Background: The relationship between cholesterol and the risk of hepatocellular carcinoma (HCC) is controversial, indicating that the relationship between cholesterol metabolism and HCC is far more complex than anticipated.Methods: FVB/NJ mice were hydrodynamically injected with c-Myc and Sleeping Beauty to induce HCC. UPLC-MS/MS was used to analyze metabolites of glycolysis and the pentose phosphate pathway (PPP). 14C-acetate sodium and 3H-thymidine were used to determine cholesterol synthesis and DNA synthesis respectively.Findings: Cholesterol synthesis was increased in HCC patients. High levels of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), the rate-limiting enzyme of cholesterol synthesis, predicted poor survival of HCC patients. Hydrodynamic injection of c-Myc into mice promoted cholesterol synthesis and HCC development. Increased metabolites of glycolysis and the PPP was observed in livers of c-Myc mice. Furthermore, a positive feedback between cholesterol synthesis and the PPP was observed. Blocking the PPP rather than glycolysis reduced c-Myc-induced HCC. Hmgcr and G6pd (glucose-6-phosphate dehydrogenase) are direct targets of microRNA-206. By targeting Hmgcr and G6pd, microRNA-206 disrupted the positive feedback between cholesterol synthesis and the PPP, thereby inhibiting DNA synthesis and hepatocyte proliferation. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth that was fully prevented by microRNA-206.Interpretation: The positive feedback loop between cholesterol synthesis and the PPP fuels HCC development in c-Myc mice. MicroRNA-206 prevents c-Myc-induced HCC by disrupting this loop.Funding Information: This work was supported by the Fund for Distinguished-Young-Scholar of Hubei University of Chinese Medicine and the American Cancer Society Research Scholar Award (IS-16-210-01- RMC).Declaration of Interests: The authors involved in this study declared that they have nothing to disclose regarding funding or conflict of interest with respect to this manuscript.Ethics Approval Statement: Mice were housed, fed, and monitored in accordance with protocols approved by the committee for animal research at the Hubei University of Chinese Medicine and the University of Minnesota.
Abstract In order to find new insect resistance genes, four homologous cDNAs, hta ‐a, hta ‐b, hta ‐c and hta ‐d with lengths of 775, 718, 784 and 752 bp, respectively (GenBank accession numbers AF477031‐AF477034), were isolated from a tuber cDNA expression library of Helianthus tuberosus L. Sequence analysis revealed that all four cDNAs contain an open reading frame of 444 bp, coding a polypeptide of 147 amino acid residues, and that the sequences of the cDNAs are very similar to those of the mannose‐binding agglutinin genes of the jacalin‐related family. In hemagglutination reactions and hapten inhibition assays, affinity‐purified HTA (Helianthus tuberosus agglutinin) from induced Escherichia coli BL21(DE3) expressing GST‐HTA shows hemagglutination ability and a higher carbohydrate‐binding ability for mannose than other tested sugars. Trypsin inhibitory activity was detected in the crude extracts of induced E. coli BL21(DE3) expressing HTA, and was further verified by trypsin inhibitory activity staining on native polyacrylamide gel. The mechanism of interaction between HTA and trypsin was studied by molecular modeling. We found that plenty of hydrogen bonds and electrostatic interactions can be formed between the supposed binding sites of HTA‐b and the active site of trypsin, and that a stable HTA/trypsin complex can be formed. The results above imply that HTA might be a bifunctional protein with carbohydrate‐binding activity and trypsin inhibitory activity. Moreover, Northern blotting analysis demonstrated that hta is predominantly expressed in tubers of H. tuberosus , very weakly expressed in stems, but not expressed at all in other tissues. Southern blotting analysis indicated that hta is encoded by a multi‐gene family. The insect resistance traits have been described in another paper. (Managing editor: Li‐Hui Zhao)
