LncRNAH19 acts as a ceRNA of let-7 g to facilitate endothelial-to-mesenchymal transition in hypoxic pulmonary hypertension via regulating TGF-ß signalling pathway.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality rates, and the efficiency of current HPH treatment strategies is unsatisfactory. Endothelial-to-mesenchymal transition (EndMT) in the pulmonary artery plays a crucial role in HPH. Previous studies have shown that lncRNA-H19 (H19) is involved in many cardiovascular diseases by regulating cell proliferation and differentiation but the role of H19 in EndMT in HPH has not been defined. METHODS: In this research, the expression of H19 was investigated in PAH human patients and rat models. Then, we established a hypoxia-induced HPH rat model to evaluate H19 function in HPH by Echocardiography and hemodynamic measurements. Moreover, luciferase reporter gene detection, and western blotting were used to explore the mechanism of H19. RESULTS: Here, we first found that the expression of H19 was significantly increased in the endodermis of pulmonary arteries and that H19 deficiency obviously ameliorated pulmonary vascular remodelling and right heart failure in HPH rats, and these effects were associated with inhibition of EndMT. Moreover, an analysis of luciferase activity indicated that microRNA-let-7 g (let-7 g) was a direct target of H19. H19 deficiency or let-7 g overexpression can markedly downregulate the expression of TGFßR1, a novel target gene of let-7 g. Furthermore, inhibition of TGFßR1 induced similar effects to H19 deficiency. CONCLUSIONS: In summary, our findings demonstrate that the H19/let-7 g/TGFßR1 axis is crucial in the pathogenesis of HPH by stimulating EndMT. Our study may provide new ideas for further research on HPH therapy in the near future.

Persistent TLR4 Activation Promotes Hepatocellular Carcinoma Growth through Positive Feedback Regulation by LIN28A/Let-7g miRNA.

Chronic inflammation caused by liver damage or infection plays an important role in the development and progression of hepatocellular carcinoma (HCC). The activation of Toll-like receptors 4 (TLR4) is involved in HCC tumorigenesis. Moreover, high TLR4 expression in HCC has been linked to poor prognosis. Although the expression of TLR4 in HCC is relatively low compared to hematopoietic cells, it is important to explore the molecular mechanism leading to the elevation of TLR4 in HCC. In this study, we aimed to investigate the positive regulating loop for TLR4 expression in HCC in response to chronic inflammation. Our results confirm that the mRNA expression of TLR4 and proinflammatory cytokines, including interleukin 6 (IL6) and C-C motif chemokine ligand 2 (CCL2), positively correlate in human HCC samples. High TLR4 expression in HCC is more susceptible to lipopolysaccharide (LPS); TLR4 activation in HCC provides growth and survival advantages and thus promotes tumorigenesis. It has been shown that the LIN28/let-7 microRNA (miRNA) axis is a downstream effector of the TLR4 signal pathway, and let-7 miRNA is a potential post-transcriptional regulator for TLR4. Thus, we investigated the correlation between TLR4 and LIN28A mRNA and let-7g miRNA in HCC clinical samples and found that the expression of TLR4 was positively correlated with LIN28A and negatively correlated with let-7g miRNA. Moreover, by culturing PLC/PRF5 (PLC5) HCC cells in low-dose LPS-containing medium to mimic chronic inflammation for persistent TLR4 activation, the mRNA and protein levels of TLR4 and LIN28A were elevated, and let-7g miRNA was decreased. Furthermore, the 3' untranslated region (3'UTR) of TLR4 mRNA was shown to be the target of let-7g miRNA, suggesting that inhibition of let-7g miRNA is able to increase TLR4 mRNA. While parental PLC5 cells have a low susceptibility to LPS-induced cell growth, long-term LPS exposure for PLC5 cells leads to increased proliferation, cytokine expression and stemness properties. In conclusion, our studies demonstrate positive feedback regulation for chronic TLR4 activation in the modulation of TLR4 expression level through the LIN28A/let-7g pathway in HCC and suggest a connection between chronic inflammation and TLR4 expression level in HCC for promoting tumorigenesis.

