Hypoxia-Challenged Pancreatic Adenocarcinoma Cell-Derived Exosomal circR3HCC1L Drives Tumor Growth Via Upregulating PKM2 Through Sequestering miR-873-5p.

Pancreatic adenocarcinoma (PAAD) is a fatal disease with poor survival. Increasing evidence show that hypoxia-induced exosomes are associated with cancer progression. Here, we aimed to investigate the function of hsa_circ_0007678 (circR3HCC1L) and hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD progression. Through the exoRBase 2.0 database, we screened for a circular RNA circR3HCC1L related to PAAD. Changes of circR3HCC1L in PAAD samples and cells were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, migration, invasion were analyzed by colony formation, cell counting, and transwell assays. Measurements of glucose uptake and lactate production were done using corresponding kits. Several protein levels were detected by western blotting. The regulation mechanism of circR3HCC1L was verified by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Exosomes were separated by differential ultracentrifugation. Animal experiments were used to verify the function of hypoxia-derived exosomal circR3HCC1L. CircR3HCC1L was upregulated in PAAD samples and hypoxic PAAD cells. Knockdown of circR3HCC1L decreased hypoxia-driven PAAD cell proliferation, migration, invasion, and glycolysis. Hypoxic PAAD cell-derived exosomes had higher levels of circR3HCC1L, hypoxic PAAD cell-derived exosomal circR3HCC1L promoted normoxic cancer cell malignant transformation and glycolysis in vitro and xenograft tumor growth in mouse models in vivo. Mechanistically, circR3HCC1L regulated pyruvate kinase M2 (PKM2) expression via sponging miR-873-5p. Also, PKM2 overexpression or miR-873-5p silencing offset circR3HCC1L knockdown-mediated effects on hypoxia-challenged PAAD cell malignant transformation and glycolysis. Hypoxic PAAD cell-derived exosomal circR3HCC1L facilitated PAAD progression through the miR-873-5p/PKM2 axis, highlighting the contribution of hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD.

COL8A1 Regulates Esophageal Squamous Carcinoma Proliferation and Invasion Through PI3K/AKT Pathway.

PURPOSE: Esophageal squamous carcinoma (ESCC) is a gastrointestinal malignancy with a high mortality, but the tumorigenesis is still unclear, restricting the target therapy development of ESCC. We explored the role of COL8A1 in ESCC development. METHODS: Tissue microarrays were used to investigate the expression level of COL8A1 in ESCC tissues. The association between COL8A1 and the overall survival of ESCC patients was assessed. The effect of differential COL8A1 expression on tumor growth was investigated by the xenograft model. The regulation of COL8A1 on tumor growth, migration, and invasion was studied by using ESCC cell lines. The signal transduction pathways involved in COL8A1 were bioinformatically profiled and validated. RESULTS: The COL8A1 was significantly expressed in cancerous tissues and was associated with poor prognosis in patients with ESCC. In vivo, the tumor growth obviously declined after inhibition of the COL8A1 expression. The abilities of cell proliferation and invasion were both decreased when the expression of COL8A1 was knockdown in ESCC cell line. Furthermore, we found the inactivation of the PI3K/AKT pathway that was mediated by knockdown of COL8A1 in ESCC cells, which was reversed with COL8A1 overexpression, whereas the cell proliferation and invasion ability were restored. CONCLUSIONS: This is the first report that COL8A1 promote ESCC progression, which hopefully will provide a theoretical basis for clinical targeting of ESCC.

Ligamentum teres hepatis wrapping of the gastroduodenal artery stump for protection in total laparoscopic pancreaticoduodenectomy: a single-center experience.

OBJECTIVE: Hemorrhage from the stump of the gastroduodenal artery (GDA) is a significant postoperative risk with pancreaticoduodenectomy (PD). Studies have shown that wrapping the GDA stump using the omentum or the falciform ligament can help prevent bleeding. We aimed to determine whether wrapping the GDA stump with the ligamentum teres hepatis (LTH) would reduce postoperative PD hemorrhage. METHODS: We retrospectively reviewed data for 148 patients who underwent laparoscopic pancreatoduodenectomy (LPD) at our hospital from November 2015 to September 2021. We compared perioperative data from 63 LPD patients without wrapping of the GDA (unwrapped group) and 85 whose GDA stumps were wrapped (wrapped group). RESULTS: There were no significant differences in the groups' baseline characteristics. The postoperative GDA stump bleeding incidence was significantly lower in the wrapped group than that in the unwrapped group (7.9% vs. 0, respectively). There was also no significant difference in the incidence of other complications (intra-abdominal infection, postoperative pancreatic fistula (POPF), biliary fistula, and gastrointestinal bleeding). CONCLUSION: Using the LTH to wrap the GDA stump during LPD can reduce bleeding from the GDA stump but not the incidence of other complications.

