Shuai Zhu

MiR-130b Is a Prognostic Marker and Inhibits Cell Proliferation and Invasion in Pancreatic Cancer through Targeting STAT3

Accumulating evidence indicates that microRNAs (miRNAs) are aberrantly expressed in human cancer and contribute to the tumorigenesis, but their roles in pancreatic cancer are still largely unknown. In this study, our data showed that miR-130b was significantly downregulated in 52 pairs of pancreatic cancer tissues and five cell lines. Furthermore, the deregulated miR-130b was correlated with worse prognosis, increased tumor size, late TNM stage, lymphatic invasion and distant metastasis. Multivariate analysis showed that miR-130b expression was a significant and independent prognostic predictor for pancreatic cancer patients. Functional studies indicated that the overexpression of miR-130b dramatically suppressed the proliferation of pancreatic cancer cells both in vitro and in vivo, which could be attributed to the induction of apoptosis and cell cycle arrest at S phase. Meanwhile, an overexpressed miR-130b remarkably inhibited the invasive ability of pancreatic cancer cells. Moreover, the dual luciferase assay revealed that STAT3 was directly targeted by miR-130b, which was further confirmed by the inverse expression of miR-130b and STAT3 in pancreatic cancer samples. Our findings suggested that miR-130b might have a considerable potential in prognosis identification and application of therapy for pancreatic cancer.

miRNA-141, Downregulated in Pancreatic Cancer, Inhibits Cell Proliferation and Invasion by Directly Targeting MAP4K4

miRNAs are associated with various types of cancer due to their ability to affect expression of genes that modulate tumorigenesis. In this study, we explored the role of miR-141 in pancreatic cancer. The analysis of clinical characteristics showed that miR-141 was significantly downregulated in tissues and cell lines of pancreatic cancer. Moreover, the decreased miR-141 level was significantly associated with tumor size and TNM stage, as well as lymph node and distant metastasis. Meanwhile, both Kaplan–Meier and multivariate survival analysis showed decreased miR-141 were associated with overall survival. Overexpression of miR-141 in pancreatic cancer cells inhibited cell proliferation, clonogenicity, and invasion; induced G1 arrest and apoptosis; and enhanced chemosensitivity. To understand how miR-141 mediates the phenotype of pancreatic cancer cells, a bioinformatics tool was used to identify MAP4K4 as a potential target of miR-141. The Dual-Luciferase reporter gene assay showed that miR-141 binds directly to the 3′-untranslated region (3′UTR) of MAP4K4 to inhibit MAP4K4 expression. Western blot and quantitative real-time PCR (qRT-PCR) analyses revealed that MAP4K4 expression was inversely correlated with miR-141 expression both in pancreatic cancer samples and cell lines. Knockdown of MAP4K4 inhibited cell proliferation, clonogenicity, and invasion, induced G1 arrest and apoptosis, and enhanced chemosensitivity. In a nude mouse xenograft model, both overexpression of miR-141 and knockdown of MAP4K4 significantly repressed pancreatic cancer cell growth. Therefore, we conclude that miR-141 targets MAP4K4, acts as a tumor suppressor in pancreatic cancer cells, and may serve as a novel therapeutic agent for miRNA-based pancreatic cancer therapy. Mol Cancer Ther; 12(11); 2569–80. ©2013 AACR.

Chronic pancreatitis and pancreatic cancer demonstrate active epithelial–mesenchymal transition profile, regulated by miR-217-SIRT1 pathway

Epithelial-mesenchymal transition (EMT) is supposed to be an inflammation induced response which may take a central role in tumorigenesis. Since recent evidence indicates that microRNAs may be involved in EMT, the present study set out to reveal the miRNA which might regulate the EMT in CP (chronic pancreatitis) and PC (pancreatic cancer) and its potential mechanism. Firstly, we provided evidence that both CP and PC tissues demonstrated active EMT profile. Consistently, miR-217 was obviously down-regulated in CP, PC and TGF-β1 treated PC cells, while negatively correlated to its direct target SIRT1. Moreover, either ectopic expression of miR-217 or inhibition of SIRT1 remarkably induced mesenchymal to epithelial transition (MET) in TGF-β1 treated PC cells. On the contrary, miR-217 inhibitor promoted EMT in PC cells but not in SIRT-knockdown PC cells. Clinical information from a cohort of 54 PC patients demonstrated that down-regulated miR-217 was positively correlated with late tumor stage, lymphatic invasion, vascular infiltration and distant metastasis. These results suggest that the overexpressed TGF-β1 in inflammation triggers the deregulation of the miR-217-SIRT1 pathway and then promotes the EMT process, which might be involved in the tumorigenesis of PC. Additionally, miR-217 may function as a novel target and predictor for PC prevention and therapy.