Long noncoding RNA H19 act as a competing endogenous RNA of Let-7g to facilitate IEC-6 cell migration and proliferation via regulating EGF.

Intestinal mucosal injury is one of the most significant complications of burns. In our previous study, it was found that autophagy could alleviate burn-induced intestinal injury, but the underlying mechanisms are still unclear. Irregular expression of long noncoding RNAs (lncRNAs) is present in many diseases, including burns. However, the relationship between lncRNAs and intestinal mucosal injury requires further elucidation. In this study, we established a burn mice model and detected the expression level of autophagy-related proteins. Then, H19 content after autophagy intervention was tested in vitro and in vivo. The interaction of H19 with Let-7g and that of Let-7g with epidermal growth factor (EGF) were verified by dual-luciferase reporter assays. We found that the expression of the autophagy-associated proteins LC3-II and Beclin-1 was raised in the intestinal tract of the burn mice model. Similarly, the transfection of H19 raised autophagy levels. H19 was elevated after autophagy intervention in vitro and in vivo. H19 overexpression was able to promote IEC-6 cell migration and proliferation. Let-7g was suppressed by the overexpression of H19 and the combination of Let-7g mimic was able to abolish the physiological effect of H19. Moreover, the suppression of Let-7g increased the expression of EGF protein, which heightened IEC-6 cell migration and proliferation. Besides this, dual-luciferase assays revealed that Let-7g was a direct target of H19 as well as the EGF gene. Taken together, autophagy-mediated H19 increases in mouse intestinal tract after severe burn and functions as a sponge to Let-7g to regulate EGF, which suggests that H19 serves as a potential therapeutic target and biomarker for intestinal mucosal injury after burns.

Long non-coding RNA NEAT1 facilitates the growth, migration, and invasion of ovarian cancer cells via the let-7 g/MEST/ATGL axis.

BACKGROUND/AIM: Growing evidence indicates a significant role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in ovarian cancer, a frequently occurring malignant tumor in women; however, the possible effects of an interplay of NEAT1 with microRNA (miRNA or miR) let-7 g in ovarian cancer are not known. The current study aimed to investigate the role of the NEAT1/let-7 g axis in the growth, migration, and invasion of ovarian cancer cells and explore underlying mechanisms. METHODS: NEAT1 expression levels were examined in clinical tissue samples and cell lines. The relationships between NEAT1, let-7 g, and MEST were then analyzed. Gain- or loss-of-function approaches were used to manipulate NEAT1 and let-7 g. The effects of NEAT1 on cell proliferation, migration, invasion, and apoptosis were evaluated. Mouse xenograft models of ovarian cancer cells were established to verify the function of NEAT1 in vivo. RESULTS: NEAT1 expression was elevated while let-7 g was decreased in ovarian cancer clinical tissue samples and cell lines. A negative correlation existed between NEAT1 and let-7 g, whereby NEAT1 competitively bound to let-7 g and consequently down-regulate let-7 g expression. By this mechanism, the growth, migration, and invasion of ovarian cancer cells were stimulated. In addition, let-7 g targeted mesoderm specific transcript (MEST) and inhibited its expression, leading to promotion of adipose triglyceride lipase (ATGL) expression and inhibition of ovarian cancer cell growth, migration, and invasion. However, the effect of let-7 g was abolished by overexpression of MEST. Furthermore, silencing of NEAT1 decreased the xenograft tumor growth by decreasing MEST while up-regulating let-7 g and ATGL. CONCLUSIONS: Cumulatively, the findings demonstrated that NEAT1 could promote malignant phenotypes of ovarian cancer cells by regulating the let-7 g/MEST/ATGL signaling axis. Therefore, NEAT1 can be regarded as an important molecular target and biomarker for ovarian cancer.

