H19 Knockdown Research Articles

LncRNA H19 Activates the RAS-MAPK Signaling Pathway via miR-140-5p/SOS1 Axis in Malignant Liver Tumors.

To study the influences of LncRNA H19 (H19) on malignant liver tumor cells and elucidate the underlying molecular mechanisms. H19 expression in liver tumor tissues, matched normal liver tissues, human liver malignant tumor cell lines and the human hepatocyte line LO2 was assessed via quantitative RT-PCR. Cell viability analysis and Matrigel invasion analysis were performed to evaluate the effects of H19 on cell proliferation and invasion. Luciferase reporter analysis was carried out to assess the interaction between miR-140-5p and SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1). The influence of H19 on the Ras-MAPK signalling pathway was evaluated by detecting key protein levels via active Ras pull-down analysis and Western blot analysis. H19 expression was lower in liver cancer samples than in matched normal liver tissue samples. H19 overexpression enhanced the proliferation and invasion of HepG2 and SMMC-7721 cells. H19 overexpression increased the level of activated Ras. The expression levels of phosphorylated Raf, phosphorylated ERK and phosphorylated MEK were increased by H19 overexpression. H19 knockdown had the opposite effect. Treatment with a MAPK inhibitor significantly reversed the influence of H19 overexpression on liver malignant tumor cell growth and invasion. The MAPK activator reversed the opposing effects of H19 silencing. H19 overexpression increased the protein level of SOS1, and miR-140-5p directly targeted SOS1. H19 can activate the Ras-MAPK signalling pathway via the miR-140-5p/SOS1 axis in malignant liver tumour cells.

Trimetazidine restores autophagy via lncRNA H19/AMPK in diabetic heart: Implications for its therapeutic value against diabetic cardiomyopathy.

Previous studies have shown that trimetazidine (TMZ) alleviates diabetes-induced cardiac dysfunction. However, the underlying mechanism for its protective effects on cardiac function remains incompletely understood. Diminished autophagy was found in diabetic hearts, and restoration of autophagy generates cardioprotective effect. This study aims to investigate whether and how TMZ produces protective effect through increasing autophagic activity in the diabetic heart. A high-fat diet and low-dose streptozotocin were applied to induce type 2 diabetes mellitus (T2DM) in male C57BL/6 mice, followed by treatment with TMZ for 14 weeks before cardiac function was evaluated. To mimic the diabetic condition, the neonatal rat cardiomyocytes (NRCMs) were exposed to high glucose/palmitic acid (HP) in the presence or absence of TMZ. We found that TMZ treatment promotes autophagic flux in cardiomyocytes, which is impaired in diabetes. We further found that the AMPK and lncRNA H19 played critical roles in mediating TMZ-induced enhancement of autophagy in cardiomyocyte. We showed that TMZ treatment restored the level of H19 and phosphorylated AMPK (p-AMPK T172) in diabetic heart and NRCMs exposed to HP. Of note, the effect of TMZ on autophagy and p-AMPK was abolished by knockdown of H19. These findings indicated that TMZ is able to recover the cardiac autophagic activity which is impaired by T2DM, and the underlying mechanism accounted for this ability is mostly likely attributed to the restored expression of H19 and AMPK activity.

Calcific aortic valve disease-associated long noncoding RNA H19 promotes the osteogenic transition of valvular interstitial cells via the JAG1/NOTCH1 axis