Correction: miR-146a Ameliorates Liver Ischemia/Reperfusion Injury by Suppressing IRAK1 and TRAF6.

[This corrects the article DOI: 10.1371/journal.pone.0101530.].

CircLMTK2 Silencing Attenuates Gemcitabine Resistance in Pancreatic Cancer by Sponging miR-485-5p and to Target PAK1.

Pancreatic cancer (PC) has a high degree of malignancy and poor prognosis, and countless patients have distant metastasis when diagnosed. Gemcitabine (GEM) chemotherapy is one of the main ways of treatment. However, PC cells have been displayed chemoresistance to GEM during treatment. Circular RNAs (circRNAs) have been demonstrated to be the most popular diagnostic and prognostic biomarkers in PC with GEM resistance. Here, we assessed the potential of circLMTK2 in the GEM resistance of PC cells. Functional assays were implemented to measure the impacts of circLMTK2 on the proliferation, migration/invasion, and apoptosis of GEM-resistant PC cells. Bioinformatics analysis and mechanical experiments displayed the underlying mechanism of circLMTK2 in GEM-resistant PC cells. We found that circLMTK2 was upregulated in PC and GEM-resistant PC tissues and cells. CircLMTK2 knockdown suppressed proliferation, invasion, migration, and enhanced apoptosis in GEM-resistant PC cells. Moreover, circLMTK2 silencing could decrease GEM resistance-associated tumor size in vivo. In terms of mechanism, circLMTK2 served as a sponge for miR-485-5p, and miR-485-5p bound to p21 (RAC1) activated kinase 1 (PAK1), which were clarified via the dual-luciferase assay in PC cell lines. We confirmed that circLMTK2 knockdown attenuated GEM-resistant PC cells by regulating PAK1 via miR-485-5p. Our study demonstrated that circLMTK2 may be a novel diagnostic and prognostic biomarker in GEM-resistant PC cells.

LncRNA LINC01857 drives pancreatic adenocarcinoma progression via modulating miR-19a-3p/SMOC2.

OBJECTIVES: Emerging evidence has demonstrated that LINC01857 exerts a pivotal function in many cancers. However, its function in Pancreatic Ductal Adenocarcinoma (PDAC) still remains unclear. This study was designed to investigate the regulatory character of LINC01857 in PDAC. METHODS: Bioinformatic tools and databases were used to seek potential miRNAs and mRNAs. Gene expression was evaluated by Reverse Transcription quantitative real-time Polymerase Chain Reaction (RT-qPCR), and western blot was used for protein level detection. A subcellular fraction assay was done to ascertain the location of LINC01857 in PANC-1 and BxPC-3 human pancreatic cancer cells. CCK-8, EdU, wound healing and Transwell assays were performed to inquire into the influence of LINC01857, and SPARC -related Modular Calcium-binding protein-2 (SMOC2) on cell viability, proliferation, migration, and invasion, respectively. The interaction between LINC01857 and its downstream genes was explored by RNA immunoprecipitation and luciferase reporter assays. RESULTS: LINC01857 levels were significantly elevated in PDAC. Knockdown of LINC01857 significantly restrained the proliferation, migration, invasion, and Epithelial-Mesenchymal Transition (EMT) process of PDAC cells. MiR-19a-3p was a downstream target of LINC01857, and miR-19a-3p levels were significantly decreased in PDAC cells. In addition, SMOC2 expression had a negative correlation with that of miR-19a-3p, and SMOC2 was a downstream target of miR-19a-3p. Furthermore, SMOC2 upregulation partially abolished the inhibitive influence of LINC01857 downregulation on cell proliferation, migration, invasion, and the EMT process. CONCLUSION: LINC01857 promotes malignant phenotypes of PDAC cells via upregulation of SMOC2 by interacting with miR-19a-3p.

LncRNA MBNL1-AS1 represses gastric cancer progression via the TGF-ß pathway by modulating miR-424-5p/Smad7 axis.

