FBXL19 promotes malignant behaviours by activating MAPK signalling and negatively correlates with prognosis in hepatocellular carcinoma.

FBXL19 is a member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases and is linked to a variety of vital biological processes, such as cell proliferation, migration, and differentiation. Previous studies have identified it as an oncogene in breast cancer and glioma. However, its role in hepatocellular carcinoma (HCC) remains unclear. To comprehensively elucidate its role in tumour biology and its underlying mechanisms, a variety of sophisticated methods, including bioinformatics analysis, RNA-sequencing technique, and in vitro cell biology experiments, were used. Here, we found that FBXL19 was upregulated in patients with HCC and correlated with poor prognosis. In in vitro experiments, the specific targeting of short hairpin RNAs via lentiviruses successfully induced the knockdown of FBXL19, resulting in notable inhibition of the proliferation, migration, and invasion of HCC cells. Furthermore, FBXL19 downregulation resulted in significant induction of G0/G1 phase cell cycle arrest. Importantly, FBXL19 knockdown inhibited tumour malignant behaviour primarily by inactivating extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinases. In conclusion, this study revealed that FBXL19 was upregulated in patients with HCC, and that its expression was negatively correlated with prognosis. Thus, FBXL19 displays oncogenic properties in HCC by activating mitogen-activated protein kinase signalling.

Complement inhibition alleviates donor brain death-induced liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase signaling.

Brain death (BD) donors are the primary source of donor organs for liver transplantation. However, the effects of BD on donor livers and outcomes after liver transplantation remain unclear. Here, we explored the role of complement and the therapeutic effect of complement inhibition in BD-induced liver injury and posttransplantation injury in a mouse BD and liver transplantation model. For complement inhibition, we used complement receptor 2 (CR2)-Crry, a murine inhibitor of C3 activation that specifically targets sites of complement activation. In the mouse model, BD resulted in complement activation and liver injury in donor livers and a cascade liver injury posttransplantation, mediated in part through the C3a-C3aR (C3a receptor) signaling pathway, which was ameliorated by treatment with CR2-Crry. Treatment of BD donors with CR2-Crry improved graft survival, which was further improved when recipients received an additional dose of CR2-Crry posttransplantation. Mechanistically, we determined that complement inhibition alleviated BD-induced donor liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase (PI3K) signaling pathways. Together, BD induced donor liver injury and cascade injury post-transplantation, which was mediated by complement activation products acting on PI3K signaling pathways. Our study provides an experimental basis for developing strategies to improve the survival of BD donor grafts in liver transplantation.

mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A.

BACKGROUND: Exploiting cancer metabolism during nutrient availability holds immense potential for the clinical and therapeutic benefits of hepatocellular carcinoma (HCC) patients. Dietary methionine is a metabolic dependence of cancer development, but how the signal transduction integrates methionine status to achieve the physiological demand of cancer cells remains unknown. METHODS: Low or high levels of dietary methionine was fed to mouse models with patient-derived xenograft or diethyl-nitrosamine induced liver cancer. RNA sequence and metabolomics were performed to reveal the profound effect of methionine restriction on gene expression and metabolite changes. Immunostaining, sphere formation assays, in vivo tumourigenicity, migration and self-renewal ability were conducted to demonstrate the efficacy of methionine restriction and sorafenib. RESULTS: We discovered that mTORC1-c-Myc-SIRT4 axis was abnormally regulated in a methionine-dependent manner and affected the HCC progression. c-Myc rewires methionine metabolism through TRIM32 mediated degradation of SIRT4, which regulates MAT2A activity by ADP-ribosylation on amino acid residue glutamic acid 111. MAT2A is a key enzyme to generate S-adenosylmethionine (SAM). Loss of SIRT4 activates MAT2A, thereby increasing SAM level and dynamically regulating gene expression, which triggers the high proliferation rate of tumour cells. SIRT4 exerts its tumour suppressive function with targeted therapy (sorafenib) by affecting methionine, redox and nucleotide metabolism. CONCLUSIONS: These findings establish a novel characterization of the signaling transduction and the metabolic consequences of dietary methionine restriction in malignant liver tissue of mice. mTORC1, c-Myc, SIRT4 and ADP ribosylation site of MAT2A are promising clinical and therapeutic targets for the HCC treatment.