Abstract Background Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease causing tremendous suffering globally. The osteogenic differentiation of the valvular interstitial cells (VICs) is a key process in the pathogenesis of CAVD leading to calcification and stiffening. Here, we focus on the contribution of long noncoding RNA H19 in the osteogenic differentiation of VICs promoting CAVD. Methods and results A human lncRNA array performed in AVS patients with coronary artery disease (CAD) revealed the differential expression of lncRNA H19. For the experiments in this study, loss of function (LOF) was performed in primary VICs isolated from the aortic valves of patients with CAVD, and commercially-available VICs. Therefore, H19 expression was effectively downregulated via RNAi. Subsequent gene expression analysis via RT-qPCR revealed that the expression of calcification markers RUNX2 and BMP2, as well as the expression of the H19 downstream targets JAG1 and NOTCH1 were unchanged after the H19 knockdown alone. Then, the VICs were additionally treated with osteogenic medium (OM) and procalcifying medium (PCM) for 7 d to induce the calcification of the cells. In the OM, inorganic phosphate is generated in an alkaline phosphatase (ALPL)-dependent manner and integrated into newly formed hydroxyapatite crystals, thus promoting cellular calcification. PCM contains inorganic phosphate to form hydroxyapatite crystals and is therefore not ALPL-dependent. Gene expression analysis via RT-qPCR and protein analysis via immunofluorescence revealed that the calcification markers BMP2 and RUNX2 were upregulated after H19 knockdown and calcification induction. This indicates that H19 regulates the expression of calcification makers promoting cellular calcification. Furthermore, RT-qPCR and immunoblotting revealed the differential expression of several potential H19 downstream targets, namely JAG1, NOTCH1, and STAT3. The analysis showed that the potential downstream targets were upregulated after H19 knockdown and calcification treatment hinting at the involvement of the NOTCH1 or STAT3 pathway in the pathogenesis of CAVD. Moreover, an Alizarin Red S staining of calcified nodules revealed reduced calcification after H19 knockdown and calcification treatment for 21 d. This demonstrates that H19 also regulates a late-stage event in the cellular calcification of VICs. Conclusion Our study showed that H19 regulates calcification marker expression and the expression of potential downstream targets JAG1, NOTCH1, and STAT3 both in healthy and AVS patient VICs. This hints at the involvement of the H19/JAG1/NOTCH1 pathway in the calcification of VICs, which might play a critical role in the pathogenesis of CAVD.

Aortic valve stenosis augments long noncoding RNA H19 to govern angiogenic responses and phenotypes of valvular endothelial cell through endothelial to mesenchymal transition

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as important regulators of cardiovascular disease, including aortic valve stenosis (AVS). Although several recent studies have reported that lncRNAs are dysregulated and might play a crucial role in (Endothelial to mesenchymal transition) EndMT, no specific role of lncRNA was reported in AVS pathogenesis. Here, we sought to investigate the role of H19 in EndMT during the pathogenesis of AVS. Methods and results High-throughput lncRNA sequencing of human aortic valve tissue explanted from patients with AVS due to severe stenosis during surgical aortic valve replacement (SAVR) demonstrated that certain lncRNAs [H19, AGAP2-AS1 (PUNISHER), GAS5, MALAT1] are significantly dysregulated compared to patients without AVS. To validate the RNA-seq data, 60 patients with AVS (n=30) or without AVS (n=30) were prospectively investigated. Among these, H19 (p=0.002), AGAP2-AS1 (p=0.002), and GAS5 (0.02) were significantly upregulated in patients with AVS, compared to non-AVS patients. Aortic valvular endothelial cells (VECs) were isolated from explanted valve tissue from AVS patients or obtained commercially. In vitro EndMT-promoting stimuli (TNFα or TGFß+IL1ß) were used for VEC stimulation for 5 days. RT-qPCR was used to quantify the expression of EndMT markers (EC markers, vWF, and NOS3, and mesenchymal markers, αSMA, SM22, Vimentin, and SNAI2). Loss of H19 led to dysregulation of the EC gene network and protein expression patterns that were analyzed by qRT-PCR, immunoblotting, and immunofluorescence, respectively. Further experiments show that silencing of H19 affects migration and proliferation, and promotes EndMT in EC. Functional assays suggested that H19 is a critical regulator of angiogenic properties. The molecular signature of angiogenesis was investigated using a RT-qPCR-based angiogenesis array to identify the regulation of angiogenesis with 84-angiogenesis-related genes. Mechanistically, the angiogenesis array revealed that H19 regulates Jagged Canonical Notch Ligand 1 (JAG1) (4.43-fold vs. control EC), an important regulator of NOTCH1 signaling, and pro-angiogenic factor in EC. Accordingly, EndMT marker expressions were dysregulated upon H19 depletion. A high-throughput luciferase reporter array demonstrated STAT3 activation upon H19 knockdown. Depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on EndMT marker expression as well as on angiogenesis. Overexpression of H19 in VEC abrogated the H19-mediated promotion of JAG1- and VEGFA gene- and protein expression. Our data suggest that H19 might play a role in EC angiogenic gene expression and EndMT. The data reveal a pivotal role of H19 in controlling EndMT in AVS via regulation of the JAG1-NOTCH axis, which could potentially be therapeutically targeted to halt EndMT during AVS.