Abstract Background and Significance: Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for the development of hepatocellular carcinoma (HCC). Even with successful surgical removal, the presence of NAFLD is associated with an increased recurrence of HCC. Despite the apparent association between NAFLD and HCC, the mechanistic pathways that link both disorders are essentially unknown and therapeutic agents for both disorders are lacking. MicroRNAs (miRNAs) play critical roles in both cancer and metabolic disorders, and present tremendous therapeutic potential. Methods: We used four mouse models including ATK/Ras HCC mice, cMyc HCC mice, xenograft HCC mice and dietary obese mice to evaluate the therapeutic effects of miR-206 on the pathogenesis of HCC and NAFLD. Mini-circle and Sleeping Beauty system were used to deliver miR-206 into livers of mice. Results: miRNA profiling revealed that hepatic fat accumulation impaired biogenesis of miR-206 that is also robustly under-expressed in livers of HCC mice and HCC patients. System administration of miR-206 completely prevented HCC development in both cMyc and AKT/Ras HCC mice, while 100% control mice died from lethal tumor burdens. Upon dissection, no tumor nodules were observed in livers of both AKT/Ras and cMyc mice treated with miR-206. Notably, liver-specific expression of miR-206 displayed the robust therapeutic effects on obesity and NAFLD in dietary obese mice and delivery of miR-206 into human hepatocytes significantly reduced intracellular lipid content. Combining bioinformatic prediction and molecular and cellular approaches, we identified cMet (Met proto-oncogene), CCND1, CDK4/6, EGFR, and PTPN1 (protein tyrosine phosphatase 1B) as essential direct targets of miR-206. It is known that PTPN1 can dephosphorylate PP2A (phosphatase 2A). Dephosphorylated PP2A (active form) can activate transcription of Srebp1c by dephosphorylating Sp1, a transcription activator of Srebp1c. As expected, through modulating PTPN1-PP2A-SP1-Srebp1c axis, miR-206 impaired Srebp1c-mediated lipogenesis in livers of dietary obese mice and in human hepatocytes treated with a fatty acid, while the prevention of the interaction between miR-206 and PTPN1 offset the inhibitory effects of miR-206 on lipogenesis and hepatic lipid accumulation in vivo and in vitro, suggesting that PTPN1 mediates the therapeutic effects of miR-206 on NAFLD. Through inhibiting expression of cMet, CCND1, CDK4/6, and EGFR, miR-206 completely prevented colony formation of HCC cell lines with divergent backgrounds and growth of xenograft HCC tumor from different HCC cells by impairing cMet signaling, EGFR signaling and cell cycle progression. Conclusion: This defines a critical role of miR-206 in preventing the pathogenesis of highly-associated NAFLD and HCC, and suggests its strong potential as a therapeutic strategy for both disorders. Citation Format: Guisheng Song, Heng Wu, Junyan Tao, Xin Chen. microRNA-206 as a potential therapeutic approach for highly-associated NAFLD and HCC. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A18.
Hepatic cholesterol accumulation and hypercholesterolemia are implicated in hepatocellular carcinoma (HCC). However, the therapeutic effects of cholesterol-lowering drugs on HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated. A positive feedback between cholesterol synthesis and the pentose phosphate pathway (PPP) rather than glycolysis was formed in tumors of c-Myc mice. Blocking the PPP prevented cholesterol synthesis and thereby HCC in c-Myc mice, while ablating glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6PD (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting Hmgcr and G6pd, microRNA-206 disrupted the positive feedback and fully prevented HCC in c-Myc mice, while 100% of control mice died of HCC. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth that was inhibited by miR-206. This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC.
