Elevated expression of LCN13 through FXR activation ameliorates hepatocellular lipid accumulation and inflammation.

BACKGROUND: Lipocalin 13 (LCN13) is a member of the lipocalin family that consists of numerous secretory proteins. LCN13 high-expression has been reported to possess anti-obesity and anti-diabetic effects. Although metabolic dysfunction-associated steatotic liver diseases (MASLD) including metabolic dysfunction-associated steatohepatitis (MASH) are frequently associated with obesity and insulin resistance, the functional role of endogenous LCN13 and the therapeutic effect of LCN13 in MASH and related metabolic deterioration have not been evaluated. METHODS: We employed a methionine-choline deficient diet model and MASH cell models to investigate the role of LCN13 in MASH development. We sought to explore the effects of LCN13 on lipid metabolism and inflammation in hepatocytes under PA/OA exposure using Western blotting, real-time RT-PCR, enzyme-linked immunosorbent assay, hematoxylin and eosin staining, oil red O staining. Using RNA sequencing, chromatin immunoprecipitation assay, and luciferase reporter assays to elucidate whether farnesoid X receptor (FXR) regulates human LCN13 transcription as a transcription factor. RESULTS: Our study found that LCN13 was down-regulated in MASH patients, MASH mouse and cell models. LCN13 overexpression in hepatocyte cells significantly inhibited lipid accumulation and inflammation in vitro. Conversely, LCN13 downregulation significantly exacerbated lipid accumulation and inflammatory responses in vivo and in vitro. Mechanistically, we provided the first evidence that LCN13 was transcriptionally activated by FXR, representing a novel direct target gene of FXR. And the key promoter region of LCN13 binds to FXR was also elucidated. We further revealed that LCN13 overexpression via FXR activation ameliorates hepatocellular lipid accumulation and inflammation in vivo and in vitro. Furthermore, LCN13-down-regulated mice exhibited aggravated MASH phenotypes, including increased hepatic lipid accumulation and inflammation. CONCLUSION: Our findings provide new insight regarding the protective role of LCN13 in MASH development and suggest an innovative therapeutic strategy for treating MASH or related metabolic disorders.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

Enterovirus A71 (EV-A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand-foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon-stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad-spectrum antiviral activity, it remains elusive whether SHFL inhibits EV-A71. In this work, we validated that EV-A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71-infected cells suggested that this factor could markedly suppress EV-A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV-A71 and promoted the degradation of 3Dpol through the ubiquitin-proteasome pathway. Furthermore, the zinc-finger domain and the 36 amino acids (164-199) of SHFL were crucial to the interaction between SHFL and EV-A71 3Dpol . Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV-A71.

Single-cell transcriptomic landscape reveals the differences in cell differentiation and immune microenvironment of papillary thyroid carcinoma between genders.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the main pathological type of thyroid carcinoma (TC). Gender is a prominent background parameter for patients with PTC. Here, we aimed to delineate the differences in cell clusters and immune microenvironment in relation to gender in PTC. METHODS: We generated 6720, 14,666, and 33,373 single-cell transcriptomes that were pooled from the tissues of four male patients with PTC, seven female patients with PTC, and three patients with nodular goiter, respectively. We performed single-cell RNA-sequencing (scRNA-seq) based on BD Rhapsody and characterized the first single-cell transcriptomic landscape of PTC involving gender. The differential cell clusters and their gene profiles were identified and analyzed via a multi-resolution network in male and female patients. The interactions of fibroblasts and endothelial cells with malignant epithelial cells and the difference in the immune infiltration of B and T lymphocytes according to gender were assessed. RESULTS: Malignant epithelial cells were divided into two distinct subsets in male and female patients with PTC. Moreover, significant differences involving inferred copy-number variations (CNVs), gene profiles, and cell differentiation were detected between male and female patients. Regarding the interactions of fibroblasts and endothelial cells with malignant epithelial cells, members of the human leukocyte antigen (HLA) family and their receptors were considered as typical in female patients with PTC, while transforming growth factor beta 1 (TGFB1) and its receptors were typical of male patients with PTC. The characteristics of B cells, including cell clusters, cell differentiation, and dominant gene sets, were significantly different between genders. CONCLUSIONS: Our data revealed the detailed differences in cell clusters and immune microenvironment in PTC according to gender at the single-cell level, which provided new insights into the understanding of the impact of gender on PTC.