Divergent regulation of long non-coding RNAs H19 and PURPL affects cell senescence in human dermal fibroblasts.

Cellular senescence is a permanent cell growth arrest that occurs in response to various intrinsic and extrinsic stimuli and is associated with cellular and molecular changes. Long non-coding RNAs (lncRNAs) are key regulators of cellular senescence by affecting the expression of many important genes involved in senescence-associated pathways and processes. Here, we evaluated a panel of lncRNAs associated with senescence for their differential expression between young and senescent human dermal fibroblasts (NHDFs) and studied the effect of a known senomorphic compound, resveratrol, on the expression of lncRNAs in senescent NHDFs. As markers of senescence, we evaluated cell growth, senescence-associated (SA)-β-Gal staining, and the expression of p21, Lamin B1 and γH2AX. We found that H19 and PURPL were the most altered lncRNAs in replicative, in doxorubicin (DOXO) and ionising radiation (IR)-induced senescence models. We then investigated the function of H19 and PURPL in cell senescence by siRNA-mediated silencing in young and senescent fibroblasts, respectively. Our results showed that H19 knockdown reduced cell viability and induced cell senescence and autophagy of NHDFs through the regulation of the PI3K/AKT/mTOR pathway; conversely, PURPL silencing reversed senescence by reducing (SA)-β-Gal staining, recovering cell proliferation with an increase of S-phase cells, and reducing the p53-dependent DNA damage response. Overall, our data highlighted the role of H19 and PURPL in the senescent phenotype and suggested that these lncRNAs may have important implications in senescence-related diseases.

Static magnetic field contributes to osteogenic differentiation of hPDLSCs through the H19/Wnt/β-catenin axis

BackgroundStatic magnetic field (SMF) as an effective physical stimulus is capable of osteogenic differentiation for multiple mesenchymal stem cells, including human periodontal ligament stem cells (hPDLSCs). However, the exact molecular mechanism is still unknown. Therefore, this study intends to excavate molecular mechanisms related to SMF in hPDLSCs using functional experiments. MethodshPDLSCs were treated with different intensities of SMF, H19 lentivirus, and Wnt/β-catenin pathway inhibitor (XAV939). Changes in osteogenic markers (Runx2, Col Ⅰ, and BMP2), Wnt/β-catenin markers (β-catenin and GSK-3β), and calcified nodules were examined using RT-qPCR, western blotting, and alizarin red staining in hPDLSCs. ResultsSMF upregulated the expression of H19, and SMF and overexpressing H19 facilitated the expression of osteogenic markers (Runx2, Col Ⅰ, and BMP2), activation of the Wnt/β-catenin pathway, and mineralized sediment in hPDLSCs. Knockdown of H19 alleviated SMF function, and treatment with XAV939 limited SMF- and H19-mediated osteogenic differentiation of hPDLSCs. Notably, the expression of hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p in hPDLSCs was regulated by SMF, and may form an endogenous competitive RNA mechanism with H19 and β-catenin. ConclusionSMF contributes to the osteogenic differentiation of hPDLSCs by mediating the H19/Wnt/β-catenin pathway, and hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p may be the key factors in it.

