Extracellular vesicles released by hypoxia-induced tumor-associated fibroblasts impart chemoresistance to breast cancer cells via long noncoding RNA H19 delivery.

Recently, extracellular vesicles (EVs) have been emphasized in regulating the hypoxic tumor microenvironment of breast cancer (BC), where tumor-associated fibroblasts (TAFs) play a significant role. In this study, we describe possible molecular mechanisms behind the pro-tumoral effects of EVs, secreted by hypoxia (HP)-induced TAFs, on BC cell growth, metastasis, and chemoresistance. These mechanisms are based on long noncoding RNA H19 (H19) identified by microarray analysis. We employed an in silico approach to identify differentially expressed lncRNAs that were associated with BC. Subsequently, we explored possible downstream regulatory mechanisms. We isolated EVs from TAFs that were exposed to HP, and these EVs were denoted as HP-TAF-EVs henceforth. MTT, transwell, flow cytometry, and TUNEL assays were performed to assess the malignant phenotypes of BC cells. A paclitaxel (TAX)-resistant BC cell line was constructed, and xenograft tumor and lung metastasis models were established in nude mice for in vivo verification. Our observation revealed that lncRNA H19 was significantly overexpressed, whereas miR-497 was notably downregulated in BC. HP induced activation of TAFs and stimulated the secretion of EVs. Coculture of HP-TAF-EVs and BC cells led to an increase in TAX resistance of the latter. HP-TAF-EVs upregulated methylation of miR-497 by delivering lncRNA H19, which recruited DNMT1, thus lowering the expression of miR-497. In addition, lncRNA H19-containing HP-TAF-EVs hindered miR-497 expression, enhancing tumorigenesis and TAX resistance of BC cells in vivo. Our study presents evidence for the contribution of lncRNA H19-containing HP-TAF-EVs in the reduction of miR-497 expression through the recruitment of DNMT1, which in turn promotes the growth, metastasis, and chemoresistance of BC cells.

Long noncoding RNA H19 acts as a miR-29b sponge to promote wound healing in diabetic foot ulcer.

Aberrant expression of long noncoding RNA (lncRNA) H19 and microRNA (miR)-29b has been implicated in the complications of diabetes mellitus (DM). As a common and important complication of DM, diabetic foot ulcer (DFU) is characterized by high incidence and poor prognosis. Herein, we explored the role of lncRNA H19 in wound healing of DFU. Differentially expressed DM-related lncRNAs were initially screened by microarray data analysis. DFU models were then induced in DM mouse models. The functional role and interaction of lncRNA H19, miR-29b and FBN1 in DFU were subsequently determined by examining the proliferation, migration, and apoptosis of fibroblasts after silencing H19, inhibiting or overexpressing miR-29b and FBN1. According to microarray-based analysis, lncRNA H19 was upregulated in DM. In the ulcerative edge tissues of DFU, high expression of lncRNA H19 and FBN1 and low expression of miR-29b were observed. FBN1 was identified to be a target gene of miR-29b. LncRNA H19 could competitively bind to miR-29b, and then, inhibited its expression, which consequently upregulating FBN1. Silencing of lncRNA H19 led to inhibited proliferation, migration, and enhanced apoptosis of fibroblasts, accompanied by downregulated FBN1 but upregulated miR-29b, which diminished the expression of TGF-ß1, Smad3, FN, and Col-1 and reduced extracellular matrix accumulation. Altogether, upregulation of lncRNA H19 can elevate the expression of FBN1 through competitively binding to miR-29b, which enhances the proliferation, migration, and inhibits apoptosis of fibroblasts, thus facilitating the wound healing of DFU.

Effects of long noncoding RNA H19 on cementoblast differentiation, mineralisation, and proliferation.