Application of da Vinci robot and laparoscopy on repeat hepatocellular carcinoma.

Background: Repeat laparoscopic liver resection has been used safely and effectively on hepatocellular carcinoma (HCC). However, few studies have been performed on repeat HCC surgery by a da Vinci robot. This study aims to evaluate the outcomes of the patients with repeat HCC treated using a da Vinci robot or laparoscopic system at a single centre. Methods: All of the patients with repeat HCC treated using a da Vinci robotic or laparoscopic system between April 2017 and April 2020 were included in this retrospective study. Results: There were 24 patients with a mean age of 56 years who underwent da Vinci robotic or laparoscopic surgery for treatment of repeat HCC who were included in this study. The operations lasted 152 ± 25 min and 142 ± 34 min. The average intraoperative blood loss was 284 ± 89 ml and 251 ± 92 ml. The average hospitalisation stay lasted 9 ± 2 days and 9 ± 3 days. The rates at which surgeons switched to open surgery were 9% and 23%. No serious perioperative or post-operative complications were encountered. Conclusion: Da Vinci robots can provide a precise dissection of the tissue under a perfect view. It is a technically feasible procedure for less rates at which surgeons switched to open surgery on repeat HCC.

Structural Insights into the Catalytic Mechanism and Ubiquitin Recognition of USP34.

Ubiquitination, an important posttranslational modification, participates in virtually all aspects of cellular functions and is reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 34 (USP34) plays an essential role in cancer, neurodegenerative diseases, and osteogenesis. Despite its functional importance, how USP34 recognizes ubiquitin and catalyzes deubiquitination remains structurally uncharacterized. Here, we report the crystal structures of the USP34 catalytic domain in free state and after binding with ubiquitin. In the free state, USP34 adopts an inactive conformation, which contains a misaligned catalytic histidine in the triad. Comparison of USP34 structures before and after ubiquitin binding reveals a structural basis for ubiquitin recognition and elucidates a mechanism by which the catalytic triad is realigned. Transition from an open inactive state to a relatively closed active state is coupled to a process by which the "fingertips" of USP34 intimately grip ubiquitin, and this has not been reported before. Our structural and biochemical analyses provide important insights into the catalytic mechanism and ubiquitin recognition of USP34.

A novel recurrence associated immune gene signature and its clinical significance in liver cancer.

Hepatocellular carcinoma (HCC) is one of the most common malignancies across nations. Although the outcome of HCC has been improved significantly with the advances in comprehensive treatment, patients remain suffered from recurrence as well as metastasis. Therefore, it is urgent to identify reliable biomarkers for predicting the recurrence of HCC, by which the treating strategy can be made to restrain tumor progress. Increasing evidence has shown the association between immune signature and prognosis of HCC. Thus, we aimed to discover an immune-related gene signature that can estimate the recurrence rates of HCC. We collected gene expression profiles and clinical information of patients from GEO and TCGA dataset. Furthermore, we conducted a lasso regression analysis and established a recurrence-related model consisting of 36 immune-related gene pairs (IRGPs) with 54 genes. We validated the IRGPs in the validation cohort and observed that the immune-related signature robustly stratified patients with HCC into high- and low-risk groups in terms of recurrence (P < 0.001). Multivariant Cox regression analysis showed the relationship between the model and recurrence outcomes (Hazard Ratio: 3.81 95% Confidence Interval: 2.90-5.00). Gene Ontology and KEGG enrichment analyses revealed that those genes were enriched in important signaling pathways. In summary, we developed a robust model based on the signature of immune-related genes for forecasting the recurrence outcome of patients with HCC, which holds the potential to assist clinical practice.

LNCAROD enhances hepatocellular carcinoma malignancy by activating glycolysis through induction of pyruvate kinase isoform PKM2.