Effects of different resuscitation fluid on severe acute pancreatitis

AIM To compare effects of different resuscitation fluid on microcirculation, inflammation, intestinal barrier and clinical results in severe acute pancreatitis (SAP). METHODS One hundred and twenty patients with SAP were enrolled at the Pancreatic Disease Institute between January 2007 and March 2010. The patients were randomly treated with normal saline (NS group), combination of normal saline and hydroxyethyl starch (HES) (SH group), combination of normal saline, hydroxyethyl starch and glutamine (SHG group) in resuscitation. The ratio of normal saline to HES in the SH and SHG groups was 3:1. The glutamine (20% glutamine dipeptide, 100 mL/d) was supplemented into the resuscitation liquid in the SHG group. Complications and outcomes including respiratory and abdominal infection, sepsis, abdominal hemorrhage, intra-abdominal hypertension, abdominal compartment syndrome (ACS), renal failure, acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), operation intervention, length of intensive care unit stay, length of hospital stay, and mortality at 60 d were compared. Moreover, blood oxygen saturation (SpO2), gastric intramucosal pH value (pHi), intra-abdominal pressure (IAP), inflammation cytokines, urine lactulose/mannitol (L/M) ratio, and serum endotoxin were investigated to evaluate the inflammatory reaction and gut barrier. RESULTS Compared to the NS group, patients in the SH and SHG groups accessed the endpoint more quickly (3.9 ± 0.23 d and 4.1 ± 0.21 d vs 5.8 ± 0.25 d, P < 0.05) with less fluid volume (67.26 ± 28.53 mL/kg/d, 61.79 ± 27.61 mL/kg per day vs 85.23 ± 21.27 mL/kg per day, P < 0.05). Compared to the NS group, incidence of renal dysfunction, ARDS, MODS and ACS in the SH and SHG groups was obviously lower. Furthermore, incidence of respiratory and abdominal infection was significantly decreased in the SH and SHG groups, while no significant difference in sepsis was seen. Moreover, less operation time was needed in the SH and SHG group than the NS group, but the difference was not significant. The mortality did not differ significantly among these groups. Blood SpO2 and gastric mucosal pHi in the SH and SHG groups increased more quickly than in the NS group, while IAP was significantly decreased in the SH and SHG group. Moreover, the serum tumor necrosis factor-α, interleukin-8 and C-reactive protein levels in the SH and SHG groups were obviously lower than in the NS group at each time point. Furthermore, urine L/M ratio and serum endotoxin were significantly lower in the SH group and further decreased in the SHG group. CONCLUSION Results indicated that combination of normal saline, HES and glutamine are more efficient in resuscitation of SAP by relieving inflammation and sustaining the intestinal barrier.

Hypoxia-induced lncRNA-NUTF2P3-001 contributes to tumorigenesis of pancreatic cancer by derepressing the miR-3923/KRAS pathway.

Recent studies indicate that long non-coding RNAs (lncRNAs) play crucial roles in numerous cancers, while their function in pancreatic cancer is rarely elucidated. The present study identifies a functional lncRNA and its potential role in tumorigenesis of pancreatic cancer. Microarray co-assay for lncRNAs and mRNAs demonstrates that lncRNA-NUTF2P3-001 is remarkably overexpressed in pancreatic cancer and chronic pancreatitis tissues, which positively correlates with KRAS mRNA expression. After downregulating lncRNA-NUTF2P3-001, the proliferation and invasion of pancreatic cancer cell are significantly inhibited both in vitro and vivo, accompanying with decreased KRAS expression. The dual-luciferase reporter assay further validates that lncRNA-NUTF2P3-001 and 3′UTR of KRAS mRNA competitively bind with miR-3923. Furthermore, miR-3923 overexpression simulates the inhibiting effects of lncRNA-NUTF2P3-001-siRNA on pancreatic cancer cell, which is rescued by miR-3923 inhibitor. Specifically, the present study further reveals that lncRNA-NUTF2P3-001 is upregulated in pancreatic cancer cells under hypoxia and CoCl2 treatment, which is attributed to the binding of hypoxia-inducible factor-1α (HIF-1α) to hypoxia response elements (HREs) in the upstream of KRAS promoter. Data from pancreatic cancer patients show a positive correlation between lncRNA-NUTF2P3-001 and KRAS, which is associated with advanced tumor stage and worse prognosis. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumor oncogene KRAS and implicate that lncRNA-NUTF2P3-001 and miR-3923 can be applied as novel predictors and therapeutic targets for pancreatic cancer.