OBJECTIVE: Cementum which is a layer of thin and bone-like mineralised tissue covering tooth root surface is deposited and mineralised by cementoblasts. Recent studies suggested long noncoding RNA H19 (H19) promotes osteoblast differentiation and matrix mineralisation, however, the effect of H19 on cementoblasts remains unknown. This study aimed to clarify the regulatory effects of H19 on cementoblast differentiation, mineralisation, and proliferation. MATERIAL AND METHODS: An immortalised murine cementoblast cell line OCCM-30 was used in this study. H19 expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) during OCCM-30 cell differentiation. OCCM-30 cells were transfected with lentivirus or siRNA to up-regulate or down-regulate H19, then the levels of runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), osteocalcin (Bglap) were tested by RT-qPCR or western blot. Alizarin red staining, ALP activity assay and MTS assay were performed to determine the mineralisation and proliferation ability of OCCM-30 cells. RESULTS: H19 was dramatically increased during OCCM-30 cell differentiation. Overexpression of H19 increased the levels of Runx2, Sp7, Alpl, Ibsp, and Bglap and enhanced ALP activity and the formation of mineral nodules. While down-regulation of H19 suppressed the above cementoblast differentiation genes and inhibited ALP activity and mineral nodule formation. However, the proliferation of OCCM-30 cells was not affected. CONCLUSIONS: H19 promotes the differentiation and mineralisation of cementoblasts without affecting cell proliferation.

Stromal cell-derived factor-1 promotes osteoblastic differentiation of human bone marrow mesenchymal stem cells via the lncRNA-H19/miR-214-5p/BMP2 axis.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1) plays an important role in the osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs), but the specific mechanism remains unclear. Our study aimed to clarify the role of the lncRNA-H19/miR-214-5p/BMP2 axis in the osteoblastic differentiation of hBMMSCs induced by SDF-1. METHODS: We used reverse-transcriptase polymerase chain reaction, western blotting, alkaline phosphatase activity test, and Alizarin red staining to evaluate the osteoblastic differentiation of primary hBMMSCs and the luciferase reporter assay to determine if lncRNA-H19 binds with miR-214-5p. RESULTS: Our results indicated that SDF-1 (50 ng/mL) promotes the osteoblastic differentiation of hBMMSCs, significantly upregulates osteoblastogenic genes (OCN, OSX, RUNX2, and ALP), and increases Alizarin red staining, alkaline phosphatase activity, and lncRNA-H19 expression. Luciferase reporter assay verified that lncRNA-H19 binds with and represses miR-214-5p, thereby upregulating BMP2 expression. Use of miR-214-5p inhibitor or overexpression of lncRNA-H19 can promote the osteoblastic differentiation of hBMMSCs, but miR-214-5p or shH19 inhibits the osteoblastic differentiation of hBMMSCs. Treatment with an miR-214-5p inhibitor could rescue the inhibitory effect of shH19 on the osteoblastic differentiation of hBMMSCs. CONCLUSIONS: Taken together, SDF-1 promotes the osteoblastic differentiation of hBMMSCs through the lncRNA-H19/miR-214-5p/BMP2 axis. Increased osteoblastic differentiation by an miR-214-5p inhibitor reveals a new possible strategy for the treatment of bone defect and osteoporosis.

Long noncoding RNA H19 promotes the apoptosis of corpus cavernsum smooth muscle cells after cavernosal nerve injury via JNK signalling pathway.

JNK/ Bcl-2/ Bax pathway participates in corpus cavernosal smooth muscle cells apoptosis during early period after cavernosal nerve (CN) crush injury (CNCI). Nevertheless, the regulation mechanisms of long noncoding RNA H19 in apoptosis during early stage after CN injury are still poorly understood. The rats in sham group were not direct injury to the CNs. The rats in CNCI group were performed to bilateral CN crush injury. The ICP/MAP rate and smooth muscle content were significantly lower than that in the sham group. Primary CCSMCs were prepared from the tissues samples after completing erectile function detection. Phosphorylated-JNK level was increased significantly, and the expression of Bax and Bcl-2 was elevated and declined in CNCI group respectively. Except for Bcl-2, the mRNA levels of H19, JNK and Bax were significantly increased in CNCI group. After H19 siRNA transfection, for the mRNA and protein levels, JNK and Bax were declined, while Bcl-2 was enhanced. LncRNA H19 might be involved in regulation of Bcl-2, Bax via JNK signalling pathway in CCSMCs apoptosis after CN injury.

Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus.

The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.

LncRNA H19/miR-194/PFTK1 axis modulates the cell proliferation and migration of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a huge challenge due to its high mortality and morbidity; gene therapy might be a promising treatment for PDAC. The critical role of Wnt-signaling pathway in cancer pathogenesis has been widely recognized; cyclin-dependent kinase 14 (CDK14, PFTK1)-induced low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) phosphorylation is an important issue in Wnt-signaling activation. Long noncoding RNA (LncRNA)-microRNA (miRNA)-messenger RNA (mRNA) modulating the pathogenesis of cancers has been regarded as a major mechanism. In the current study, upregulated lncRNAs positively correlated with PFTK1 were analyzed and selected using The Cancer Genome Atlas (TCGA) database. Of them, lncRNA H19 can activate Wnt signaling in cancers. In PDAC tissues, the expression of H19 and PFTK1 were upregulated; H19 knockdown suppressed the cell proliferation and migration of PDAC, while PFTK1 overexpression partially attenuated the suppressive effect of H19 knockdown. As analyzed by TCGA and predicted by online tools, miR-194 was negatively correlated with PFTK1 and might bind to both H19 and PFTK1, which was further confirmed by luciferase reporter and RNA immunoprecipitation assays. Moreover, the effect of H19 knockdown on PFTK1 protein and the cell proliferation and migration could be partially reversed by miR-194 inhibition; H19/miR-194 axis modulated PDAC cell proliferation and migration through PFTK1 downstream Wnt signaling. Results suggested that rescuing miR-194 expression in PDAC can inhibit lncRNA H19 and PFTK1 expression, subsequently suppressing PDAC cell proliferation and migration. Due to the complexity of the lncRNA-miRNA-mRNA network, further in vivo experiments examining potential side effects are needed in future study to explore the clinical application of these findings.

LncRNA PTCSC3 inhibits triple-negative breast cancer cell proliferation by downregulating lncRNA H19.

Long noncoding RNA (lncRNA) PTCSC3 (hereafter PTCSC3 is used to represent lncRNA PTCSC3) inhibits glioma and thyroid cancer, indicating its potential tumor suppression function in other types of cancers. We explored the potential involvement of PTCSC3 in triple-negative breast cancer (TNBC). In the current study, we found that PTCSC3 was downregulated in tumor tissues of patients with TNBC. PTCSC3 expression was positively correlated with plasma levels of PTCSC3. LncRNA H19 was upregulated and was inversely correlated with PTCSC3 in tumor tissues. PTCSC3 overexpression led to downregulated H19 in TNBC cells, while H19 overexpression did not affect PTCSC3 expression. PTCSC3 inhibited and H19 promoted proliferation of TNBC cells. H19 overexpression attenuated the effects of PTCSC3 overexpression. Cancer cell migration and invasion were not significantly affected by PTCSC3 overexpression. Therefore, lncRNA PTCSC3 inhibits TNBC cell proliferation by downregulating lncRNA H19.

Association of long noncoding RNA H19 polymorphisms with the susceptibility and clinical features of ischemic stroke in southern Chinese Han population.