LncRNA H19 accelerates renal fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis

BackgroundTransforming growth factor-beta1 (TGF-β1)-mediated renal fibrosis is a critical pathological process of chronic kidney disease worsening to end-stage renal disease. Recent studies have shown that long noncoding RNA H19 (lncRNA H19) is widely involved in the formation and progression of fibrosis in multiple organs. However, its molecular events in renal fibrosis remain to be elucidated. MethodsRats were treated with adenine intragastrically and HK-2 cells were induced by TGF-β1 to construct renal fibrosis models in vivo and in vitro, respectively. Renal histopathological examination was performed using HE and Masson staining. Gene expression levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), TGF-β1, fibronectin (Fn), alpha-smooth muscle actin (α-SMA), H19, let-7b-5p, TGF-β receptor 1 (TGF-βR1), and type I collagen (COL1A1) were detected by qRT-PCR. Immunohistochemistry, immunofluorescence, and western blot analysis were used to evaluate the expression of renal fibrosis biomarkers. Dual-luciferase reporter assay was used to verify the presence of binding sites between H19 and let-7b-5p, and between let-7b-5p and TGF-βR1 and COL1A1. ResultsH19 was overexpressed in both in vivo and in vitro renal fibrosis models. H19 knockdown significantly reversed TGF-β1-induced upregulation of fibronectin, COL1A1, and α-SMA and downregulation of E-cadherin in HK-2 cells, accompanied by an increase in let-7b-5p. Let-7b-5p was bound to H19 in HK-2 cells, and its overexpression inhibited TGF-β1-induced HK-2 cell fibrosis. Further experiments determined that let-7b-5p directly targets TGF-βR1 and COL1A1 in HK-2 cells. In addition, inhibition of let-7b-5p reversed the reduction in HK-2 cell fibrosis induced by H19 knockdown. Finally, knockdown of H19 alleviated renal fibrosis in vivo and was associated with regulation of the let-7b-5p/TGF-βR1/COL1A1 axis. ConclusionOur results indicate that knockdown of H19 inhibits renal tubular epithelial fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis, which may provide new mechanistic insights into CRF progression.

Long noncoding RNA H19 knockdown promotes angiogenesis via IMP2 after ischemic stroke.

This study aimed to explore the effects of long noncoding RNA (lncRNA) H19 knockdown on angiogenesis and blood-brain barrier (BBB) integrity following cerebral ischemia/reperfusion (I/R) and elucidate their underlying regulatory mechanisms. A middle cerebral artery occlusion/reperfusion model was used to induce cerebral I/R injury. The cerebral infarct volume and neurological impairment were assessed using 2,3,5-triphenyl-tetrazolium chloride staining and neurobehavioral tests, respectively. Relevant proteins were evaluated using western blotting and immunofluorescence staining. Additionally, a bioinformatics website was used to predict the potential target genes of lncRNA H19. Finally, a rescue experiment was conducted to confirm the potential mechanism. Silencing of H19 significantly decreased the cerebral infarct volume, enhanced the recovery of neurological function, mitigated BBB damage, and stimulated endothelial cell proliferation following ischemic stroke. Insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) is predicted to be a potential target gene for lncRNA H19. H19 knockdown increased IMP2 protein expression and IMP2 inhibition reversed the protective effects of H19 inhibition. Downregulation of H19 enhances angiogenesis and mitigates BBB damage by regulating IMP2, thereby alleviating cerebral I/R injury.

Mechanosensitive lncRNA H19 promotes chondrocyte autophagy, but not pyroptosis, by targeting miR-148a in post-traumatic osteoarthritis