BACKGROUND: Mounting evidence has suggested the essential role of long non-coding RNAs (lncRNAs) in a plethora of malignant tumors, including hepatocellular carcinoma. However, the underlyling mechanisms of lncRNAs remain unidentified in HCC. The present work was aimed to explore the regulatory functions and mechanisms of LncRNA LNCAROD in HCC progression and chemotherapeutic response. METHODS: The expression of LNCAROD in HCC tissues and cell lines were detected by quantitative reverse transcription PCR (qPCR). Cancer cell proliferation, migration, invasion, and chemoresistance were evaluated by cell counting kit 8 (CCK8), colony formation, transwell, and chemosensitivity assays. Methylated RNA immunoprecipitation qRCR (MeRIP-qPCR) was used to determine N6-methyladenosine (m6A) modification level. RNA immunoprecipitation (RIP) and RNA pull down were applied to identify the molecular sponge role of LNCAROD for modulation of miR-145-5p via the competing endogenous RNA (ceRNA) mechanism, as well as the interaction between LNCAROD and serine-and arginine-rich splicing factor 3 (SRSF3). The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and LNCAROD was also identified by RIP assay. Gain- or-loss-of-function assays were used to identify the function and underlying mechanisms of LNCAROD in HCC. RESULTS: We found that LNCAROD was significantly upregulated and predicted a poorer prognosis in HCC patients. LNCAROD upregulation was maintained by increased m6A methylation-mediated RNA stability. LNCAROD significantly promoted HCC cell proliferation, migration, invasion, and chemoresistance both in vitro and in vivo. Furthermore, mechanistic studies revealed that pyruvate kinase isoform M2 (PKM2)-mediated glycolysis enhancement is critical for the role of LNACROD in HCC. According to bioinformatics prediction and our experimental data, LNCAROD directly binds to SRSF3 to induce PKM switching towards PKM2 and maintains PKM2 levels in HCC by acting as a ceRNA against miR-145-5p. The oncogenic effects of LNCAROD in HCC were more prominent under hypoxia than normoxia due to the upregulation of hypoxia-triggered hypoxia-inducible factor 1α. CONCLUSIONS: In summary, our present study suggests that LNCAROD induces PKM2 upregulation via simultaneously enhancing SRSF3-mediated PKM switching to PKM2 and sponging miR-145-5p to increase PKM2 level, eventually increasing cancer cell aerobic glycolysis to participate in tumor malignancy and chemoresistance, especially under hypoxic microenvironment. This study provides a promising diagnostic marker and therapeutic target for HCC patients.

LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3.

BACKGROUND: Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. METHODS: QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. RESULTS: We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. CONCLUSION: LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

Tetramethylpyrazine ameliorates hepatic fibrosis through autophagy-mediated inflammation.

BACKGROUND: Imbalanced immune response and hepatic fibrosis are key factors related to the progression of chronic liver diseases. Tetramethylpyrazine (TMP), a natural alkaloid, has been widely used for treating liver injury. In this study, we explored the effect of TMP on hepatic fibrosis and the related mechanisms regulating autophagy. METHODS: A rat model of hepatic fibrosis and a model using an hepatic stellate cell line (HSC-T6) were created using CCl4 and platelet-derived growth factor (PDGF). Staining with haematoxylin and eosin (HE), Masson's stain, and TUNEL were performed for pathological diagnosis. ELISA, Western blotting, and immunofluorescence analyses were conducted to determine the expression levels of the specific markers for fibrosis, autophagy, inflammation, and signalling pathways. RESULTS: TMP treatment significantly rescued pathological injury and hepatic fibrosis. It also alleviated imbalances in the immune system, accumulation of extracellular matrix, and autophagy signals in hepatic fibrosis. At the same time, we found that application of the autophagy inducer rapamycin enhanced the therapeutic effect of TMP, whereas the autophagy inhibitor 3-methyladenine, PI3K pathway inhibitor LY294002, and AKT pathway agonist SC79 did the opposite. CONCLUSIONS: TMP exerts therapeutic effects in hepatic fibrosis mainly through promoting autophagy to ameliorate inflammation by inhibiting the AKT-mTOR signalling pathway, providing a new perspective for the treatment of chronic liver diseases.

Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis.