Stroke is the leading cause of death in China. Previous studies have demonstrated that long noncoding RNAs play important roles in ischemic stroke (IS). This study aimed to investigate long noncoding RNA H19 (lncRNA H19) expression in IS cases and the association between lncRNA H19 variants and IS risk and IS-related risk factors. A total of 550 IS cases and 550 controls were recruited for this study. LncRNA H19 expression was detected using quantitative real-time polymerase chain reaction. Genotyping was conducted by the Sequenom MassARRAY technology. LncRNA H19 level in peripheral blood of IS cases was significantly upregulated compared with healthy controls (P = 0.046). No significant association was observed between lncRNA H19 rs217727 and rs4929984 polymorphisms with IS risk in all genetic models, and rs217727-rs4929984 haplotypes are not associated with IS susceptibility. Further meta-analysis also implied that the rs217727 and rs4929984 polymorphisms were not associated with IS in Chinese population. However, rs4929984 is significantly associated with the diastolic blood pressure level of IS patients (additive model: Padj = 0.007; dominant model: Padj = 0.013), whereas rs217727 is associated with international normalized ratio (additive model: Padj = 0.019; recessive model: Padj = 0.004), prothrombin time activity level (additive model: Padj = 0.026; recessive model: Padj = 0.004), and homocysteine level (recessive model: Padj = 0.048) in patients with IS. Our findings suggest that lncRNA H19 level may affect the occurrence of IS, and lncRNA H19 variants may influence blood pressure, coagulation function, and homocysteine metabolism of patients with IS in the southern Chinese Han population.

Effects of long non-coding RNA H19 and microRNA let7a expression on thyroid cancer prognosis.

This study aims to explore the effects of long non-coding RNA H19 (lncRNA H19) and microRNA let7a (miRNA let7a) expression on the prognosis of thyroid cancer (TC). This may aid in the discovery of more effective treatment and prognosis approaches for TC. Between January 2008 and January 2011, 131 TC tissues and adjacent tissues were obtained from TC patients. An additional 122 normal thyroid tissues were also collected as normal controls from patients with benign thyroid lesions. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect lncRNA H19 and miRNA let7a mRNA expression. Five-year follow-ups were conducted. A Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic values of lncRNA H19 and miRNA let7a in TC. The Kaplan-Meier method was applied to analyze the 5-year survival rate of TC patients. Univariate and multivariate factor analyses were employed to analyze the prognostic factors of TC. The lncRNA H19 mRNA expression was higher while the miRNA let7a mRNA expression was lower in TC tissues than, in the normal thyroid tissues and adjacent tissues. The area under the ROC curve (AUC) of lncRNA H19 and miRNA let7a were 0.801 and 0.116, with sensitivity at 72.5% and 84%, as well as specificity 75.4% and 77%, respectively. In TC patients with tumor diameters≥1.0cm, lncRNA H19 mRNA expression was elevated, but miRNA let7a mRNA expression was reduced. This was also evident in TC patients with TNM stages III+IV and those with lymph node metastasis. TC patients with a lower 5-year survival rate showed upregulated levels of lncRNA H19 expression and, downregulated levels of miRNA let7a expression. LncRNA H19 and miRNA let7a expression, tumor diameter, TNM stage and lymph node metastasis were independent prognostic factors of TC. This study demonstrated that increased lncRNA H19 and decreased miRNA let7a expression levels are associated with poor prognosis in TC patients. An inverse relationship between lncRNA H19 and miRNA let7a expression levels was exhibited.

Mesenchymal stem cells-derived extracellular vesicle-incorporated H19 attenuates cardiac remodeling in rats with heart failure.

Cardiac remodeling is manifested by hypertrophy and apoptosis of cardiomyocytes, resulting in the progression of cardiovascular diseases. Long noncoding RNAs (lncRNAs) serve as modifiers of cardiac remodeling. In this study, we aimed to explore the molecular mechanism of H19 shuttled by mesenchymal stem cells (MSC)-derived extracellular vesicles (EV) in cardiac remodeling upon heart failure (HF). Using the GEO database, H19, microRNA (miR)-29b-3p, and CDC42 were screened out as differentially expressed biomolecules in HF. H19 and CDC42 were elevated, and miR-29b-3p was decreased after MSC-EV treatment in rats subjected to ligation of the coronary artery. MSC-EV alleviated myocardial injury in rats with HF. H19 downregulation exacerbated myocardial injury, while miR-29b-3p inhibitor alleviated myocardial injury. By contrast, CDC42 downregulation aggravated the myocardial injury again. PI3K/AKT pathway was activated by MSC-EV. These findings provide insights into how H19 shuttled by EV mitigates cardiac remodeling through a competitive endogenous RNA network regarding miR-29b-3p and CDC42.