ObjectiveInvestigating whether mechanosensitive lncRNA H19 can directly target miR-148a to alleviate cartilage damage in post-traumatic osteoarthritis (PTOA). MethodsThirty-two female rats were randomly divided into four groups: Sham-operated group (Sham group, n = 8), treadmill running group (R group, n = 8), anterior cruciate ligament transection (ACLT) group (ACLT group, n = 8), and ACLT + treadmill running group (ACLT + R group, n = 8). Histological evaluation was performed to observe the pathological changes in the cartilage of the rat knee. Micro-CT was performed to detect the bone morphological changes in the subchondral bone. RT-qPCR and Western-Blot were performed to detect changes in mRNA and protein levels of metabolic and inflammatory factors as well as changes in the expression of lncRNA H19 and miR-148a in cartilage. The Flexcell 5000™ Tension System was used to further validate that lncRNA H19 has mechanosensitivity in vitro. Finally, cell transfection techniques were used to knock down the expression of lncRNA H19 in chondrocytes to validate the regulatory role of lncRNA H19/miR-148a in cartilage metabolism. ResultsACLT combined with treadmill running aggravated the abnormal hyperplasia of subchondral bone in the lateral tibial plateau of the rat knee joint, disturbed the balance of cartilage metabolism, induced cartilage inflammatory response and chondrocyte pyroptosis, which eventually led to cartilage damage and PTOA. Importantly, we found that the expression of lncRNA H19 was significantly downregulated in the cartilage of the ACLT + R group. Bioinformatics analysis revealed that miR-148a may be a direct target of lncRNA H19. Subsequently, we focused on the mechanosensitive of lncRNA H19. Subsequently, moderate-intensity mechanical tension stress reversed the expression of lncRNA H19 and autophagy-related factors in inflammatory chondrocytes, while miR-148a showed an opposite expression trend, demonstrating that mechanosensitive lncRNA H19 may be involved in regulating the chondrocyte inflammatory response by targeting miR-148a and activating autophagy. Cell transfection experiments revealed that lncRNA H19 knockdown upregulated miR-148a expression and significantly inhibited the autophagy level of chondrocytes without significant alteration of chondrocyte pyroptosis, which in turn exacerbated the inflammatory response of chondrocytes. ConclusionsMechanosensitive lncRNA H19 can promote chondrocyte autophagy rather than pyroptosis by targeting miR-148a, thus alleviating cartilage damage in PTOA. LncRNA H19 may be a potential therapeutic target for PTOA.

LncRNA H19 knockdown promotes neuropathologic and functional recovery via the Nrf2/HO-1 axis after traumatic brain injury.

Traumatic brain injury (TBI) stands as a significant concern in public health, frequently leading to enduring neurological deficits. Long non-coding RNA H19 (lncRNA H19) exerts a potential regulator role in the pathology of brain injury. This study investigates the effects of lncRNA H19 knockdown (H19-KD) on the pathophysiology of TBI and its potential neuroprotective mechanisms. Controlled cortical impact was employed to establish a stable TBI mouse model. The expression levels of various genes in perilesional cortex and striatum tissue after TBI was detected by RT-qPCR. AAV9-shRNA-H19 was injected into the lateral ventricle of mice to knockdown the expression of lncRNA H19. Various behavioral tests were performed to evaluate sensorimotor and cognitive functions after TBI. Immunofluorescence and Nissl staining were performed to assess brain tissue damage and neuroinflammation. The Nrf2 and HO-1 expression was performed by Western blot. After TBI, the expression of lncRNA H19 was elevated in perilesional tissue and gradually reverted to baseline. Behavioral tests demonstrated that H19-KD significantly promoted the recovery of sensorimotor and cognitive functions after TBI. Besides, H19-KD reduced brain tissue loss, preserved neuronal integrity, and ameliorated white matter damage at the histological level. In addition, H19-KD restrained the pro-inflammatory and facilitated anti-inflammatory phenotypes of microglia/macrophages, attenuating the neuroinflammatory response after TBI. Furthermore, H19-KD promoted activation of the Nrf2/HO-1 axis after TBI, while suppression of Nrf2 partially abolished the neuroprotective effect. H19-KD exerts neuroprotective effects after TBI in mice, partially mediated by the activation of the Nrf2/HO-1 axis.

A prominent role of LncRNA H19 in H. pylori CagA induced DNA damage response and cell malignancy

Helicobacter pylori (H. pylori), together with its CagA, has been implicated in causing DNA damage, cell cycle arrest, apoptosis, and the development of gastric cancer. Although lncRNA H19 is abundantly expressed in gastric cancer and functions as a pro-oncogene, it remains unclear whether lncRNA H19 contributes to the oncogenic process of H. pylori CagA. This study investigates the role of H19 in the DNA damage response and malignancy induced by H. pylori. It was observed that cells infected with CagA+H. pylori strain (GZ7/cagA) showed significantly higher H19 expression, resulting in increased γH2A.X and p-ATM expression and decreased p53 and Rad51 expression. Faster cell migration and invasion was also observed, which was reversed by H19 knockdown in H. pylori. YWHAZ was identified as an H19 target protein, and its expression was increased in H19 knockdown cells. GZ7/cagA infection responded to the increased YWHAZ expression induced by H19 knockdown. In addition, H19 knockdown stimulated cells to enter the G2-phase and attenuated the effect of GZ7/cagA infection on the cellular S-phase barrier. The results suggest that H. pylori CagA can upregulate H19 expression, participate in the DNA damage response and promote cell migration and invasion, and possibly affect cell cycle arrest via regulation of YWHAZ.