Tumour-associated macrophages as a novel target of VEGI-251 in cancer therapy.

Tumour-associated macrophages (TAMs), which possess M2-like characters and are derived from immature monocytes in the circulatory system, represent a predominant population of inflammatory cells in solid tumours. TAM infiltration in tumour microenvironment can be used as an important prognostic marker in many cancer types and is a potential target for cancer prevention or treatment. VEGI-251 not only is involved in the inhibition of tumour angiogenesis, but also participates in the regulation of host immunity. This work aimed to investigate the involvement of VEGI-251 in the regulation of specific antitumour immunity. We found that recombinant human VEGI-251(rhVEGI-251) efficiently mediated the elimination of TAMs in tumour tissue in mice, and induced apoptosis of purified TAMs in vitro. During this process, caspase-8 and caspase-3 were activated, leading to PARP cleavage and apoptosis. Most importantly, we further elucidated the mechanism underlying VEGI-251-triggered TAM apoptosis, which suggests that ASK1, an intermediate component of the VEGI-251, activates the JNK pathway via TRAF2 in a potentially DR3-dependent manner in the process of TAM apoptosis. Collectively, our findings provide new insights into the basic mechanisms underlying the actions of VEGI-251 that might lead to future development of antitumour therapeutic strategies using VEGI-251 to target TAMs.

The Association between a 24-Hour Blood Pressure Pattern and Circadian Change in Plasma Aldosterone Concentration for Patients with Aldosterone-Producing Adenoma.

The absence of nocturnal blood pressure (BP) decline is associated with hypertensive complications. Data regarding circadian BP patterns in patients with aldosterone-producing adenoma (APA) are limited and equivocal. We evaluated the circadian BP profile in patients with APA and its relationship with the circadian aldosterone rhythm. BP in patients with APA and in those with essential hypertension (EH) were assessed through in-hospital 24-h ambulatory blood pressure monitoring. Over a 24-h in-hospital period, plasma aldosterone levels taken at midnight, 0400, 0800, 1200, 1600, and 2000 h were measured. To evaluate a correlation between BP and hormone rhythm, we included 27 patients with APA (APA group) and 27 patients with EH (EH group). Both groups had similar age, sex ratio, body mass index, duration of hypertension, family history of hypertension, and lipid profiles. The day-night BP differences in both patient groups were similar, whether expressed as absolute values or percentages. The proportions of patients with dipping BP profiles were also comparable (APA group, 5 of 27; EH group, 7 of 27; χ2 = 0.429; P = 0.513). At each time point, APA group plasma aldosterone concentrations (PACs) were higher than those of the EH group. A circadian change in relation to PAC was observed in both groups. A correlation between PAC and BP was statistically nonsignificant in most study patients in either group. Our data indicated that the circadian BP pattern was not associated with a change in PAC levels in patients with APA.

Long Noncoding RNA LOC100129940-N Is Upregulated in Papillary Thyroid Cancer and Promotes the Invasion and Progression.

Thyroid cancer is the most common endocrine malignancy, and its incidence has increased rapidly in recent decades worldwide. Papillary thyroid cancer (PTC) is the most common type of all thyroid cancers. The molecular mechanisms underlying the disease still need to be further investigated. Long noncoding RNAs (lncRNAs), a class of noncoding RNAs (ncRNAs) longer than 200 nucleotides, are aberrantly expressed in malignant diseases, including PTC. Here, we identified a novel isoform of LOC100129940 and designated it as LOC100129940-N. We demonstrated that the expression level of LOC100129940-N was elevated in PTC, indicating that LOC100129940-N may be involved in PTC development and progression. Moreover, our results showed that overexpression of LOC100129940-N promoted, whereas silencing of LOC100129940-N suppressed, PTC cell proliferation, invasion, and migration. Mechanistically, LOC100129940-N played an important role in activating Wnt/ß-catenin signaling and upregulating downstream target genes. Taken together, we demonstrate that LOC100129940-N promotes the activation of Wnt/ß-catenin signaling, which in turn regulates the downstream target genes, thereby enhancing invasion and progression of PTC.