[Long noncoding RNA H19 promotes vascular calcification by repressing the Bax inhibitor 1/optic atrophy 1 pathway].

OBJECTIVE: To investigate whether long noncoding RNA H19 (lncRNA H19) induces vascular calcification by promoting calcium deposition, osteogenic differentiation and apoptosis via inhibiting the Bax inhibitor 1/optic atrophy 1 (BI-1/ OPA1) pathway. METHODS: ß-glycerophosphate and calcium chloride were used to induce calcification in rat vascular smooth muscle cells (VSMCs), and the effects of siH19, alone or in combination with BI-1 or OPA1 knockdown, on calcification of the cells were investigated. Osteogenic differentiation was assessed by measuring Runt-related transcription factor 2 (Runx-2) and bone morphogenetic protein 2 (BMP-2) expression with Western blotting, and cell apoptosis was evaluated by TUNEL staining and Western blotting. An ApoE-/- diabetic mouse model with high-fat feeding for 32 weeks were given an intraperitoneal injection of siH19, and the changes in calcium deposition in the aortic arch were examined using Alizarin red S staining and von Kossa staining. RESULTS: In rat VSMCs with calcification, the expression of lncRNA H19 was significantly increased, and the expressions of BI- 1 and OPA1 were significantly decreased. Downregulation of lncRNA H19 significantly increased the expressions of BI-1 and OPA1 proteins in the cells, and BI-1 knockdown further reduced OPA1 expression (P<0.001). The cells treated with siH19 showed total disappearance of the calcified nodules with significantly reduced expressions of Runx-2, BMP-2 and cleaved caspase-3 and a lowered cell apoptosis rate (P<0.001). Calcified nodules were again observed in the cells with lncRNA H19 knockdown combined with BI-1 or OPA1 knockdown, and the expressions of Runx-2, BMP-2, cleaved-caspase-3 and cell apoptosis rate all significantly increased (P<0.001). In the diabetic mouse model with high-fat feeding, siH19 treatment significantly reduced the calcification area and increased mRNA expressions of BI-I and OPA1 in the aortic arch. CONCLUSION: LncRNA H19 promotes vascular calcification possibly by promoting calcium deposition, osteogenic differentiation and cell apoptosis via inhibiting the BI-1/OPA1 pathway.

The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells.

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

[Effect of moxibustion on autophagy lysosome function mediated by mTOR/TFEB pathway and lncRNA H19 expression in APP/PS1 double transgenic mice].