Epidermal growth factor regulation by autophagy-mediated lncRNA H19 in murine intestinal tract after severe burn.

To investigate the regulation of epidermal growth factor (EGF) by autophagy-mediated long non-coding RNA (lncRNA) H19 in the intestinal tracts of severely burned mice. C57BL/6J mice received third-degree burns to 30% of the total body surface area. Rapamycin and 3-methyladenine (3-MA) were used to activate and inhibit autophagy, and the changes in LC3 and Beclin1 levels were assessed by Western blotting. The effect of autophagy on lncRNA H19 was detected by qRT-PCR. Adenovirus-mediated overexpression of lncRNA H19 in IEC-6 cells was used to assess the effects of lncRNA H19 on EGF and let-7g via bioinformatics analysis, Western blotting and qRT-PCR. let-7g mimic/inhibitor was used to overexpress/inhibit let-7g, and qRT-PCR and Western blotting were used to detect the effects of let-7g on EGF. The expression levels of LC3-II, Beclin1 and lncRNA H19 were increased in intestinal tissues and IEC-6 cells after rapamycin treatment but were reversed after 3-MA treatment. LC3-II, Beclin1 and lncRNA H19 levels increased in intestinal tissues after the burn, and these increases were more significant after rapamycin treatment but decreased after 3-MA treatment. The lncRNA H19 overexpression in IEC-6 cells resulted in increased and decreased expression levels of EGF and let-7g, respectively. Furthermore, overexpression and inhibition of let-7g resulted in decreased and increased expression of EGF, respectively. Taken together, intestinal autophagy is activated after a serious burn, which can increase the transcription level of lncRNA H19. lncRNA H19 may regulate the repair of EGF via let-7g following intestinal mucosa injury after a burn.

Verbascoside inhibits progression of glioblastoma cells by promoting Let-7g-5p and down-regulating HMGA2 via Wnt/beta-catenin signalling blockade.

Glioblastoma (GBM) continues to show a poor prognosis despite advances in diagnostic and therapeutic approaches. The discovery of reliable prognostic indicators may significantly improve treatment outcome of GBM. In this study, we aimed to explore the function of verbascoside (VB) in GBM and its effects on GBM cell biological processes via let-7g-5p and HMGA2. Differentially expressed GBM-related microRNAs (miRNAs) were initially screened. Different concentrations of VB were applied to U87 and U251 GBM cells, and 50 µmol/L of VB was selected for subsequent experiments. Cells were transfected with let-7g-5p inhibitor or mimic, and overexpression of HMGA2 or siRNA against HMGA2 was induced, followed by treatment with VB. The regulatory relationships between VB, let-7g-5p, HMGA2 and Wnt/ß-catenin signalling pathway were determined. The results showed that HMGA2 was a direct target gene of let-7g-5p. VB treatment or let-7g-5p overexpression inhibited HMGA2 expression and the activation of Wnt/ß-catenin signalling pathway, which further inhibited cell viability, invasion, migration, tumour growth and promoted GBM cell apoptosis and autophagy. On the contrary, HMGA2 overexpression promoted cell viability, invasion, migration, tumour growth while inhibiting GBM cell apoptosis and autophagy. We demonstrated that VB inhibits cell viability and promotes cell autophagy in GBM cells by up-regulating let-7g-5p and down-regulating HMGA2 via Wnt/ß-catenin signalling blockade.