BackgroundsThe incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing due to the prevalence of obesity. NAFLD is a major risk factor of hepatocellular carcinoma (HCC). Even with successful surgical removal, the presence of NAFLD is associated with an increased recurrence of HCC. Despite the extensive study of NAFLD, its underlying mechanism(s) remains essentially unknown and there are no FDA-approved drugs for its treatment. Alterations in microRNA (miR) expression have been observed in human fatty livers. However, regulatory mechanism(s) of miRNA biogenesis and their role in regulating the development of NAFLD is poorly described.MethodsWe used immunohistochemistry, luciferase assays and immunoblotting to study the regulatory mechanism of miR-378 biogenesis. Wild-type mice kept on a high fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378 to study loss and gain-of functions of miR-378.ResultsmiR-378 was significantly increased in fatty livers of dietary obese mice and human hepatoma HepG2 cells with accumulated lipid. Further studies identified NRF1 (Nuclear receptor factor 1), a key regulator of fatty acid oxidation (FAO), as a direct target of miR-378. Overexpression of miR-378 impaired FAO and promoted lipid accumulation in murine hepatoma Hepa1-6 cells. In contrast, knockdown of miR-378 using its ASO (anti-sense oligo) improved FAO and reduced intracellular lipid content in Hepa1-6 cells. Liver-specific expression of miR-378 impaired FAO, which subsequently promoted the development of hepatosteatosis. Antagonizing miR-378 via injecting miR-378-ASO into HFD-treated mice led to increased expression of Nrf1, improved FAO and decreased hepatosteatosis. Additional knockdown of up-regulated Nrf1 offset the effects of miR-378-ASO, suggesting that Nrf1 mediated the inhibitory effect of miR-378-ASO on hepatosteatosis. Furthermore, Nrf1 was identified as a transcriptional repressor of miR-378. Ablation of Nrf1 using its shRNA in livers led to increased miR-378, which subsequently resulted in reduced FAO and elevated hepatic lipid content.ConclusionsThese findings identified a negative feedback loop between miR-378 and Nrf1 that promotes the pathogenesis of hepatosteatosis, and suggests the use of miR-378 as a potential therapeutic target for NAFLD.
MicroRNAs play significant roles in development, metabolism and carcinogenesis, however, limited information is available about their primary transcripts and the transcriptional regulation of the microRNA genes. We report here the cloning of two primary miRNAs (pri-miR-433 and pri-miR-127) encoded by the miR-433-127 locus. Using both database mining and experimental methods, we isolated the full-length primary transcripts of the mouse miR-433 and miR-127 and demonstrated that they overlapped in a 5′-3′ unidirectional way. These two miRNA genes are expressed in a compact space by using overlapping coding regions. This is the first report to identify an economical structure for miR-433 and miR-127 genes, which may be a novel way of miRNA gene to maximize the genetic information in order to fit the complex physiological function of mammalian organism.
Rationale: Hyperlipidemia is a major risk factor of atherosclerosis and cardiovascular diseases (CVD).As a standard-of-care approach for hyperlipidemia, statins only reduce the risk of coronary artery disease by 20-40%, underscoring the importance of identifying molecular pathways for the design of drugs against this disorder.Alterations in microRNA (miRNA) expression have been reported in patients with hyperlipidemia and CVD.This study was designed to determine the mechanism of dysregulated miR-378a-3p under the status of hyperlipidemia and evaluate how miR-378a-3p regulates hepatic secretion of VLDL.Methods: Wild-type mice kept on a high fat diet were injected with miR-378a-3p inhibitor or a mini-circle expression system containing miR-378a precursor to study loss and gain-of functions of miR-378a-3p.Mice were treated with Triton WR1339 and 35 S-methionine/cysteine to determine the effect of miR-378a-3p on hepatic secretion of VLDL.Database mining, luciferase assay, and ChIP (chromatin immunoprecipitation) were used to study the mechanism of dysregulated miR-378a-3p biogenesis.Results: miR-378a-3p expression is significantly increased in livers of hyperlipidemic mice.Sort1 (sortilin 1) was identified as a direct target of miR-378a-3p.By inhibiting the function of sortilin 1 as a transmembrane trafficking receptor, miR-378a-3p stabilized ApoB100 and promoted ApoB100 secretion in vitro.Liver-specific expression of miR-378a-3p stabilized ApoB100 and facilitated hepatic secretion of VLDL, which subsequently increased levels of VLDL/LDL cholesterol as well as triglycerides.In contrast, antagonizing miR-378a-3p using its inhibitor increased hepatic expression of Sort1 and reduced hepatic export of VLDL with its consequent effects of serum lipid levels.Additional knockdown of up-regulated Sort1 in livers of mice offset the effects of miR-378a-3p inhibitor, suggesting that Sort1 was indispensable for miR-378a-3p to promote secretion of VLDL and thereby high levels of circulating VLDL/LDL cholesterol and triglycerides.Furthermore, oncogenic E2F1 (E2F transcription factor 1) was identified as a transcriptional activator of miR-378a-3p.E2f1 knockdown, through reducing miR-378a-3p, impaired secretion of VLDL and reduced levels of VLDL/LDL cholesterol and triglycerides.Conclusions: This study defines a novel pathway of E2F1-miR-378a-3p-SORT1-ApoB100 that controls levels of circulating VLDL/LDL cholesterol and triglycerides by modulating degradation and secretion of ApoB100, and suggests the use of miR-378a-3p as a potential therapeutic target for dyslipidemia.