OBJECTIVE: To observe the effect of moxibustion (Moxi) at acupoints of Governor Vessel on autophagy lysosomal function and lncRNA H19 in amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic Alzheimer's disease (AD) mice, so as to explore its underlying mechanisms in relieving AD. METHODS: Fifty two male APP/PS1 double transgenic AD mice were randomly divided into model, Moxi, Moxi+inhibitor and medication (rapamycin) groups, with 13 mice in each group. Other 13 male C57BL/6J mice of the same age were selected as the control group. The mice of the Moxi group received aconite cake-separated Moxi stimulation at "Baihui" (GV20), "Dazhui"(GV14) and "Fengfu" (GV16), for 15 min, those of the Moxi+inhibitor group received intraperitoneal injection of 3-methyladenine (an inhibitor of PI3K for suppressing autophagy) 1.5 mg· kg-1 · d-1 on the basis of Moxi, and those of the medication group received intraperitoneal injection of rapamycin 2 mg· kg-1 · d-1. The treatment was conducted once daily for 2 weeks. The mouse's learning-memory ability was detected by Morris water maze tests. The hippocampus tissue was sampled for observing the formation of autophagy by using transmission electron microscope, detecting the expression of Aß_(1-42) protein with immunohistochemical staining, and for detecting the expression levels of long noncoding RNA H19 (lncRNA H19), mammalian target of rapamycin kinase (mTOR), nuclear transcription factor EB (TFEB), Cathepsin D and lysosome associated membrane protein-1 (LAMP1) genes and proteins as well as microtubule associated protein 1 light chain 3B (LC3B)-Ⅱ/LC3B-Ⅰand autophagy protein p62 protein by quantitative real-time PCR and Western blot, respectively. RESULTS: In contrast to the control group, the model group had an evident increase in the escape latency of Morris water maze test, and in the expression levels of Aß_(1-42) protein, lncRNA H19 mRNA, mTOR mRNA and protein, and p62 protein (P<0.05), and a significant decrease in the expression levels of TFEB, Cathepsin D, LAMP1 mRNAs and proteins and LC3B-Ⅱ/LC3B-Ⅰ (P<0.05). After the treatment and relevant to the model and Moxi+inhibitor groups, both the Moxi and medication groups had an obvious down-regulation in the levels of latency of Morris water maze, expression levels of Aß_(1-42) protein, lncRNA H19 mRNA, mTOR mRNA and protein, and p62 protein (P<0.05), and a significant up-regulation in the levels of TFEB, Cathepsin D, LAMP1 mRNAs and proteins and LC3B-Ⅱ/LC3B-Ⅰ (P<0.05). CONCLUSION: Moxi at acupoints of Governor Vessel can improve cognitive function of AD mice, which may be associated with its functions in inhibiting mTOR/TFEB pathway by down-regulating the expression of lncRNA H19, improving autophagy lysosomal function, promoting autophagy and clearing away Aß1-42 in the hippocampus.

The Long Noncoding RNA-H19 Mediates the Progression of Fibrosis from Acute Kidney Injury to Chronic Kidney Disease by Regulating the miR-196a/Wnt/ß-Catenin Signaling.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been reported to be involved in the occurrence and development of various diseases. This study was to investigate the role of lncRNA-H19 in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) and its underlying mechanism. METHODS: Bilateral renal pedicle ischemia-reperfusion injury (IRI) was used to establish the IRI-AKI model in C57BL/6 mice. The expression levels of lncRNA-H19, miR-196a-5p, α-SMA, collagen I, Wnt1, and ß-catenin in mouse kidney tissues and fibroblasts were determined by quantitative real-time PCR and Western blotting. The degree of renal fibrosis was evaluated by hematoxylin and eosin staining. The interaction between lncRNA-H19 and miR-196a-5p was verified by bioinformatics analysis and luciferase reporter assay. Immunohistochemistry and immunofluorescence were used to evaluate the expression of α-SMA and collagen I in kidney tissues and fibroblasts of mice. RESULTS: lncRNA-H19 is upregulated, and miR-196a-5p is downregulated in kidney tissues of IRI mice. Moreover, miR-196a-5p is a direct target of lncRNA-H19. lncRNA-H19 overexpression promotes kidney fibrosis and activates fibroblasts during AKI-CKD development, while miR-196a-5p overexpression reversed these effects in vitro. Furthermore, lncRNA-H19 overexpression significantly upregulates Wnt1 and ß-catenin expression in kidney tissues and fibroblasts of IRI mice, while miR-196a-5p overexpression downregulates Wnt1 and ß-catenin expression in kidney tissues and fibroblasts of IRI mice. CONCLUSION: lncRNA-H19 induces kidney fibrosis during AKI-CKD by regulating the miR-196a-5p/Wnt/ß-catenin signaling pathway.