Favorable surgical outcomes of aldosterone-producing adenoma based on lateralization by CT imaging and hypokalemia: a non-AVS-based strategy.

PURPOSE: To test the efficacy of a strategy based on CT imaging and clinical characteristics on lateralizing origin of excess aldosterone secretion in primary aldosteronism. PATIENTS AND METHODS: Consecutive patients with diagnosed primary hyperaldosteronism from June 2006 to July 2012 in our center underwent adrenal surgeries without pre-operational adrenal venous sampling (AVS) if all the three criteria were met: (1) round- or oval-shaped occupational lesion of low density after contrast enhancement with diameter >1 cm on CT scan was located in one adrenal gland; (2) unequivocally normal contralateral adrenal gland; (3) serum potassium level lower than 3.5 mmol/L. Subjects who had received operation were taken into analysis and follow-ups. RESULTS: One hundred and twenty-five patients fulfilled the criteria and were recruited into our research. One hundred and twenty-two operated patients (97.6%) experienced complete resolution of hypokalemia as well as resolution or improvement in hypertension with reduction in antihypertensive medication, while 3 patients (2.4%) failed to obtain normal kalemia and continued on spironolactone therapy. At a median of 65-month (range 21-93) follow-up of these 122 subjects, 27 patients dropped out (22.1%). The 95 responding patients reported no episodes of paralysis or confirmed hypokalemia or any supplementation of potassium. Multivariate linear correlation analysis showed that plasma potassium level was correlated inversely with tumor diameter (r = -0.258, 95% CI -0.076, -0.514, p = 0.037) and basal plasma aldosterone level (r = -0.251, 95% CI -0.040, -0.464, p = 0.042). CONCLUSIONS: Most patients with typical unilateral adrenal macroadenomas, normal contralateral glands and hypokalemia could attain favorable surgical therapeutic outcomes without pre-operational AVS lateralization.

Trop2 enhances invasion of thyroid cancer by inducing MMP2 through ERK and JNK pathways.

BACKGROUND: Mounting evidence has showed that Tumor-associated calcium signal transducer 2 (Trop2) is upregulated in various kinds of human cancers and plays important roles in tumorigenesis. However, the expression status and functional significance of Trop2 in thyroid cancer are largely unknown. METHODS: We first determined the expression of Trop2 by using RNAseqV2 data sets for thyroid cancer deposited on The Cancer Genome Atlas (TCGA) website. The expression of Trop2 was then confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry assays. Cell invasion and migration were assessed by conducting Transwell and wound healing assays. Furthermore, we explored the underlying mechanisms by using real-time RT-PCR, Western blot, zymography, and luciferase reporter assays. RESULTS: In this study, we demonstrated that the expression of Trop2 was significantly elevated in thyroid cancer and that its expression level was correlated with the tumor-node-metastasis (TNM) staging and N classification. Dysregulation of Trop2 altered the invasive capability of thyroid cancer cells. Further mechanistic study revealed that MMP2 expression was upregulated by Trop2. Moreover, we found that the effects of Trop2 were dependent on ERK and JNK pathways. The results from clinical specimens showed that Trop2 expression correlated with MMP2 expression in primary thyroid cancer. CONCLUSION: The current study suggests that elevated expression of Trop2 may represent an important molecular hallmark that is biologically and clinically relevant to the progression of thyroid cancer.

Transmembrane protease serine 4 promotes thyroid cancer proliferation via CREB phosphorylation.