All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.MethodsMCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student’s t-test or one-way ANOVA was used to analyze data from replicates.ResultsOur results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.ConclusionsTo the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Long non-coding RNA H19 mediates osteogenic differentiation of bone marrow mesenchymal stem cells through the miR-29b-3p/DKK1 axis.

Single immobilization theory cannot fully account for the extensive bone loss observed after spinal cord injury (SCI). Bone marrow mesenchymal stem cells (BMSCs) are crucial in bone homeostasis because they possess self-renewal capabilities and various types of differentiation potential. This study aimed to explore the molecular mechanism of long non-coding RNA H19 in osteoporosis after SCI and provide new research directions for existing prevention strategies. We used small interfering RNA to knockdown H19 expression and regulated miR-29b-2p expression using miR-29b-3p mimetics and inhibitors. Western blotting, real-time fluorescence quantitative PCR, Alizarin red staining, alkaline phosphatase staining and double-luciferase reporter gene assays were used to assess gene expression, osteogenic ability and binding sites. lncRNA H19 was upregulated in BMSCs from the osteoporosis group, whereas miR-29b-3p was downregulated. We identified the binding sites between miR-29b-3p and lncRNAs H19 and DKK1. H19 knockdown promoted BMSCs' osteogenic differentiation, whereas miR-29b-3p inhibition attenuated this effect. We discovered potential binding sites for miR-29b-3p in lncRNAs H19 and DKK1. Our findings suggest that long non-coding RNA H19 mediates BMSCs' osteogenic differentiation in osteoporosis after SCI through the miR-29b-3p/DKK1 axis and by directly inhibiting the β-catenin signalling pathway.

Knockdown H19 Accelerated iPSCs Reprogramming through Epigenetic Modifications and Mesenchymal-to-Epithelial Transition.

H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.

Exercise promotes osteogenic differentiation by activating the long non-coding RNA H19/microRNA-149 axis

BACKGROUND Regular physical activity during childhood and adolescence is beneficial to bone development, as evidenced by the ability to increase bone density and peak bone mass by promoting bone formation. AIM To investigate the effects of exercise on bone formation in growing mice and to investigate the underlying mechanisms. METHODS 20 growing mice were randomly divided into two groups: Con group (control group, n = 10) and Ex group (treadmill exercise group, n = 10). Hematoxylin-eosin staining, immunohistochemistry, and micro-CT scanning were used to assess the bone formation-related indexes of the mouse femur. Bioinformatics analysis was used to find potential miRNAs targets of long non-coding RNA H19 (lncRNA H19). RT-qPCR and Western Blot were used to confirm potential miRNA target genes of lncRNA H19 and the role of lncRNA H19 in promoting osteogenic differentiation. RESULTS Compared with the Con group, the expression of bone morphogenetic protein 2 was also significantly increased. The micro-CT results showed that 8 wk moderate-intensity treadmill exercise significantly increased bone mineral density, bone volume fraction, and the number of trabeculae, and decreased trabecular segregation in the femur of mice. Inhibition of lncRNA H19 significantly upregulated the expression of miR-149 and suppressed the expression of markers of osteogenic differentiation. In addition, knockdown of lncRNA H19 significantly downregulated the expression of autophagy markers, which is consistent with the results of autophagy-related protein changes detected in mouse femurs by immunofluorescence. CONCLUSION Appropriate treadmill exercise can effectively stimulate bone formation and promote the increase of bone density and bone volume in growing mice, thus enhancing the peak bone mass of mice. The lncRNA H19/miR-149 axis plays an important regulatory role in osteogenic differentiation.

Hypomethylation of the LncRNA H19 promoter accelerates osteogenic differentiation of vascular smooth muscle cells by activating the Erk1/2 pathways.