Long Noncoding RNA Hypoxia-Inducible Factor-1 Alpha-Antisense RNA 1 Regulates Vascular Smooth Muscle Cells to Promote the Development of Thoracic Aortic Aneurysm by Modulating Apoptotic Protease-Activating Factor 1 and Targeting let-7g.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a severe threat that is characterized by the increased aortic diameter. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of TAA. Previous research indicated that long noncoding RNAs (lncRNAs) were involved in the development of TAA. This article aimed to explore the role of lncRNA hypoxia-inducible factor-1 alpha-antisense RNA 1 (HIF1A-AS1) and potential action mechanisms in VSMCs. METHODS: The expression of HIF1A-AS1, collagen I, collagen III, microRNA let-7g (let-7g) and apoptotic protease-activating factor 1 (APAF1) was detected by quantitative real-time polymerase chain reaction. Cell proliferation and cell apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assays, respectively. The protein levels of proliferating cell nuclear antigen, Cleaved caspase-3 (Cleaved-cas3), B cell lymphoma/leukemia-2 (Bcl-2), Collagen I, Collagen III, and APAF1 were quantified by Western blot. The relationship between let-7g and HIF1A-AS1 or APAF1 was predicted by the online bioinformatics tool and verified by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: HIF1A-AS1 was upregulated in TAA tissues and was a valuable diagnostic marker of TAA. HIF1A-AS1 overexpression suppressed proliferation, induced apoptosis, and reduced the expression of extracellular matrix proteins in VSMCs. let-7 g was a target of HIF1A-AS1, and its inhibition functioned the same role as HIF1A-AS1 overexpression. APAF1 was a target of let-7g, and its knockdown played the opposite role with HIF1A-AS1 overexpression. The reintroduction of let-7g or APAF1 knockdown reversed the effects of HIF1A-AS1 overexpression in VSMCs. CONCLUSIONS: HIF1A-AS1 regulated the proliferation, apoptosis ,and the activity of extracellular matrix proteins in VSMCs through modulating APAF1 by targeting let-7g, leading to the development of TAA.

A single nuclear polymorphism in let-7g binding site affects the doubling time of thyroid nodule by regulating KRAS-induced cell proliferation.

As an indicator for the malignancy of thyroid nodules (TN), the doubling time of TN was studied in this study to evaluate the effect of rs712 polymorphism on the progression of TN. In addition, we aimed to study the potential molecular mechanisms underlying the pathological effect of rs712 polymorphism upon TN. A Taqman method was used to genotype the patients according to their rs712 polymorphism. Real-time polymerase chain reaction, western blot, Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was conducted to study the correlation between KRAS expression and the pathological effect of rs712 polymorphism. In-silicon analysis and luciferase assay were utilized to establish the regulatory relationship between let-7g and KRAS. KRAS messenger RNA (mRNA)/protein levels in the GG group were upregulated with a decreased apoptosis index. KRAS mRNA was validated to be a virtual target of let-7g. In addition, the mRNA/protein level of KRAS as well as cell proliferation index was decreased in primary thyroid cancer cells genotyped as TT/TG and transfected with KRAS small interfering RNA (siRNA)/let-7g precursors. The cell apoptosis index was evidently elevated in the KRAS siRNA/let-7g precursors group compared with that in the scramble controls. Moreover, KRAS mRNA/protein only showed slight reduction when GG-genotyped primary thyroid cancer cells were transfected by let-7g precursors. Additionally, let-7g precursors exhibited no significant effect on cell proliferation index or cell apoptosis in GG cells. Rs712 polymorphism T>G in the 3'-untranslated region of KRAS interrupts the interactions between let-7g and KRAS mRNA, leading to a higher cell proliferation index and reduced doubling time of TN.

Kallistatin attenuates endothelial senescence by modulating Let-7g-mediated miR-34a-SIRT1-eNOS pathway.

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H2 O2 -induced senescence in human endothelial cells, as indicated by reduced senescence-associated-ß-galactosidase activity, p16INK4a and plasminogen activator inhibitor-1 expression, and elevated telomerase activity. Kallistatin blocked H2 O2 -induced superoxide formation, NADPH oxidase levels and VCAM-1, ICAM-1, IL-6 and miR-34a synthesis. Kallistatin reversed H2 O2 -mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)-2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti-senescence and anti-oxidant effects were attributed to SIRT1-mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up-regulated Let-7g, whereas Let-7g inhibitor abolished kallistatin's effects on miR-34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium-specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild-type mouse endothelial cells, and H2 O2 treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let-7g, SIRT1, eNOS, catalase and SOD-1 mRNA levels, and elevated miR-34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let-7g-mediated miR-34a-SIRT1-eNOS pathway.

microRNA let-7g suppresses PDGF-induced conversion of vascular smooth muscle cell into the synthetic phenotype.