Complement is known to play a role in alcoholic fatty liver disease (AFLD), but the underlying mechanisms are poorly understood, thereby constraining the development of a rational approach for therapeutic intervention in the complement system. C3 deficiency has been shown to impart protective effects against ethanol-induced hepatic steatosis and inflammation. Here we demonstrate a protection effect in wild-type mice by treatment with CR2-Crry, a specific inhibitor of C3 activation. The expression of glycine transfer (t) RNA-derived fragments (Gly-tRFs) is upregulated in ethanol-fed mice and inhibition of Gly-tRFs in vivo decreases chronic ethanol feeding-induced hepatosteatosis without affecting inflammation. The expression of Gly-tRF was downregulated in C3-deficient or CR2-Crry-treated mice, but not in C5-deficient mice; Gly-tRF expression was restored by the C3 activation products C3a or Asp (C3a-des-Arg) via the regulation of CYP2E1. Transcriptome profiling of hepatic tissues showed that Gly-tRF inhibitors upregulate the expression of sirtuin1 (Sirt1) and subsequently affect downstream lipogenesis and ß-oxidation pathways. Mechanistically, Gly-tRF interacts with AGO3 to downregulate Sirt1 expression via sequence complementarity in the 3' UTR. Notably, the expression levels of C3d, CYP2E1 and Gly-tRF are upregulated, whereas Sirt1 is decreased in AFLD patients compared to healthy controls. Collectively, our findings suggest that C3 activation products contribute to hepatosteatosis by regulating the expression of Gly-tRF. Complement inhibition at the C3 activation step and treatment with Gly-tRF inhibitors may be potential and precise therapeutic approaches for AFLD.

CBX8 exhibits oncogenic properties and serves as a prognostic factor in hepatocellular carcinoma.

Polycomb group family is a class of proteins that have important roles in both physiological and pathological processes, and its family member Chromobox homolog 8 (CBX8) regulates cell differentiation, aging, and cell cycle progression in numerous carcinomas; however, the effects and underlying mechanisms of CBX8 in hepatocellular carcinoma (HCC) are rarely reported. We found that CBX8 expression in clinical HCC specimens correlates inversely with patient survival. In HCC cells, we found that enforced overexpression of CBX8 induces epithelial-mesenchymal transition, invasive migration, and stem cell-like traits, which are associated with increased tumor growth and metastasis in mice. Conversely, CBX8 silencing inhibits the aggressive phenotype of HCC cells that have high CBX8 expression. Mechanistically, CBX8 modulates H3K27me3 in the gene promoter of bone morphogenetic protein 4 (BMP4), which is associated with active BMP4 transcription and, consequently, the activation of Smads and mitogen-activated protein kinases. BMP4 expression reverses the effects of CBX8 silencing in inhibiting epithelial-mesenchymal transition, stemness, and metastasis. Our results establish CBX8 as a critical driver of HCC stem cell-like and metastatic behaviors and characterize its role in modulating BMP4 expression. These findings have implications for the targeting of CBX8 as an approach to HCC prognosis and treatment.

Lnc-PDZD7 contributes to stemness properties and chemosensitivity in hepatocellular carcinoma through EZH2-mediated ATOH8 transcriptional repression.

BACKGROUND: Hepatocellular carcinoma (HCC) with stemness features are pivotal for tumorigenesis, chemoresistance, and progression. Long non-coding RNAs have been implicated in the regulation of HCC stemness features; however, their mechanisms remain largely unknown. Here, we found that Lnc-PDZD7 is a potential oncogene. We systematically analyzed the clinical significance and mechanism of Lnc-PDZD7 in stemness and chemosensitivity regulation. METHODS: We analyzed the Lnc-PDZD7 expression levels in liver cancer tissues and cell line by qRT-PCR and In situ hybridization. Gain- and loss-of-function experiments were conducted to investigate the biological functions of Lnc-PDZD7 in stemness and chemosensitivity regulation. Bioinformatics analysis, dual-luciferase reporter assays were performed to validate that Lnc-PDZD7 competitively regulates EZH2, Moreover, chromatin immunoprecipitation assays, bisulfite genomic sequencing and Western blot were performed to evaluate the mechanisms of EZH2 repressing ATOH8. RESULTS: Lnc-PDZD7 is frequently upregulated in HCC tissues. Patients with high Lnc-PDZD7 expression had poorer prognoses and a poor response to adjuvant TACE therapy. Lnc-PDZD7 could promote stemness features and suppress the sensitivity of HCC cells to anticancer drugs in vitro and in vivo. Mechanistically, Lnc-PDZD7 functioned as a molecular sponge for miR-101, antagonizing its ability to repress EZH2 expression. Subsequently, EZH2 can further inhibit the expression of the stemness regulator ATOH8 via elevating its H3K27 trimethylation and DNA methylation. CONCLUSION: Lnc-PDZD7 promotes stemness properties and suppresses chemosensitivity though the miR-101/EZH2/ATOH8 pathway, providing new biomarkers for diagnosis and potential drug targets for HCC.