BACKGROUND: Transmembrane protease serine 4 (TMPRSS4), one of the type II transmembrane serine proteases (TTSPs), is elevated in various cancers and is associated with multiple malignant phenotypes. However, the expression pattern and biologic significance of TMPRSS4 in thyroid cancer are largely unknown. In this study, we investigated the expression of TMPRSS4 in thyroid cancer and assessed the pro-proliferative role of TMPRSS4 in thyroid cancer. METHODS: Immunohistochemistry and real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to assess the expression of TMPRSS4 in thyroid cancer. We evaluated in vitro cell proliferation using MTT, colony formation, anchorage-independent growth, flow cytometry analysis, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. Western blot, real-time RT-PCR, and luciferase assays were conducted to reveal the underlying mechanisms. RESULTS: TMPRSS4 is overexpressed in thyroid cancer and is associated with the grade of malignancy. Depletion of TMPRSS4 in thyroid cancer cells significantly suppressed proliferation. Moreover, the proliferation of thyroid cancer cells with TMPRSS4 overexpression was significantly enhanced. We also show that cyclic adenosine monophosphate response element-binding protein (CREB)-cyclin D1 signaling mediates, at least partially, the role of TMPRSS4 in thyroid cancer cell proliferation. CONCLUSIONS: TMPRSS4 is overexpressed in thyroid cancer and TMPRSS4-CREB signaling is needed to sustain thyroid cancer cell proliferation.

Downregulation of Bcl-2 expression by miR-34a mediates palmitate-induced Min6 cells apoptosis.

Recent studies have demonstrated that the expression of miR-34a is significantly upregulated and associated with cell apoptosis in pancreatic ß -cell treated with palmitate. Nevertheless, the underlying detailed mechanism is largely unknown. Here, we showed that miR-34a was significantly induced in Min6 pancreatic ß -cell upon palmitate treatment. Elevated miR-34a promoted Min6 cell apoptosis. Intriguingly, ectopic expression of miR-34a lowered the expression of Bcl-2, an antiapoptotic protein. Luciferase reporter assay indicated the direct interaction of miR-34a with the Bcl-2 3'-UTR. Moreover, downregulated expression of Bcl-2 induced by palmitate could be restored by inhibition of miR-34a. We conclude that direct suppression of Bcl-2 by miR-34a accounts for palmitate-induced increased apoptosis rate in pancreatic ß -cell.

miR-101 inhibits cell proliferation by targeting Rac1 in papillary thyroid carcinoma.

Accumulating evidence suggests that some microRNAs (miRNAs) are involved in papillary thyroid carcinoma (PTC) progression. However, it remains necessary to elucidate the underlying molecular mechanisms involved. In the present study, we investigated the role of microRNA-101 (miR-101) in PTC via targeting of Ras-related C3 botulinum toxin substrate 1 (Rac1). The results showed that miR-101 was significantly downregulated in PTC tissues compared with adjacent normal tissues. Restoration of miR-101 expression significantly inhibited cell proliferation in the K1 PTC cell line. Moreover, algorithm-based and experimental strategies verified Rac1 as a direct target of miR-101 in the K1 cell line. Taken together, these findings suggest that miR-101 inhibited PTC growth via the downregulation of Rac1 expression, providing a better understanding of miRNA-modulated signaling networks for future cancer therapeutics.

Bezafibrate prevents palmitate-induced apoptosis in osteoblastic MC3T3-E1 cells through the NF-κB signaling pathway.