Studies over the past decades have implicated lncRNAs in promoting the development, migration and invasion of gastric cancer (GC). However, the role and mechanism of lncRNA MBNL1-AS1 in GC promotion are poorly understood. In this research, qRT-PCR showed that MBNL1-AS1 was down-regulated in GC tissues and cells. Cell experiments and the animal study demonstrated that MBNL1-AS1 knockdown accelerated GC cell proliferation, migration, and invasion, thus restraining cell apoptosis. Meanwhile, overexpression of MBNL1-AS1 repressed GC cell promotion. Bioinformatics analysis confirmed that MBNL1-AS1 binds to miR-424-5p via negative modulation. Rescue experiments showed that decreased miR-424-5p level inhibited GC cell promotion by silencing MBNL1-AS1. Furthermore, Smad7 was identified as a target of miR-424-5p that could reverse the promotion of GC cell growth mediated by miR-424-5p. Western blot results proved that MBNL1-AS1 affected TGF-ß/SMAD pathways by regulating the miR-424-5p/Smad7 axis. Collectively, MBNL1-AS1 restrained GC growth via the miR-424-5p/Smad7 axis and thus could be a promising target for GC therapy. These findings illustrate that lncRNA MBNL1-AS1, as a tumor suppressor gene, participates in GC progression by regulating miR-424-5p/Smad7 axis, thus activating TGF-ß/EMT pathways. The evidence may provide a potential marker for GC patients.

LncRNA LINC01857 drives pancreatic adenocarcinoma progression via modulating miR-19a-3p/SMOC2

Abstract Objectives Emerging evidence has demonstrated that LINC01857 exerts a pivotal function in many cancers. However, its function in Pancreatic Ductal Adenocarcinoma (PDAC) still remains unclear. This study was designed to investigate the regulatory character of LINC01857 in PDAC. Methods Bioinformatic tools and databases were used to seek potential miRNAs and mRNAs. Gene expression was evaluated by Reverse Transcription quantitative real-time Polymerase Chain Reaction (RT-qPCR), and western blot was used for protein level detection. A subcellular fraction assay was done to ascertain the location of LINC01857 in PANC-1 and BxPC-3 human pancreatic cancer cells. CCK-8, EdU, wound healing and Transwell assays were performed to inquire into the influence of LINC01857, and SPARC -related Modular Calcium-binding protein-2 (SMOC2) on cell viability, proliferation, migration, and invasion, respectively. The interaction between LINC01857 and its downstream genes was explored by RNA immunoprecipitation and luciferase reporter assays. Results LINC01857 levels were significantly elevated in PDAC. Knockdown of LINC01857 significantly restrained the proliferation, migration, invasion, and Epithelial-Mesenchymal Transition (EMT) process of PDAC cells. MiR-19a-3p was a downstream target of LINC01857, and miR-19a-3p levels were significantly decreased in PDAC cells. In addition, SMOC2 expression had a negative correlation with that of miR-19a-3p, and SMOC2 was a downstream target of miR-19a-3p. Furthermore, SMOC2 upregulation partially abolished the inhibitive influence of LINC01857 downregulation on cell proliferation, migration, invasion, and the EMT process. Conclusion LINC01857 promotes malignant phenotypes of PDAC cells via upregulation of SMOC2 by interacting with miR-19a-3p. HIGHLIGHTS LINC01857 is upregulated in PAAD and promotes malignant cellular behaviors. LINC01857 interacts with miR-19a-3p to regulate SMOC2 expression. LINC01857 promotes malignant cellular phenotypes by upregulating SMOC2.

HDAC6 inhibitor, ACY1215 suppress the proliferation and induce apoptosis of gallbladder cancer cells and increased the chemotherapy effect of gemcitabine and oxaliplatin.

As an anti-tumor agent, histone deacetylases (HDACs) inhibitors have attracted wide attention. ACY1215 is a highly effective selective inhibitor of HDAC6, which can inhibit many kinds of tumors. Whether the expression of HDAC6 and its new inhibitor ACY1215 can inhibit the proliferation of gallbladder cancer cells and induce their apoptosis remains to be further studied. The purpose of this study was to explore the effects of ACY1215 on the gallbladder cancer cells. Cell proliferation of GBC-SD and SGC-996 was assessed by cell counting kit-8 assay and colony formation assay. Flow cytometry was used to detect the apoptosis of gallbladder cancer cells. Western blot was used to detect the expressions of PCNA，KI67, and apoptosis-related proteins of gallbladder cancer cells. The HDAC6 inhibitor ACY1215 suppressed the proliferation of GBC-SD and SDC-996 cells and promoted the apoptosis of gallbladder cancer cells. The HDAC6 inhibitor ACY1215 increases the chemotherapy effect of gemcitabine and oxaliplatin. ACY1215 could suppress cell proliferation and induce apoptosis of GBC-SD and SGC-996, and increased the chemotherapy effect of gemcitabine and oxaliplatin, which provides a rationale for the combination of HDAC6 selective inhibitors with other anticancer agents in treating gallbladder cancer.