Vascular calcification is a common chronic kidney disease complication. This study aimed to investigate the function of long non-coding RNA (LncRNA) H19 in vascular calcification to explore new therapeutic strategies. We induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) using β-glycerophosphate. Then, we detected the LncRNA H19 promoter methylation status and Erk1/2 pathways using methylation-specific polymerase chain reaction and western blotting, respectively. Compared with the control group, high phosphorus levels induced VSMC calcification, accompanied by increases in LncRNA H19 and the osteogenic marker Runx2 and reduction of the contractile phenotype marker SM22a. LncRNA H19 knockdown inhibited osteogenic differentiation and calcification of VSMCs. However, the suppressed role of VSMC calcification caused by shRNA H19 was partially reversed by simultaneous activation of the Erk1/2 pathways. Mechanically, we found that the methylation rate of CpG islands in the LncRNA H19 promoter region was significantly lower in the high-phosphorus group, and the hypomethylation state elevated LncRNA H19 levels, which in turn regulated phosphorylated Erk1/2 expression. LncRNA H19 promoted osteogenic differentiation and calcification of VSMCs by regulating the Erk1/2 pathways. Additionally, hypomethylation of LncRNA H19 promoter CpG islands upregulated LncRNA H19 levels and subsequently activated Erk1/2 phosphorylation.

LncRNA H19 Participates in Leukemia Inhibitory Factor Mediated Stemness Promotion in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy and the third leading cause of cancer-related death worldwide. Cancer stem cells (CSCs) were considered to play important roles in the genesis and development of many tumors. In recent years, it has been observed that leukemia inhibitory factor (LIF) might be involved in the regulation of stemness in cancer cells. In this study, we observed that LIF could increase the spheroid formation and stemness marker expression (inculding Nanog and SOX2) in CRC cell lines, such as HCT116 and Caco2 cells. Meanwhile, we also observed that LIF could upregulate LncRNA H19 expression via PI3K/AKT pathway. Knockdown of the expression of LncRNA H19 could decrease the spheroid formation and SOX2 expression in LIF-treated HCT116 and Caco2 cells, and thereby LncRNA H19 knockdown could compensate for the stemness enhancement effects induced by LIF. Our results indicated that LncRNA H19 might participate in the stemness promotion of LIF in CRC cells.

LncRNA H19 Promotes Gastric Cancer Metastasis via miR-148-3p/SOX-12 Axis.

Gastric cancer (GC) is the most common malignant tumor and ranks third in the world. LncRNA H19 (H19), one of the members of lncRNA, is overexpressed in various tumors. However, many undetermined molecular mechanisms by which H19 promotes GC progression still need to be further investigated. Methodology. A series of experiments was used to confirm the undetermined molecular mechanism including wound healing and transwell assays. Key Results. In this study, a significant upregulation of H19 expression was detected in GC cells and tissues. The poor overall survival was observed in GC patient with high H19 expression. Overexpression of H19 promoted the migration of GC cells, while knockdown of H19 significantly inhibited cell migration. Moreover, miR-148a-3p had a certain negative correlation with H19. Luciferase reporter assay confirmed that H19 could directly bind to miR-148a-3p. As expected, miR-148a mimics inhibited cell migration and invasion induced by H19 overexpression. The above findings proved that H19 functions as a miRNA sponge and verified that miR-148a-3p is the H19-associated miRNA in GC. We also confirmed that SOX-12 expression was upregulated in GC patient's samples. SOX-12 expression was positively correlated with expression of H19 and was able to directly bind to miR-148a-3p. Importantly, in vitro wound healing assay showed that knockout of SOX-12 could reverse the promoting effect of H19 overexpression on cell migration. In conclusion, H19 has certain application value in the diagnosis and prognosis of GC. Specifically, H19 accelerates GCs to migration and metastasis by miR-138a-3p/SOX-12 axis.

Aucubin Promotes Osteogenic Differentiation and Facilitates Bone Formation through the lncRNA-H19 Driven Wnt/β-Catenin Signaling Regulatory Axis.