The Potential Utility of Circulating Oncofetal H19 Derived miR-675 Expression versus Tissue lncRNA-H19 Expression in Diagnosis and Prognosis of HCC in Egyptian Patients.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Interestingly, lncRNA-H19 acts independently in HCC and influences miR-675 expressions. We aimed to assess the potential utility of tissue lncRNA-H19 versus miR-675 expressions as a non-invasive biomarker for HCC diagnosis and prognosis in Egyptian patients. Ninety-one HCC patients and 91 controls included in this study were investigated for expression of lncRNA-H19 and miR675 using RT-qPCR. Our results showed that the expression of lncRNA-H19 and microRNA-675 were higher in patients than in controls (p < 0.001 for both). Additionally, lncRNA-H19 expression was higher in tumorous than in non-tumorous tissue (p < 0.001). Linear regression revealed that miR-675 expression was a significantly higher positive predictor than lncRNA-H19 for tumor size, pathologic grade, and AFP level; similarly, for cyclin D1 and VEGF protein expression. By using the ROC curve, the sensitivity of miR-675 was higher than lncRNA-H19 for discriminating HCC from controls (95-89%, respectively) and the sensitivity of lncRNA-H19 was higher in tumorous than in non-tumorous tissues (76%). The high expressions of both were associated with low OS (p < 0.001, 0.001, respectively). Oncofetal H19-derived miR-675 expression could be considered a potential noninvasive diagnostic and prognostic biomarker, outstanding the performance of the expression of tissue lncRNA-H19 for HCC.

Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor.

AIMS: Osteoarthritis (OA) is a common degenerative joint disease characterized by chronic inflammatory articular cartilage degradation. Long noncoding RNAs (lncRNAs) have been previously indicated to play an important role in inflammation-related diseases. Herein, the current study set out to explore the involvement of lncRNA H19 in OA. METHODS: Firstly, OA mouse models and interleukin (IL)-1ß-induced mouse chondrocytes were established. Expression patterns of IL-38 were determined in the synovial fluid and cartilage tissues from OA patients. Furthermore, the targeting relationship between lncRNA H19, tumour protein p53 (TP53), and IL-38 was determined by means of dual-luciferase reporter gene, chromatin immunoprecipitation, and RNA immunoprecipitation assays. Subsequent to gain- and loss-of-function assays, the levels of cartilage damage and proinflammatory factors were further detected using safranin O-fast green staining and enzyme-linked immunosorbent assay (ELISA) in vivo, respectively, while chondrocyte apoptosis was measured using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) in vitro. RESULTS: IL-38 was highly expressed in lentivirus vector-mediated OA mice. Meanwhile, injection of exogenous IL-38 to OA mice alleviated the cartilage damage, and reduced the levels of proinflammatory factors and chondrocyte apoptosis. TP53 was responsible for lncRNA H19-mediated upregulation of IL-38. Furthermore, it was found that the anti-inflammatory effects of IL-38 were achieved by its binding with the IL-36 receptor (IL-36R). Overexpression of H19 reduced the expression of inflammatory factors and chondrocyte apoptosis, which was abrogated by knockdown of IL-38 or TP53. CONCLUSION: Collectively, our findings evidenced that upregulation of lncRNA H19 attenuates inflammation and ameliorates cartilage damage and chondrocyte apoptosis in OA by upregulating TP53, IL-38, and by activating IL-36R.Cite this article: Bone Joint Res 2022;11(8):594-607.

LncRNA H19 activates cell pyroptosis via the miR-22-3p/NLRP3 axis in pneumonia.

OBJECTIVE: Pneumonia is an infectious pulmonary disease with a high morbidity and mortality. It has been reported that multiple long noncoding RNAs (LncRNAs) are involved in the progression of pneumonia, such as LncRNA SNHG16. However, the role and underlying mechanism of LncRNA H19 in the pyroptosis of pneumonia has not been elucidated. The purpose of this research was to explore the mechanism by which LncRNA H19 regulates LPS-induced pneumonia in WI-38 cells. METHODS: An LPS induced pneumonia model in WI-38 cells was established. Total RNA extracted from WI-38 cells was analyzed using RT-qPCR, and the total proteins isolated from the WI-38 cells were analyzed using Western blotting. MTT assays, TUNEL staining, bioinformatics, and luciferase reporter assays were subsequently conducted. RESULTS: In the LPS induced pneumonia model, LncRNA H19 silences inhibited LPS-induced WL-38 cell pyroptosis, and LncRNA H19 overexpression promotes LPS-induced WL-38 cell pyroptosis. Also, LncRNA H19 acts as a sponge of miR-22-3p, which targets NLRP3, and NLRP3 attenuates the effect of LncRNA H19 silencing on LPS-induced WL-38 cell pyroptosis. CONCLUSION: Our data demonstrated the roles and potential mechanisms of LncRNA H19 in the regulation of pneumonia cell pyroptosis, indicating that LncRNA H19 is an efficient predictive and curative target for pneumonia.