Platelet-derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let-7g in phenotypic switching. Bioinformatics prediction was used to find let-7g target genes in the PDGF/mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/Krüppel-like factor-4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let-7g transfection were used to confirm let-7g target genes. Two contractile proteins alpha-smooth muscle actin (α-SMA) and calponin were VSMC-specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let-7g gene was injected to apolipoprotein E knockout (apoE-/- ) mice to confirm let-7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF-BB can inhibit α-SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let-7g binding sites, which were confirmed by our reporter assays. Transfection of let-7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let-7g decreased phosphorylated-ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC-specific gene expression by preventing myocardin-serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let-7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let-7g can increase α-SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let-7g gene increased let-7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE-/- mice. We demonstrated that let-7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.

MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease.

Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis and conveys a significant health burden globally. Critical limb ischaemia encompasses the most severe consequence of PAD. Our previous studies indicate that microRNA let-7g prevents atherosclerosis and improves endothelial functions. This study aimed to investigate whether and how let-7g therapy may improve blood flow to ischaemic limbs. The present study shows that let-7g has multiple pro-angiogenic effects on mouse ischaemic limb model and could be a potential therapeutic agent for PAD. Mice receiving intramuscular injection of let-7g had more neovascularization, better local perfusion and increased recruitment of endothelial progenitor cells after hindlimb ischaemia. The therapeutic effects of let-7g's on angiogenesis are mediated by multiple regulatory machinery. First, let-7g increased expression of vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2) through targeting their upstream regulators HIF-3α and TP53. In addition, let-7g affected the splicing factor SC35 which subsequently enhanced the alternative splicing of VEGF-A from the anti-angiogenic isoform VEGF-A165b towards the pro-angiogenic isoform VEGF-A164a . The pleiotropic effects of let-7g on angiogenesis imply that let-7g may possess a therapeutic potential in ischaemic diseases.

Folate deficiency disturbs hsa-let-7 g level through methylation regulation in neural tube defects.

Folic acid deficiency during pregnancy is believed to be a high-risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid-regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa-let-7 g methylation level in NTD cases. Hypomethylation of hsa-let-7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa-let-7 g methylation directly regulates its own expression. Overexpression of hsa-let-7 g, along with its target genes, disturbed the migration and proliferation of SK-N-SH cells, implying that hsa-let-7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa-let-7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.

Negative feedback regulation between microRNA let-7g and LOX-1 mediated hypoxia-induced PASMCs proliferation.

BACKGROUND: Pulmonary hypertension (PH) is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Our lately study demonstrated that let-7g inhibited hypoxia-induced proliferation of PASMCs via repressing c-myc-Bmi-1-p16 signaling pathway. However, the upstream of let-7g has not yet been fully defined. Previous studies have shown that LOX-1, a target of let-7g, could also regulate the expression of let-7g in human aortic endothelial cells. In this present study, we aimed to investigate whether there is a negative feedback regulation between microRNA let-7g and LOX-1 in hypoxia-induced proliferation of PASMCs. METHODS: SD Rats were exposed to hypoxia (10% O2, 3 weeks) to induce PH. HE staining was used to evaluate pulmonary artery remodeling. in situ hybridization and immunohistochemistry were performed to assess the expression and distribution of let-7g and LOX-1, respectively. MTS, EDU and flow cytometry were performed to evaluate PASMCs proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were conducted to assess the expression of let-7g, LOX-1, calpain-1,-2,-4, and OCT-1. RESULTS: The expression of let-7g was significantly down-regulated in pulmonary arteries of hypoxia-induced PH rats accompanied by pulmonary vascular remodeling, whereas let-7g mimic inhibited hypoxia-induced proliferation of PASMCs and up-regulation of LOX-1 expression. LOX-1 blocking reversed hypoxia-induced down-regulation of let-7g expression. Calpains, protein kinase C and OCT-1 were involved in negative feedback regulation between let-7g and LOX-1. CONCLUSION: Negative feedback regulation between let-7g and LOX-1 mediated hypoxia-induced proliferation of in PASMCs.

