LARP4B promotes hepatocellular carcinoma progression and impairs sorafenib efficacy by activating SPINK1-mediated EGFR pathway.

La-related proteins (LARPs) regulate gene expression by binding to RNAs and exhibit critical effects on disease progression, including tumors. However, the role of LARP4B and its underlying mechanisms in the progression of hepatocellular carcinoma (HCC) remain largely unclear. In this study, we found that LARP4B expression is upregulated and correlates with poor prognosis in patients with HCC. Gain- and loss-of-function assays showed that LARP4B promotes stemness, proliferation, metastasis, and angiogenesis in vitro and in vivo. Furthermore, LARP4B inhibition enhances the antitumor effects of sorafenib and blocks the metastasis-enhancing effects of low sorafenib concentrations in HCC. Mechanistically, LARP4B expression is upregulated by METTL3-mediated N6-methyladenosine (m6A)-IGF2BP3-dependent modification in HCC. RNA- and RNA immunoprecipitation (RIP)- sequencing uncovered that LARP4B upregulates SPINK1 by binding to SPINK1 mRNA via the La motif and maintaining mRNA stability. LARP4B activates the SPINK1-mediated EGFR signaling pathway, which supports stemness, progression and sorafenib resistance in HCC. Additionally, a positive feedback loop with the LARP4B/SPINK1/p-AKT/C/EBP-ß axis is responsible for the sorafenib-therapeutic benefit of LARP4B depletion. Overall, this study demonstrated that LARP4B facilitates HCC progression, and LARP4B inhibition provides benefits to sorafenib treatment in HCC, suggesting that LARP4B might be a potential therapeutic target for HCC.

WDR4 promotes HCC pathogenesis through N7-methylguanosine by regulating and interacting with METTL1.

BACKGROUND: The N7-methylguanosine (m7G), a modification at defined internal positions within tRNAs and rRNAs, is correlated with tumor progression. Methyltransferase like 1 (METTL1)/ WD repeat domain 4 (WDR4) mediated tRNA m7G modification, which could alter many oncogenic mRNAs translation to promote progress of multiple cancer types. However, whether and how the internal mRNA m7G modification is involved in tumorigenesis remains unclear. METHODS: The immunohistochemistry assay was conducted to detect the expression of WDR4 and METTL1 in hepatocellular carcinoma (HCC) and the expression of both genes whether contributes to the prognosis of the survival rate of HCC patients. Then, CCK8, colony formation assays and tumor xenograft models were conducted to determine the effects of WDR4 on HCC cells in vitro and vivo. Besides, dot blot assay, m7G-MeRIP-seq and RNA-seq analysis were conducted to determine whether WDR4 contributes to m7G modification and underlying mechanism in HCC cells. Finally, rescue and CO-IP assay were conducted to explore whether WDR4 and METTL1 proteins form a complex in Huh7 cells. RESULTS: WDR4 modulates m7G modification at the internal sites of tumor-promoting mRNAs by forming the WDR4-METTL1 complex. WDR4 knockdown downregulated the expression of mRNA and protein levels of METTL1 gene and thus further modulate the formation of WDR4-METTL1 complex indirectly. METTL1 expression was markedly correlated with WDR4 expression in HCC tissues. HCC patients with high expression of both genes had a poor prognosis. CONCLUSIONS: WDR4 may contribute to HCC pathogenesis by interacting with and regulating the expression of METTL1 to synergistically modulate the m7G modification of target mRNAs in tumor cells.