Diagnostic and prognostic value of lncRNA cancer susceptibility candidate 9 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant gastrointestinal tumor. There are currently few clinical diagnostic and prognostic markers for HCC. LncRNA cancer susceptibility candidate 9 (CASC9) is a long-chain non-coding RNA discovered in recent years, and previous studies have found that lncRNA CASC9 participates in the occurrence and development of HCC, but its clinical value remains unclear. AIM: To determine the expression of lncRNA CASC9 in HCC and its diagnostic and prognostic value. METHODS: Data on CASC9 expression in patients with HCC were collected from the Cancer Genome Atlas (TCGA) database to analyze the relationship between CASC9 and patient survival. A total of 80 HCC patients treated in The First Affiliated Hospital of Guangxi Medical University from May 2012 to January 2014 were enrolled in the patient group, and 50 healthy subjects were enrolled in the control group during the same period. CASC9 expression in the two groups was determined using quantitative real-time polymerase chain reaction, and its diagnostic and prognostic value was analyzed based on the CASC9 data and pathological data in these HCC patients. The relationship between CASC9 and patient survival was assessed during the 5-year follow-up period. RESULTS: Analysis of data from TCGA database revealed that control samples showed significantly lower CASC9 expression than carcinoma tissue samples (P < 0.001); the low CASC9 expression group had a higher survival rate than the high CASC9 expression group (P = 0.011), and the patient group showed significantly increased expression of serum CASC9, with the area under the curve (AUC) of 0.933. CASC9 expression was related to tumor size, combined hepatitis, tumor, node, metastasis (TNM) staging, lymph node metastasis, differentiation and alpha fetoprotein, and the high CASC9 expression group showed lower 1-year, 3-year and 5-year survival rates than the low CASC9 expression group (all a P < 0.05). Multivariate Cox regression analysis revealed that TNM staging, lymph node metastasis, differentiation, alpha fetoprotein and CASC9 were independent factors affecting the prognosis of patients. Stage I+II patients with lymph node metastasis, low differentiation, and alpha fetoprotein > 200 ng/mL had a poor 5-year survival rate. CONCLUSION: High CASC9 expression is beneficial in the prognosis of HCC patients. CASC9 is expected to be a potential diagnostic and prognostic indicator of HCC.

Competitive control of endoglucanase gene engXCA expression in the plant pathogen Xanthomonas campestris by the global transcriptional regulators HpaR1 and Clp.

Transcriptional regulators are key players in pathways that allow bacteria to alter gene expression in response to environmental conditions. However, work to understand how such transcriptional regulatory networks interact in bacterial plant pathogens is limited. Here, in the phytopathogen Xanthomonas campestris, we demonstrate that the global transcriptional regulator HpaR1 influences many of the same genes as another global regulator Clp, including the engXCA gene that encodes extracellular endoglucanase. We demonstrate that HpaR1 facilitates the binding of RNA polymerase to the engXCA promoter. In addition, we show that HpaR1 binds directly to the engXCA promoter. Furthermore, our in vitro tests characterize two binding sites for Clp within the engXCA promoter. Interestingly, one of these sites overlaps with the HpaR1 binding site. Mobility shift assays reveal that HpaR1 has greater affinity for binding to the engXCA promoter. This observation is supported by promoter activity assays, which show that the engXCA expression level is lower when both HpaR1 and Clp are present together, rather than alone. The data also reveal that HpaR1 and Clp activate engXCA gene expression by binding directly to its promoter. This transcriptional activation is modulated as both regulators compete to bind to overlapping sites on the engXCA promoter. Bioinformatics analysis suggests that this mechanism may be used broadly in Xanthomonas campestris pv. campestris (Xcc) and is probably widespread in Xanthomonads and, potentially, other bacteria. Taken together, these data support a novel mechanism of competitive activation by two global regulators of virulence gene expression in Xcc which is probably widespread in Xanthomonads and, potentially, other bacteria.

HIF1α-siRNA and gemcitabine combination-based GE-11 peptide antibody-targeted nanomedicine for enhanced therapeutic efficacy in pancreatic cancers.