Traditional Chinese medicine Cortex Eucommiae has been used to treat bone fracture for hundreds of years, which exerts a significant improvement in fracture healing. Aucubin, a derivative isolated from Cortex Eucommiae, has been demonstrated to possess anti-inflammatory, immunoregulatory, and antioxidative potential. In the present study, our aim was to explore its function in bone regeneration and elucidate the underlying mechanism. The effects of Aucubin on osteoblast and osteoclast were examined in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) and RAW 264.7 cells, respectively. Moreover, the lncRNA H19 and Wnt/β-catenin signaling were detected by qPCR examination, western blotting, and luciferase activity assays. Using the femur fracture mice model, the in vivo effect of Aucubin on bone formation was monitored by X-ray, micro-CT, histomorphometry, and immunohistochemistry staining. In the present study, Aucubin was found to significantly promote osteogenic differentiation in vitro and stimulated bone formation in vivo. Regarding to the underlying mechanism, H19 was found to be obviously upregulated by Aucubin in MSCs and thus induced the activation of Wnt/β-catenin signaling. Moreover, H19 knockdown partially reversed the Aucubin-induced osteogenic differentiation and successfully suppressed the activation of Wnt/β-catenin signaling. We therefore suggested that Aucubin induced the activation of Wnt/β-catenin signaling through promoting H19 expression. Our results demonstrated that Aucubin promoted osteogenesis in vitro and facilitated fracture healing in vivo through the H19-Wnt/β-catenin regulatory axis.

Long noncoding RNA H19 is elevated in aortic valve stenosis and governs antigenic response and valvular endothelial cell phenotypes through endothelial to mesenchymal transition

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as important regulators of cardiovascular disease, including aortic valve stenosis (AVS). Endothelial to mesenchymal transition (EndMT) is a transdifferentiation process that is associated with the development of AVS in the elderly population. Although several recent studies have reported that lncRNAs are dysregulated and might play a crucial role in EndMT, no specific role of lncRNA was reported in AVS pathogenesis. Here, we sought to investigate the role of H19 in EndMT during the pathogenesis of AVS. Methods and results High-throughput lncRNA sequencing of human aortic valve tissue explanted from patients with AVS due to severe stenosis during surgical aortic valve replacement (SAVR) demonstrated that certain lncRNAs [H19, AGAP2-AS1 (PUNISHER), GAS5, MALAT1] are significantly dysregulated compared to patients without AVS. To validate the RNA-seq data, 60 patients with AVS (n=30) or without AVS (n=30) were prospectively investigated. Among these, H19 (p=0.002), AGAP2-AS1 (p=0.002), and GAS5 (0.02) were significantly upregulated in patients with AVS, compared to non-AVS patients. Aortic valvular endothelial cells (VECs) were isolated from explanted valve tissue from AVS patients or obtained commercially. In vitro EndMT-promoting stimuli (TNFα or TGFß+IL1ß) were used for VEC stimulation for 5 days. RT-qPCR was used to quantify the expression of EndMT markers (EC markers, vWF, and NOS3, and mesenchymal markers, αSMA, SM22, Vimentin, and SNAI2). Loss of H19 led to dysregulation of the EC gene network and protein expression patterns that were analyzed by qRT-PCR, immunoblotting, and immunofluorescence, respectively. Further experiments show that silencing of H19 affects migration and proliferation, and promotes EndMT in EC. Functional assays suggested that H19 is a critical regulator of angiogenic properties. The molecular signature of angiogenesis was investigated using a RT-qPCR-based angiogenesis array to identify the regulation of angiogenesis with 84-angiogenesis-related genes. Mechanistically, the angiogenesis array revealed that H19 regulates Jagged Canonical Notch Ligand 1 (JAG1) (4.43-fold vs. control EC), an important regulator of NOTCH1 signaling, and pro-angiogenic factor in EC. Accordingly, EndMT marker expressions were dysregulated upon H19 depletion. A high-throughput luciferase reporter array demonstrated STAT3 activation upon H19 knockdown. Depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on EndMT marker expression as well as on angiogenesis. Overexpression of H19 in VEC abrogated the H19-mediated promotion of JAG1- and VEGFA gene- and protein expression.