Shizhifang ameliorates pyroptosis of renal tubular epithelial cells in hyperuricemia through inhibiting NLRP3 inflammasome.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) Compound Shizhifang (SZF), consisting of the seeds of four Chinese herbs, has been used in Shanghai Shuguang Hospital in China for more than 20 years and has proven its clinical safety and efficacy in lowering uric acid and protecting kidney function. AIM OF THE STUDY: Hyperuricemia (HUA)-induced pyroptosis of renal tubular epithelial cells serves as a significant cause of tubular damage. SZF proves to be effective in alleviating renal tubular injury and inflammation infiltration of HUA. However, the inhibiting effect of SZF on pyroptosis in HUA still remains elusive. This study aims to verify whether SZF could ameliorate pyroptosis in tubular cells induced by uric acid (UA). MATERIALS AND METHODS: Quality control analysis and chemical and metabolic identification for SZF and SZF drug serum were performed by using UPLC-Q-TOF-MS. In vitro, human renal tubular epithelial cells (HK-2) stimulated by UA were treated with SZF or NLRP3 inhibitor (MCC950). HUA mouse models were induced by intraperitoneal injection of potassium oxonate (PO). Mice were treated with SZF, allopurinol or MCC950. We focused on evaluated the effect of SZF on the NLRP3/Caspase-1/GSDMD pathway, renal function, pathologic structure and inflammation. RESULTS: SZF significantly restrained the activation of the NLRP3/Caspase-1/GSDMD pathway in vitro and in vivo induced by UA. SZF was better than allopurinol and MCC950 in reducing pro-inflammatory cytokine levels, attenuating tubular inflammatory injury, inhibiting interstitial fibrosis and tubular dilation, maintaining tubular epithelial cell function, and protecting kidney. Furthermore, 49 chemical compounds of SZF and 30 metabolites in serum after oral administration were identified. CONCLUSIONS: SZF inhibits UA-induced renal tubular epithelial cell pyroptosis via by targeting NLRP3 to inhibit tubular inflammatory and prevent the progression of HUA-induced renal injury effectively.

Reactive oxygen species induced by uric acid promote NRK­52E cell apoptosis through the NEK7­NLRP3 signaling pathway.

Increasing uric acid (UA) could induce renal tubular epithelial cell (NRK­52E) injury. However, the specific mechanism by which UA induces renal tubular epithelial cell injury remains unknown. It was hypothesized that UA induces renal tubular epithelial cell injury through reactive oxygen species (ROS) and the Never in mitosis gene A (NIMA)­related kinase 7 (NEK7)/NLR family pyrin domain containing 3 (NLRP3) signaling pathway. TUNEL assay and flow cytometry were applied to measure apoptosis, and the results of the present study showed that UA treatment induced apoptosis of NRK­52E cells in a concentration­dependent manner. Western blotting was performed to determine the expression levels of cleaved caspase­3, Bax and Bcl­xl, it was found that levels were significantly increased after UA treatment in NRK­52E cells. ROS and apoptosis were predominantly induced in NRK­52E cells and there was an association between ROS and apoptosis. Enhanced expression of NEK7, NLRP3, apoptosis­associated speck­like and caspase­1 were observed in NRK­52E cells treated with UA. The ROS inhibitor, N­acetyl­l­cysteine, exerted a protective effect on the UA­induced apoptosis of tubular epithelial cells by reducing excess ROS production, which significantly inhibited NEK7 and NLRP3 inflammasome activation. These results indicated that UA activates ROS and induces apoptosis of NRK­52E cells. The mechanism might be related to the regulation of the NEK7/NLRP3 signaling pathway.

Panx1 promotes invasion-metastasis cascade in hepatocellular carcinoma.

Background: The molecular function of pannexin1 (Panx1) in different tumor types has been remained equivocal. Until now, there is no study focused on the function of panx1 in hepatocellular carcinoma (HCC). This study aimed to explore the role of Panx1 in the invasion and metastasis of HCC. Methods: The expressions of Panx1 in 126 cases of HCC were analyzed by immunohistochemistry (IHC). The effects of Panx1 on HCC cell metastasis and invasion were observed by transwell. The expression levels of Panx1 and epithelial-mesenchymal transition (EMT) related proteins in HCC cells and tissues were detected by western blot and IHC. The tumor metastatic abilities were compared between Panx1 knockout mice and nude mice. Results: The higher expression of Panx1 in HCC was positively correlated with tumor lymph node metastasis, TNM (tumor, node, metastasis) classification and poor prognosis (overall survival, hazard ratio [HR] 2.769, 95% confidence interval [95%CI] 1.528-5.017, P=0.001; disease-free survival, HR=2.344, 95%CI 1.473-3.730, P<0.001). Overexpression of Panx1 promoted invasion and migration of HCC cells through modulation of EMT in vitro and in vivo. Conclusions: Our results suggest that the high expression of Panx1 is associated with poor HCC prognosis, providing a new clue for effective intervention for HCC metastasis.

