregulated in miR-19b silenced cells on Western blotting,in combined with decreased number of tumor spheres on sphere formation assay.However,the miR-19b upregulated PDAC cells exihibited enhanced expression of CSCs marker on Western blotting,and the number of tumor spheres on sphere formation assay was also increased accordingly.These results indicated miR-19b could promoting PDAC metastasis through mediating its sternness.2.SOX2,PTEN and ABCG2 were selected as the predicted targeting genes of miR-19b based on the results of microRNA.org and TargetScan and previous reports.Western blotting further revealed only the expression of PTEN was downregulated in miR-19b overexpressed PDAC cells,which implicated PTEN might be the target gene of miR-19b in regulating PDAC CSCs.3.It was displayed on Western blotting assay that the upregulation of PDAC CSCs markers(CD133,c-Met and FOXM1)in miR-19b overexpressed PDAC cells was reversed following PTEN overexpression,and the increased self-renewal ability in miR-19b overexpressed PDAC cells was also reversed through PTEN upregulation.These results demonstrated miR-19b could promoting PDAC CSCs by targeting PTEN.4.Transwell assay and Matrigel assay showed that the migration and invasion abilities enhanced by miR-19b overexpression were reduced by PTEN overexpression,indicating miR-19b could promoting PDAC metastasis by targeting PTEN.[Conclusions]1.miR-19b could increase PDAC metastasis by promoting its sternness;2.PTEN is the target of miR-19b in regulating PDAC CSCs and metastasis.