MiR-652 inhibits acidic microenvironment-induced epithelial-mesenchymal transition of pancreatic cancer cells by targeting ZEB1

Recent evidences suggest that the acidic microenvironment might facilitate epithelial mesenchymal transition (EMT) of tumor cells, while the effects of acidity on EMT of pancreatic cancer (PC) remain undefined. The present study demonstrated that acidity suppressed miR-652 expression, which further promoted EMT process by absenting inhibition on the transcriptional factor ZEB1 expression. At first, we found that acidity remarkably enhanced invasion ability of PC cells accompanying with increased mesenchymal and decreased epithelial markers. Meanwhile, miRNAs-microarray showed that miR-652, the potential regulator of ZEB1, was distinctly decreased in acidity-treated PC cells. Furthermore, restoration of miR-652 reversed acidity-induced EMT by inhibiting ZEB1 expression, while miR-652 inhibitor induced EMT in normal PC cells through promoting ZEB1 expression. Nevertheless, knockdown of ZEB1 significantly suppressed acidity-induced EMT in PC cells, but ZEB1 overexpression rescued the EMT which was inhibited by miR-652 overexpression. The in vivo results showed that the tumor growth and liver metastasis were remarkably retarded by both miR-652 overexpression and ZEB1 knockdown. The clinical samples further revealed that miR-652 was decreased in PC tissues and antagonistically correlated with ZEB1 expression, associating with late tumor stage, lymphatic invasion and metastasis. In conclusion, our study indicated a novel acidity/miR-652/ZEB1/EMT axis in the tumorigenesis of PC.

MiR-548an, Transcriptionally Downregulated by HIF1α/HDAC1, Suppresses Tumorigenesis of Pancreatic Cancer by Targeting Vimentin Expression

Hypoxic microenvironments contribute to the tumorigenesis of numerous cancers by regulating the expression of a subset of miRNAs called “hypoxiamiRs.” However, the function and mechanism of these deregulated miRNAs in hypoxic microenvironments within pancreatic cancers remain undefined. This study demonstrates that miR-548an is significantly downregulated in pancreatic cancer tissues and correlates with increased tumor size, advanced TNM stage, distant metastasis, and poor prognosis. Moreover, the overexpression of miR-548an significantly inhibited the proliferation and invasion of pancreatic cancer cells in vitro and in vivo. We further revealed that hypoxia-induced factor-1α (HIF-1α) induces the downregulation of miR-548an in pancreatic cancer cells during hypoxia. Our co-IP and ChIP assays revealed that HIF-1α and histone deacetylase 1 (HDAC1) form a complex and bind to the hypoxia response elements (HRE) on the miR-548an promoter. In addition, inhibition of HDAC1 with trichostatin A antagonizes the suppression of miR-548 by hypoxia. Our dual luciferase assay validated that miR-548an directly binds to the 3′ untranslated region of vimentin mRNA. The downregulation of vimentin suppresses the proliferation and invasion of pancreatic cancer cells in vitro and in vivo. In addition, vimentin was inversely correlated with miR-548an expression in pancreatic cancer samples. In conclusion, our findings suggest that the HIF-1α–HDAC1 complex transcriptionally inhibits miR-548an expression during hypoxia, resulting in the upregulation of vimentin that facilitates the pancreatic tumorigenesis. Mol Cancer Ther; 15(9); 2209–19. ©2016 AACR.

ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca2|[plus]||[sol]|RhoA pathway

Extracellular acid can have important effects on cancer cells. Acid-sensing ion channels (ASICs), which emerged as key receptors for extracellular acidic pH, are differently expressed during various diseases and have been implicated in underlying pathogenesis. This study reports that ASIC1 and ASIC3 are mainly expressed on membrane of pancreatic cancer cells and upregulated in pancreatic cancer tissues. ASIC1 and ASIC3 are responsible for an acidity-induced inward current, which is required for elevation of intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ASIC1 and ASIC3 with siRNA or pharmacological inhibitor significantly decreased [Ca2+]i and its downstream RhoA during acidity and, thus, suppressed acidity-induced epithelial–mesenchymal transition (EMT) of pancreatic cancer cells. Meanwhile, downregulating [Ca2+]i with calcium chelating agent BAPTA-AM or knockdown of RhoA with siRNA also significantly repressed acidity-induced EMT of pancreatic cancer cells. Significantly, although without obvious effect on proliferation, knockdown of ASIC1 and ASIC3 in pancreatic cancer cells significantly suppresses liver and lung metastasis in xenograft model. In addition, ASIC1 and ASIC3 are positively correlated with expression of mesenchymal marker vimentin, but inversely correlated with epithelial marker E-cadherin in pancreatic cancer cells. In conclusion, this study indicates that ASICs are master regulator of acidity-induced EMT. In addition, the data demonstrate a functional link between ASICs and [Ca2+]i/RhoA pathway, which contributes to the acidity-induced EMT.