Pancreatic cancer is one of the deadliest cancers across the world with an average 5-year survival rate of less than <6%. In this study, gemcitabine (GEM) and HIF1α-siRNA loaded GE-11 peptide conjugated liposome was successfully prepared and evaluated for its antitumor efficacy in pancreatic cancer cells. The GE11 increased the targeting specificity of liposome carrier and increased the intracellular concentrations in the cancer cells. Furthermore, synergistic combination of GEM and HIF1a-siRNA exhibited remarkable improvement in the declining of cancer cell proliferations. siRNA could effectively decrease the expression of HIF1a gene in the cancer cells. Importantly, GE-11 peptide-conjugated GEM/siRNA-loaded liposomes (GE-GML/siRNA) increased the total amount of apoptosis cells with higher proportion of cells in late apoptosis phase. GE-GML induced remarkable apoptosis of cancer cells and induced chromatin condensation and nuclear fragmentation which are considered to be typical features of apoptosis and cell death. GE-GML/siRNA showed a significant reduction in the tumour burden suggesting the superior anticancer efficacy of this formulation. GE-GML/siRNA showed four-fold reduction in tumour compared to control and two-fold reduction compared to GE-GML, respectively. Overall, present work lays foundation for the combination of GEM and HIF1a-siRNA loaded in a targeted nanocarrier system as a unique therapeutic option in pancreatic cancer treatment.

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.

MicroRNA-1179 inhibits the proliferation, migration and invasion of human pancreatic cancer cells by targeting E2F5.

Pancreatic cancer is one of deadly cancers and is responsible for significant mortality and morbidity across the globe. The unavailability of the efficient chemotheruptic drugs and the potent thereuprtic targets forms a bottleneck in the treatment of pancreatic cancer. In this study we explored the potential of MicroRNA-1179 as the therapeutic target for the treatment of pancreatic cancer. The results of this study indicated that the expression of miR-1179 was significantly downregulated in the pancreatic cancer cell lines as compared to the normal pancreatic cells. To unveil the potential role of miR-1179, it was overexpressed in the pancreatic cancer cells. It was observed that ectopic expression of miR-1179 caused reduction in the proliferation of pancreatic cancer cells by triggering G0/G1 cell cycle arrest. Further, overexpression of miR-1179 caused inhibition of the cell migration and invasion of the pancreatic cancer cells. To find out the potential target of miR-1179 in pancreatic cancer cells, we carried out bioinformatic analysis, the results showed that miR-1179 targets E2F transcription factor 5. This was also confirmed by western blotting analysis wherein in overexpression of miR-1179 was associated with the downregulation of the expression E2F5. Conversely, silencing of E2F5 had similar effects as that of miR-1179 suppression. Further, E2F5 overexpression could also nullify the effect on cell proliferation, migration and invasion in pancreatic cancer cells. Finally, miR-1179 overexpression could also inhibit tumor growth in vivo by suppressing the expression of E2F5. Taken together, we conclude that miR-1179 overexpression may prove beneficial for the treatment of pancreatic cancer.

Characterization of the GntR family regulator HpaR1 of the crucifer black rot pathogen Xanthomonas campestris pathovar campestris.

The GntR family transcription regulator HpaR1 identified from Xanthomonas campestris pv. campestris has been previously shown to positively regulate the genes responsible for hypersensitive reaction and pathogenicity and to autorepress its own expression. Here, we demonstrated that HpaR1 is a global regulator that positively regulates diverse biological processes, including xanthan polysaccharide production, extracellular enzyme activity, cell motility and tolerance to various stresses. To investigate the regulatory mechanisms of HpaR1, we began with xanthan polysaccharide production, which is governed by a cluster of gum genes. These are directed by the gumB promoter. Disruption of HpaR1 significantly reduced gumB transcription and an electrophoretic mobility shift assay demonstrated that HpaR1 interacts directly with gumB promoter. DNase I footprint analysis revealed that HpaR1 and RNA polymerase were bound to the sequences extending from -21 to +10 and -41 to +29 relative to the transcription initiation site of gumB, respectively. Furthermore, in vitro transcription assays showed that HpaR1 facilitated the binding of RNA polymerase to gumB promoter, leading to an enhancement of its transcription. These results suggest that HpaR1 regulates gumB transcription via a mechanism similar but different to what was found, until now, to only be used by some MerR family transcription activators.

Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris.

The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.