Enhanced expression of ten-eleven translocation 1 reverses gemcitabine resistance in cholangiocarcinoma accompanied by a reduction in P-glycoprotein expression.

Increasing evidence revealed that ten-eleven translocation 1 (TET1) plays an important role in tumorigenesis and chemoresistance, but its functions in gemcitabine resistance in cholangiocarcinoma (CCA) remain unknown. This study aims to investigate the effect of TET1 on gemcitabine resistance in CCA and the possible effect on P-glycoprotein (P-gp) expression encoded by multidrug resistance (MDR) genes. We established two kinds of gemcitabine-resistant CCA cell lines and confirmed its specific features. The expression of TET1 and P-gp was evaluated in gemcitabine-resistant CCA cells and their parental cells at mRNA and protein level by quantitative RT-PCR and western blot analysis. After transfecting the gemcitabine-resistant CCA cell lines with TET1 gene or siRNA, the cell viability test was obtained to verify the effect of TET1 on the sensitivity of CCA cells to gemcitabine. And then, the possible effect of TET1 on the expression of P-gp was examined by western blot analysis. Xenograft tumor experiment was conducted to confirm the association between TET1 and P-gp expression under gemcitabine chemoresistance. The associations between clinical outcomes of CCA patients with chemotherapy and TET1 expression were analyzed in 82 patients. The results showed that TET1 expression was significantly decreased, and P-gp expression was increased in gemcitabine-resistant CCA cells. Additionally, overexpression of TET1 augmented the sensitivity of CCA cells to gemcitabine and induced the decreased expression of P-gp in gemcitabine-resistant CCA cells. Furthermore, multivariate Cox regression analysis indicated that TET1 expression and TNM stage were independent risk factors (P < 0.001) for the clinical outcomes of CCA patients with chemotherapy. Additionally, Kaplan-Meier survival and the log-rank test showed that decreased expression of TET1 was associated with poorer prognosis of CCA patients with chemotherapy. These findings suggest that TET1 expression reverses gemcitabine resistance in CCA accompanied by a reduction in P-gp expression. Thus, TET1 may be a promising target to overcome chemoresistance in CCA.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 3050 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 106 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

Donor Polymorphisms of Toll-like Receptor 4 rs1927914 Associated with the Risk of Hepatocellular Carcinoma Recurrence Following Liver Transplantation.

BACKGROUND: Hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT) severely restricts the long-term survival of patients. Toll-like receptor 4 (TLR4) has been considered to be involved in hepatocarcinogenesis and metastasis. Additionally, there is a study demonstrating the significant association between TLR4 gene rs1927914 polymorphism and HCC, but no study investigated the association of the TLR4 rs1927914 polymorphism with the risk of HCC recurrence following LT. AIM: The purpose of this study was to assess the potential association between the TLR4 gene rs1927914 polymorphism of donors and recipients and hepatocellular carcinoma recurrence after LT. METHODS: Eighty-three patients with HCC undergoing LT from July 2006-June 2015 were identified for this analysis. We genotyped a single-nucleotide polymorphism (rs1927914) in both donors and recipients and evaluated the association between the polymorphism and risk of tumor recurrence. RESULTS: The donor TLR4 rs1927914 polymorphism was found to be significantly associated with HCC recurrence following LT. In multivariate logistic regression analysis, Milan criteria, microvascular invasion and donor TLR4 rs1927914 genotype were confirmed to be independent risk factors for HCC recurrence. Kaplan-Meier survival curves showed that patients carrying donors homozygous TT had a significantly lower recurrence-free survival and overall survival than CC/CT patients. Cox proportional hazards modeling indicated that TNM stage or Milan criteria, microvascular invasion, and donor TLR4 rs1927914 genotype were independent factors for the clinical outcomes of LT patients. CONCLUSIONS: Donor TLR4 rs1927914 polymorphism is associated with an increased risk of HCC recurrence following LT and has a potential clinical value for the prediction of HCC recurrence after LT.