Mother's nutritional miRNA legacy: Nutrition during pregnancy and its possible implications to develop cardiometabolic disease in later life.

Maternal nutrition during pregnancy and lactation influences the offspring's health in the long-term. Indeed, human epidemiological studies and animal model experiments suggest that either an excess or a deficit in maternal nutrition influence offspring development and susceptibility to metabolic disorders. Different epigenetic mechanisms may explain in part the way by which dietary factors in early critical developmental steps might be able to affect the susceptibility to develop metabolic diseases in adulthood. microRNAs are versatile regulators of gene expression and play a major role during tissue homeostasis and disease. Dietary factors have also been shown to modify microRNA expression. However, the role of microRNAs in fetal programming remains largely unstudied. This review evaluates in vivo studies conducted to analyze the effect of maternal diet on the modulation of the microRNA expression in the offspring and their influence to develop metabolic and cardiovascular disease in later life. In overall, the available evidence suggests that nutritional status during pregnancy influence offspring susceptibility to the development of cardiometabolic risk factors, partly through microRNA action. Thus, therapeutic modulation of microRNAs can open up new strategies to combat - later in life - the effects of nutritional insult during critical points of development.

MicroRNA let-7g links foam cell formation and adipogenic differentiation: A key regulator of Paeonol treating atherosclerosis-osteoporosis.

BACKGROUD: High comorbidity rates have been reported in patients with atherosclerosis and osteoporosis, posing a serious risk to the health and well-being of elderly patients. To improve and update clinical practice regarding the joint treatment of these two diseases, the common mechanisms of atherosclerosis and osteoporosis need to be clarified. MicroRNAs (miRNAs), are importance molecules in the pathogenesis of human diseases, including in cardiovascular and orthopedic fields. They have garnered interest as potential targets for novel therapeutic strategies. However, the key miRNAs involved in atherosclerosis and osteoporosis and their precise regulation mechanisms remain unknown. Paeonol (Pae), an active ingredient in Cortex Moutan, has shown promising results in improving both lipid and bone metabolic abnormalities. However, it is uncertain whether this agent can exert a cotherapeutic effect on atherosclerosis and osteoporosis. OBJECTIVE: This study aimed to screen important shared miRNAs in atherosclerotic and osteoporotic complications, and explore the mechanism of the protective effects of Pae against atherosclerosis and osteoporosis in high-fat diet (HFD)-fed ApoE-/- mice. METHODS: An experimental atherosclerosis and osteoporosis model was established in 40-week-old HFD ApoE-/- mice. Various techniques such as Oil Red O staining, HE staining and micro-CT were used to confirm the co-occurrence of these two diseases and efficacy of Pae in addition to the associated biochemical changes. Bioinformatics was used to screen key miRNAs in the atherosclerosis and osteoporosis model, and gene involvement was assessed through serum analyses, qRT-PCR, and western blot. To investigate the effect of Pae on the modulation of the miR let-7g/HMGA2/CEBPß pathway, Raw 264.7 cells were cocultured with bone marrow mesenchymal stem cells (BMSCs) and treated with an miR let-7g mimic/inhibitor. RESULTS: miR let-7g identified using bioinformatics was assessed to evaluate its participation in atherosclerosis-osteoporosis. Experimental analysis showed reduced miR let-7g levels in the atherosclerosis-osteoporosis mice model. Moreover, miR let-7g was required for BMSC - Raw 264.7 cell crosstalk, thereby promoting foam cell formation and adipocyte differentiation. Treatment with Pae significantly reduced plaque accumulation and foam cell number in the aorta while increasing bone density and improving trabecular bone microarchitecture in HFD ApoE-/- mice. Pae also increased the level of miR let-7g in the bloodstream of model mice. In vitro studies, Pae enhanced miR let-7g expression in BMSCs, thereby suppressing the HMGA2/CEBPß pathway to prevent the formation of foam cells and differentiation of adipocytes induced by oxidized low-density lipoprotein (ox-LDL). CONCLUSION: The study results suggested that miR let-7g participates in atherosclerosis -osteoporosis regulation and that Pae acts as a potential therapeutic agent for preventing atherosclerosis-osteoporosis through regulatory effects on the miR let-7g/HMGA2/CEBPß pathway to hinder foam cell formation and adipocyte differentiation.