The Relationship between Long Noncoding RNA H19 Polymorphism and the Epidermal Growth Factor Receptor Phenotypes on the Clinicopathological Characteristics of Lung Adenocarcinoma.

The aim of the current study is to investigate potential associations among Long Noncoding RNA (LncRNA) H19 single nucleotide polymorphism (SNP) and epidermal growth factor receptor (EGFR) phenotypes on the clinicopathological characteristics of lung adenocarcinoma (LADC). Five loci of LncRNA H19 SNPs (rs217727, rs2107425, rs2839698, rs3024270, and rs3741219) were genotyped by using TaqMan allelic discrimination in 223 LADC patients with wild-type EGFR phenotype and 323 LADC individuals with EGFR mutations. After the statistical analyses, patients with the EGFR mutation were related to a higher distribution frequency of rs217727 SNP CT heterozygote (p = 0.030), and the female population with EGFR mutation demonstrated a higher distribution frequency of rs217727 SNP CT heterozygote (p < 0.001) and rs2107425 CT heterozygote (p = 0.002). In addition, the presence of LncRNA H19 SNP rs217727 T allele (CT + TT) in patients with EGFR wild-type was associated to higher tumor T status (stage III or IV, p = 0.037) and poorer cell differentiation status (poor differentiation, p = 0.012) compared to those EGFR wild-type individuals with LncRNA H19 SNP rs217727 CC allele. Besides, a prominently higher tumor T status was found in subjects with LncRNA H19 SNP rs2107425 T allele (CT + TT) (stage III or IV, p = 0.007) compared to EGFR wild-type LADC individuals with LncRNA CC allele in EGFR wild-type patients. Our findings suggest that the presence of LncRNA H19 SNP rs217727 is related to the EGFR mutation in LADC patients, and the LncRNA H19 SNP rs217727 and rs2107425 are associated with progressed tumor status for LADC patients with EGFR wild-type.

Long noncoding RNA H19 regulates the therapeutic efficacy of mesenchymal stem cells in rats with severe acute pancreatitis by sponging miR-138-5p and miR-141-3p.

BACKGROUND: Patients with severe acute pancreatitis (SAP), which is characterized by high morbidity and mortality, account for an increasing medical burden worldwide. We previously found that mesenchymal stem cells (MSCs) could attenuate SAP and that expression of long noncoding RNA H19 (LncRNA H19) was upregulated in rats receiving MSCs. In the present study, we investigated the mechanisms of LncRNA H19 regulating the therapeutic efficacy of MSCs in the alleviation of SAP. METHODS: MSCs transfected with LncRNA H19 overexpression and knockdown plasmids were intravenously injected into rats 12 h after sodium taurocholate (NaT) administration to induce SAP. RESULTS: Overexpressing LncRNA H19 in MSCs significantly enhanced the anti-inflammatory capacity of the MSCs, inhibited autophagy via promotion of focal adhesion kinase (FAK)-associated pathways, and facilitated cell proliferation by increasing the level of ß-catenin in rats with SAP. LncRNA H19 functioned as a competing endogenous RNA by sponging miR-138-5p and miR-141-3p. Knocking down miR-138-5p in MSCs increased the expression of protein tyrosine kinase 2 (PTK2, encoding FAK) to suppress autophagy, while downregulating miR-141-3p enhanced the level of ß-catenin to promote cell proliferation. CONCLUSIONS: In conclusion, LncRNA H19 effectively increased the therapeutic efficacy of MSCs in rats with SAP via the miR-138-5p/PTK2/FAK and miR-141-3p/ß-catenin pathways.