MicroRNA-497 regulates cell proliferation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. MicroRNA-497 (miR-497) is known to be downregulated in several types of human cancer; however, the expression, function and underlying mechanisms of miR-497 in HCC remain unclear. Therefore, the present study investigated miR-497 expression in HCC samples and HCC-derived cell lines using reverse transcription-quantitative polymerase chain reaction. The protein expression of one of the predicted common targets of miR-497, insulin-like growth factor-1 receptor (IGF-1R), was assessed using western blot analyses and immunohistochemistry. The role of miR-497 in regulating the proliferation of HCC-derived cells was also investigated in vitro and in vivo. Of 60 paired specimens from HCC patients, miR-497 was downregulated in 42 cancer specimens compared with adjacent non-cancer tissues. Western blotting and immunohistochemical analyses revealed that IGF-1R expression was significantly increased in HCC compared to control tissues. In addition, overexpression of miR-497 was observed to inhibit colony formation and tumor growth in MHCC-97H human HCC cells. Conversely, SMMC-7721 human HCC cells transfected with a miR-497 inhibitor exhibited enhanced colony formation and tumor growth. Finally, IGF-1R protein, phosphoinositide 3-kinase/Akt signaling pathway-associated proteins and cyclin pathway-associated proteins were differentially expressed between miR-497-overexpressing cells and miR-497-silenced cells. These results indicate that miR-497 may be a potentially effective gene therapy target.

The microRNA-146a/b attenuates acute small-for-size liver graft injury in rats.

BACKGROUND & AIMS: A critical role of the Toll-like receptor (TLR)-4 and its downstream mediators in the pathogenesis of small-for-size liver graft injury has been documented. Recently, the microRNA-146 (miR-146) was identified as a potent negative regulator of the TLR4 signalling pathway. In this study, the role of miR-146a and miR-146b in the attenuation of TLR-4 signalling and small-for-size liver graft injury was investigated. METHODS: The expression levels of miR-146a and miR-146b during small-for-size liver graft injury were studied in vivo. In addition, the effects of miR-146a and miR-146b on the expression of IRAK1 and TRAF6 in the rat macrophage cell line NR8383 and rat liver kupffer cells were studied in vitro. The in vivo effect of miR-146a and miR-146b on small-for-size liver graft injury was studied by the tail vein injection of miR-146a mimics and miR-146b mimics. RESULTS: The levels of miR-146a and miR-146b decreased with a small-for-size liver graft. MiR-146a and miR-146b inhibited IRAK1 and TRAF6 expression by binding to the 3'UTR of IRAK1 or TRAF6, respectively, in the rat macrophage cell line NR8383. The administration of miR-146a mimics and miR-146b mimics prevented liver graft injury in small-for-size liver graft injury via the inactivation of IRAK1 and TRAF6 in vivo. CONCLUSIONS: miR-146a and miR-146b prevent liver injury in small-for-size liver graft injury via the inactivation of IRAK1 and TRAF6.

miR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.

Latexin exhibits tumor suppressor potential in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and latexin is downregulated in several types of human cancer. However, latexin expression in HCC remains unknown. mRNA expression of latexin in HCC samples and HCC-derived cell lines was detected by semi­quantitative PCR and real-time PCR, while protein expression was assessed by immunohistochemistry. The role of latexin in the regulation of the proliferation of HCC-derived cells was investigated both in vitro and in vivo. Flow cytometry was used to differentiate cell cycle distribution in SK-hep-1 and YY-8103. In a total of 60 paired HCC specimens, compared with adjacent non-cancer tissues, latexin mRNA was downregulated in 42 specimens. Immunohistochemical analysis showed a significant reduction in latexin expression in HCC compared to control tissues. Overexpression of latexin inhibited SK-hep-1 and HepG2 cellular colony formation and tumor growth. Conversely, YY­8103 and Focus cells transfected with shRNA enhanced colony formation and tumor growth. Latexin overexpression promoted cell cycle arrest in the G0/G1 phase in SK-hep-1 and silencing of latexin promoted the cell cycle transition from G0/G1 phase to S phase in YY-8103. The cyclin-dependent kinase inhibitors (CDKIs) (p21Cip1, p27Kip1, p15INK4B), cyclin D1 and cyclin E were shown to be differentially expressed in latexin-overexpressed cells and latexin-silenced cells. These results indicated that latexin may be an effective target for gene therapy.