let-7g sensitized liver cancer cells to 5-fluorouracil by downregulating ABCC10 expression.

Patients with advanced liver cancer may benefit from 5-fluorouracil (5-FU) therapy. However, most of them eventually faced drug resistance, resulting in a poor prognosis. The present study aims to explore the potential mechanism of let-7g/ABCC10 axis in the regulation of 5-FU resistance in liver cancer cells. Huh-7 cells were used to construct 5-FU resistant Huh-7/4X cells. CCK8, flow cytometry, and TUNEL staining were used to detect the characterization of Huh-7 cells and Huh-7/4X cells. Double luciferase report, PCR, and western blot analyses were used to detect the regulatory effects between let-7g and ABCC10. The levels of biomarkers related to cell cycle progression and apoptosis were detected by western blot assays. The role of let-7g in 5-FU sensitivity of liver cancer cells was evaluated in nude mice. Compared with LX-2 cells, the expression of let-7g was decreased in Hep3B, HepG2, Huh-7, and SK-Hep1 cells, with the lowest expression in Huh-7 cells. The sensitivity of Huh-7 cell to 5-FU was positively correlated with let-7g expression. Transfection of let-7g mimics inhibited the viability of Huh-7/4X cells by prolonging the G1 phase, with the downregulation of ABCC10, PCNA, Cyclin D1, and CDK4. Meanwhile, let-7g promoted apoptosis to increase 5-FU sensitivity of Huh-7/4X by downregulating ABCC10, Bcl-XL as well as upregulating Bax, C-caspase 3, and C-PARP. Dual-luciferase assay further confirmed that let-7g inhibited ABCC10 expression by binding to the ABCC10 3'-UTR region. Furthermore, let-7g increased the sensitivity of Huh-7/4X to 5-FU in vitro and in vivo, which can be reversed by ABCC10 overexpression. In conclusion, let-7g sensitized liver cancer cells to 5-FU by downregulating ABCC10 expression.

Sodium butyrate alleviates right ventricular hypertrophy in pulmonary arterial hypertension by inhibiting H19 and affecting the activation of let-7g-5p/IGF1 receptor/ERK.

Pulmonary arterial hypertension (PAH) is a complex and fatal cardio-pulmonary vascular disease. Decompensated right ventricular hypertrophy (RVH) caused by cardiomyocyte hypertrophy often leads to fatal heart failure, the leading cause of mortality among patients. Sodium butyrate (SB), a compound known to reduce cardiac hypertrophy, was examined for its potential effect and the underlying mechanism of SB on PAH-RVH. The in vivo study showed that SB alleviated RVH and cardiac dysfunction, as well as improved life span and survival rate in MCT-PAH rats. The in vivo and in vitro experiments showed that SB could attenuate cardiomyocyte hypertrophy by reversing the expressions of H19, let-7g-5p, insulin-like growth factor 1 receptor (IGF1 receptor), and pERK. H19 inhibition restored the level of let-7g-5p and prevented the overexpression of IGF1 receptor and pERK in hypertrophic cardiomyocytes. In addition, dual luciferase assay revealed that H19 demonstrated significant binding with let-7g-5p, acting as its endogenous RNA. Briefly, SB attenuated PAH-RVH by inhibiting the H19 overexpression, restoring the level of let-7g-5p, and hindering IGF1 receptor/ERK activation.