Abstract Purpose The causality between social determinants of health (SDoH) and mortality in patients with chronic heart failure (CHF) is uncertain. Herein, we assessed the causality using inverse probability weighting (IPW) of marginal structural models (MSMs) during the course of CHF. Method A multicenter, prospective cohort study of 1377 patients with CHF were enrolled from September 2017. The social domain and two dimensions of Chronic Heart Failure Patient-Reported Outcomes Measure (CHF-PROM) was used to assess the SDoH, social support, and support utilization of patients with CHF. CHF-PROM and mortality information were obtained at 1, 3, and 6 months following patient discharge, and every 6 months thereafter at regular follow-ups. Logistic regression and IPW of MSMs were applied to analyze the SDoH, social support, and support utilization on mortality in patients with CHF. Results Logistic regression showed that at baseline, the SDoH, social support, and support utilization were not associated with mortality in patients with CHF. After adjusting for confounders, MSMs showed that the SDoH and social support were not associated with mortality at baseline. In contrast, low support utilization at baseline and unfavorable SDoH, low social support, and low support utilization during follow-up increased the risk of death in patients with CHF. Conclusion Through follow-up data and MSMs analysis, we found that the long-term out-of-hospital causal effects, but not one-time effects of SDoH, are risk factors for CHF mortality. SDoH should be taken seriously during the entire CHF process to prolong patients’ survival. Trial registration: The cohort number registered in the China Clinical Trial Registry is ChiCTR2100043337. URL: https://www.chictr.org.cn/showproj.html?proj=64980 Registered on February 11, 2021
Orphan receptor small heterodimer partner (SHP, NROB2) has been shown to be a metabolic regulator in pathways associated with several major aspects of the metabolic syndrome. However, the significance and transcriptional regulatory role of SHP in adipocyte differentiation remain unclear. Transcriptional profiles of 3T3-L1 preadipocytes and early differentiating preadipocytes in response to SHP were systemically surveyed using Affymetrix Genome Array representing well-characterized 14,000 genes. Analysis revealed about 963 genes that were up- or down-regulated by more than 2-fold during differentiation and/or by the overexpression of SHP. These genes were organized into 4 clusters that demonstrated concerted changes in expression of genes controlling various aspects of the cellular events and metabolism. Quantitative PCR was employed to further characterize gene expression and led to the identification of several key regulators and stimulators of the adipogenic program as potential new SHP targets. Overexpression of SHP inhibited the differentiation process as well as the accumulation of neutral lipids within the cells. Our data suggests that SHP may function as a molecular switch that governs adipogenesis and a potent adipogenic suppressor that maintains preadipocytes in an undifferentiated state through inhibition of the adipogenic transcription factors and stimulators. Developing SHP agonist may promise a future treatment for obesity.
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