Osteoporosis is a bone condition defined by low bone mass and increase of fracture risk due to imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Low bone mass is likely to be due to the alteration of the osteoclast and osteoblast lifespan through regulated apoptosis. Saturated fatty acid (SFA) intake is negatively associated with bone mineral density (BMD). Furthermore, SFA induces apoptosis in osteoblastic cell lines. Bezafibrate could increase bone mass in intact male rats principally through increasing periosteal bone formation. At present, it is unknown whether bezafibrate attenuates palmitate-induced apoptosis in MC3T3-E1 cells. In the present study, we found that palmitate stimulated the degradation of IκBα and NF-κB translocation, as well as up-regulation of NF-κB-mediated Fas expression in obsteoblastic MC3T3-E1 cells. Furthermore, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could restore palmitate-induced caspase-3 decrease and inhibit palmitate-induced cleaved caspase-3 increase. We observed that bezafibrate, a dual ligand for the peroxisome proliferator-activated receptors α (PPARα) and PPARδ, significantly attenuated the palmitate-induced cytotoxicity as determined by the MTT assay and inhibited the palmitate-induced apoptosis as determined by a flow cytometry assay using Annexin V-FITC/PI and assessment of the activity of caspase-3. Pre-treatment of bezafibrate prevented palmitate-induced NF-κB activation. Therefore, these findings indicate that bezafibrate inbibits palmitate-induced apoptosis via the NF-κB signaling pathway. Our results point to bezafibrate as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions.

[The expression and significance of human telomerase reverse transcriptase protein and gene in bile duct carcinomas and their adjacent tissues].

OBJECTIVE: To detect the expression of human telomerase reverse transcriptase (hTERT) protein and mRNA in bile duct carcinomas and the adjacent tissues and to elucidate its role in bile duct carcinogenesis. METHODS: The expression of hTERT protein and hTERT mRNA in the formalin-fixed paraffin-embedded specimens of 71 cases of bile duct cancers and 39 cases of adjacent tissues was detected by streptavidin-peroxidase immunostaining and in situ hybridization. The correlation was analysed statistically between the expression of hTERT protein and mRNA and clinicopathological parameters bile duct carcinomas. RESULTS: The positive rate of hTERT protein expression and mRNA expression in malignant specimens was 78.9% (56/71) and 67.6% (48/71), while that in the adjacent tissues was 35.9% (14/39) and 23.1% (9/39), respectively. All the positive signals were found in the hyperplastic biliary epithelia. No significant correlation was established between hTERT expression and clinicopathological parameters. CONCLUSION: hTERT gene transcription and protein expression is most likely involved in the proliferation and malignant transformation of bile epithelia and the malignant progression of bile duct carcinomas. The detection of hTERT expression may serve elucidating the carcinogenesis of bile duct.

[In situ nucleic acid detection of HBV X gene in extrahepatic biliary tract carcinomas and its clinicopathological significance].

OBJECTIVE: To detect the expression of HBV X gene (HBx mRNA) in extrahepatic biliary tract carcinomas and the adjacent non-cancerous tissues, and to analyzed the relationship between HBV infection and incidence of biliary tract carcinomas, thereby to elucidate the possible role of HBx in the carcinogenesis of biliary tract. METHODS: The plasmid pSPX46 was digested by appropriate restriction enzyme. HBx fragment was obtained through gel extraction kit. The digoxigenin-labeled DNA probes for HBx mRNA were prepared by a random prime technique. The expression of HBx mRNA was detected in formalin-fixed- paraffin-embedded specimens from 71 cases of biliary tract carcinomas and 39 specimens of non-cancerous tissues adjacent to cancer by in situ hybridization. The correlations between HBx mRNA expression and clinicopathological parameters were statistically analysed in 71 cases of biliary duct carcinomas. RESULTS: Forty-three of 71 malignant specimens had detectable HBx mRNA expression with a positive rate being 61%. Only 7 of 39 specimens of non-cancerous tissues adjacent to cancer had weak HBx mRNA expression, with a positive rate being 18%, and all these positive signals were found in the hyperplastic biliary epithelium. No significant correlation was found between HBx mRNA expression and clinicopathological parameters, but a strong positive correlation was found between HBx mRNA and protein expression. CONCLUSION: There is a high frequency of HBx mRNA expression in extrahepatic biliary tract carcinomas. HBV infection and its gene integration might play a role to certain extent in the development of biliary tract carcinomas.