Let-7g Upregulation Attenuated the KRAS-PI3K-Rac1-Akt Axis-Mediated Bioenergetic Functions.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K-Rac1-Akt-BCL-2/BCL-xL-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K-Rac1-Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS.

Regulatory and Interacting Partners of PDLIM7 in Thyroid Cancer.

Enigma protein, encoded by the PDLIM7 gene, is overexpressed in thyroid cancer in a stage-dependent manner, suggesting a potential involvement in the initiation and progression of thyroid cancer. The Enigma interacts with several cellular pathways, including PI3K/AKT, MDM2, and BMP-1. The Enigma is regulated by microRNAs. Specifically, we showed that the Enigma protein upregulation corresponds to the downregulation of Let-7 family genes. There is limited research on the interactions and regulation of the Enigma with other proteins/genes in thyroid cancer tissues, indicating a gap in current knowledge. Our aim is to establish the Enigma as a biomarker. We also aim to study the interacting partners of the Enigma signaling pathways and their probable miRNA regulation in thyroid cancer progression. Using Western blotting, densitometric analysis, immunoprecipitation (IP), and reverse IP, we detected the protein expression and protein-protein interactions in the corresponding papillary thyroid carcinomas (PTCs). Utilizing real-time qPCR assay and Pearson's correlation test, we highlighted the correlation between PDLIM7 and Let-7g gene expression in the same tissues. The results showed the differential upregulations of the Enigma protein in different stages of PTCs compared to benign tissues along with AKT, VDR, BMP-1, and MDM2 proteins. Loss of DBP was observed in a subset of PTCs. Strong interactions of the Enigma with PI3K/AKT and MDM2 were noted, along with a weaker BMP-1 interaction. Pearson's correlation coefficient analysis between PDLIM7 and let-7g gene expression was significant (p < 0.05); however, there was a weak inverse correlation (r = -0.27). The study suggests the potential utility of the PDLIM7-qPCR assay as a biomarker for thyroid cancer. The Enigma's interactions with key signaling pathways may provide valuable insights into the development of thyroid cancer. The study contributes to understanding the molecular mechanisms involving the Enigma protein in thyroid cancer and highlights its potential as a biomarker.

Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells.

Thymic epithelial cells (TECs) are essential for T cell development in the thymus, yet the mechanisms governing their differentiation are not well understood. Lin28, known for its roles in embryonic development, stem cell pluripotency, and regulating cell proliferation and differentiation, is expressed in endodermal epithelial cells during embryogenesis and persists in adult epithelia, implying postnatal functions. However, the detailed expression and function of Lin28 in TECs remain unknown. In this study, we examined the expression patterns of Lin28 and its target Let-7g in fetal and postnatal TECs and discovered opposing expression patterns during postnatal thymic growth, which correlated with FOXN1 and MHCII expression. Specifically, Lin28b showed high expression in MHCIIhi TECs, whereas Let-7g was expressed in MHCIIlo TECs. Deletion of Lin28a and Lin28b specifically in TECs resulted in reduced MHCII expression and overall TEC numbers. Conversely, overexpression of Lin28a increased total TEC and thymocyte numbers by promoting the proliferation of MHCIIlo TECs. Additionally, our data strongly suggest that Lin28 and Let-7g expression is reliant on FOXN1 to some extent. These findings suggest a critical role for Lin28 in regulating the development and differentiation of TECs by modulating MHCII expression and TEC proliferation throughout thymic ontogeny and involution. Our study provides insights into the mechanisms underlying TEC differentiation and highlights the significance of Lin28 in orchestrating these processes.