MiRNA-646-mediated reciprocal repression between HIF-1α and MIIP contributes to tumorigenesis of pancreatic cancer

Migration and invasion inhibitory protein (MIIP) is recently identified as an inhibitor in tumor development. However, the regulatory mechanism and biological contributions of MIIP in pancreatic cancer (PC) have been not elucidated. In this study, we demonstrated a negative feedback of MIIP and hypoxia-induced factor-1α (HIF-1α), which was mediated by a hypoxia-induced microRNA. Compared with paracarcinoma tissues, MIIP was downregulated in PC tissues. Overexpression of MIIP significantly impeded the proliferation and invasion of PC cells both in vitro and in mouse xenograft models. We further verified MIIP was downregulated under hypoxia in a HIF-1α-mediated manner. Interestingly, although MIIP promoter containing two putative hypoxia response elements (HREs), the chromatin immunoprecipitation (ChIP) and luciferase reporter assays did not support an active interaction between HIF-1α and MIIP promoter. Meanwhile, microRNA array revealed a hypoxia-induced microRNA, miR-646, impaired stability of MIIP mRNA and consequently inhibited its expression by targeting the coding sequence (CDS). Coincidently, knockdown of miR-646 significantly repressed proliferation and invasion ability of PC cells both in vitro and in vivo by upregulating MIIP expression. Besides, ChIP and luciferase reporter assays further validated that HIF-1α activated transcription of miR-646 in hypoxia condition. Therefore, these results suggested HIF-1α indirectly regulated MIIP expression in post-transcriptional level through upregulating miR-646 transcription. Conversely, our results further revealed that MIIP suppressed deacetylase ability of histone deacetylase 6 (HDAC6) to promote the acetylation and degradation of HIF-1α, by which impairing HIF-1α accumulation. What is more, a specific relationship between downregulated MIIP and upregulated miR-646 expression was validated in PC samples. Moreover, the dysregulated miR-646 and MIIP expression was correlated with advanced tumor stage, lymphatic invasion, metastasis and shorter overall survival in PC patients. Together, our results highlight that the reciprocal loop of HIF-1α/miR-646/MIIP might be implemented as an applicable target for pancreatic cancer therapy.

Reciprocal loop of hypoxia‐inducible factor‐1α (HIF‐1α) and metastasis‐associated protein 2 (MTA2) contributes to the progression of pancreatic carcinoma by suppressing E‐cadherin transcription

Metastasis‐associated protein 2 (MTA2) is overexpressed in certain malignancies, and plays important roles in tumour metastasis and progression. The present study highlights the function of MTA2 in pancreatic carcinoma through its role as a deacetylator of hypoxia‐inducible factor‐1α (HIF‐1α) and a cotranscriptional factor for E‐cadherin expression. We found that overexpression of MTA2 promoted, and knockdown of MTA2 inhibited, the invasion and proliferation of pancreatic carcinoma cells both in vitro and in xenograft models in vivo. We also found that MTA2 is transcriptionally upregulated by HIF‐1α through a hypoxia response element (HRE) of the MTA2 promoter in response to hypoxia. Reciprocally, MTA2 deacetylates HIF‐1α and enhances its stability through interacting with histone deacetylase 1 (HDAC1). Consequently, HIF‐1α recruits MTA2 and HDAC1 to the HRE of the E‐cadherin promoter, by which E‐cadherin transcription is repressed. In agreement with these experimental results, MTA2 is positively associated with HIF‐1α, but inversely correlated with E‐cadherin, in pancreatic carcinoma samples. Moreover, data from The Cancer Genome Atlas on 172 pancreatic carcinomas indicate an association between high expression of MTA2 and short overall survival. Taken together, our study identifies MTA2 as a critical hub and potential therapeutic target to inhibit the progression and metastasis of pancreatic carcinoma. Copyright