LncRNA MALAT1 Knockdown Alleviates Fibrogenic Response in Human Endometrial Stromal Cells Via the miR-22-3p/TGFßR1/Smad2/3 Pathway.

Intrauterine adhesion (IUA) resulting from irreversible fibrotic repair of endometrium is the main cause of secondary infertility in women, and current therapeutic approaches to IUA are limited. Increasing evidence has suggested the important role of competitive endogenous RNA (ceRNA) in IUA pathologies. This study aimed to investigate the long noncoding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1)-associated ceRNA in IUA development. We harvested endometrial tissues from patients with or without IUA and extracted endometrial stromal cells (ESCs) from normal endometrial tissues. Transforming growth factor ß1 (TGF-ß1) was used to induce fibrosis in ESCs. The expression of transforming growth factor ß receptor 1 (TGFßR1), α-smooth muscle actin, phosphorylated suppressor of mother against decapentaplegic (p-Smad)2/3, collagen type I alpha 1, MALAT1, and microRNA (miR)-22-3p in endometrial tissues and ESCs was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blotting. Pearson's correlation analysis was conducted to assess the correlation between miR-22-3p expression or TGFßR1 and MALAT1 expression in endometrial tissues. The expression of TGFßR1 in ESCs was also evaluated by immunofluorescence staining. The location of MALAT1 was examined by fluorescence in situ hybridization. Luciferase reporter assays were performed to verify the binding relationship between MALAT1 or TGFßR1 and miR-22-3p. Cell viability was assessed via cell counting kit-8 assays. Our findings revealed that lncRNA MALAT1 and TGFßR1 were upregulated while miR-22-3p was downregulated in IUA endometrial tissues or TGF-ß1-stimulated ESCs, and lncRNA MALAT1 expression was negatively correlated with miR-22-3p expression while being positively correlated with TGFßR1 expression in IUA endometrial tissues. Additionally, lncRNA MALAT1 was mainly located in the cytoplasm of ESCs and directly targeted miR-22-3p to regulate TGFßR1 expression. Moreover, knockdown of lncRNA MALAT1 exerted anti-fibrotic effects on ESCs by targeting miR-22-3p, and miR-22-3p overexpression inhibited the fibrosis of ESCs by binding to TGFßR1 3'untranslated region. Collectively, lncRNA MALAT1 promotes endometrial fibrosis by sponging miR-22-3p to regulate TGFßR1 and Smad2/3, and inhibition of MALAT1 may represent a promising therapeutic option for suppressing endometrial fibrosis.

Pericardial Fluid Accumulates microRNAs That Regulate Heart Fibrosis after Myocardial Infarction.

Pericardial fluid (PF) has been suggested as a reservoir of molecular targets that can be modulated for efficient repair after myocardial infarction (MI). Here, we set out to address the content of this biofluid after MI, namely in terms of microRNAs (miRs) that are important modulators of the cardiac pathological response. PF was collected during coronary artery bypass grafting (CABG) from two MI cohorts, patients with non-ST-segment elevation MI (NSTEMI) and patients with ST-segment elevation MI (STEMI), and a control group composed of patients with stable angina and without previous history of MI. The PF miR content was analyzed by small RNA sequencing, and its biological effect was assessed on human cardiac fibroblasts. PF accumulates fibrotic and inflammatory molecules in STEMI patients, namely causing the soluble suppression of tumorigenicity 2 (ST-2), which inversely correlates with the left ventricle ejection fraction. Although the PF of the three patient groups induce similar levels of fibroblast-to-myofibroblast activation in vitro, RNA sequencing revealed that PF from STEMI patients is particularly enriched not only in pro-fibrotic miRs but also anti-fibrotic miRs. Among those, miR-22-3p was herein found to inhibit TGF-ß-induced human cardiac fibroblast activation in vitro. PF constitutes an attractive source for screening diagnostic/prognostic miRs and for unveiling novel therapeutic targets in cardiac fibrosis.

miR-22 negatively regulating NOD-like receptor protein 3 gene in the proliferation, invasion, and migration of malignant melanoma cells.

Introduction: Malignant melanoma (MM) is a highly aggressive skin tumour. Aim: To investigate whether miR-22 is involved in the proliferation, invasion, and migration of melanoma cells (MCs) by negatively regulating NOD-like receptor protein 3 (NLRP3) gene. Material and methods: Human MCs (WM239a) and human epidermal melanocytes (HEM) were used as study material. The expression levels of miR-22 and NLRP3 were detected by qRT-PCR. The expression of NLRP3 protein was determined by Western blot (WB) analysis. The effects of miR-22 and NLRP3 on the proliferation, invasion, and migration of MCs were evaluated by cell counting kit-8 (CCK-8), Transwell cell invasion assay, and scratch assay. Results: The expression of miR-22 was clearly lower in WM239a than in HEM. Up-regulation of miR-22 expression in WM239a clearly raised the expression of miR-22, Caspase-1, and E-cadherin and the apoptotic rate of WM239a; however, the levels of interleukin-1ß (IL-1ß) and NLRP3, cell proliferation activity, invasion and migration ability were clearly decreased. The negative regulation of NLRP3 by miR-22 may play a major role in activities of MM. Conclusions: Further studies will help to reveal the molecular details of this regulatory mechanism and provide new therapeutic strategies.

MiR-22-3p Inhibits 5-Fluorouracil Resistance in Cholangiocarcinoma Cells Through PTEN/PI3K/AKT Axis.

Cholangiocarcinoma (CCA) is a prevalent and highly lethal form of cancer globally. Although microRNAs (miRNAs) have been implicated in the advancement of CCA, their potential influence on 5-fluorouracil (5-Fu) resistance in CCA remains to be fully elucidated. Here, in this study, we investigated the impact of miR-22-3p on CCA resistance. Our investigation involved bioinformatics analysis, which revealed an association between miR-22-3p and the progression, diagnosis, and patient survival of CCA. Furthermore, we validated a notable downregulation of miR-22-3p expression in CCA cell lines. Elevated levels of miR-22-3p inhibit the activity and proliferation of 5-Fu-resistant CCA cell lines. In addition, we confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a target gene of miR-22-3p, and its expression correlates with the survival of CCA patients. Reduced PTEN expression enhances apoptosis in 5-Fu-resistant CCA cells. Meanwhile, we verified the existence of the miR-22-3p/PTEN/phosphatidylinositol-3 kinase (PI3K)/Protein kinase B (AKT) regulatory networks in CCA, influencing the sensitivity of CCA cells to 5-Fu. In conclusion, our findings suggest that miR-22-3p acts as a tumor suppressor. Its overexpression inhibits the PTEN/PI3K/AKT axis, promoting cell apoptosis and enhancing CCA sensitivity to 5-Fu.

Mir22hg facilitates ferritinophagy-mediated ferroptosis in sepsis by recruiting the m6A reader YTHDC1 and enhancing Angptl4 mRNA stability.

BACKGROUND: Ferritinophagy-mediated ferroptosis plays a crucial role in fighting pathogen aggression. The long non-coding RNA Mir22hg is involved in the regulation of ferroptosis and aberrantly overexpression in lipopolysaccharide (LPS)-induced sepsis mice, but whether it regulates sepsis through ferritinophagy-mediated ferroptosis is unclear. METHODS: Mir22hg was screened by bioinformatics analysis. Ferroptosis was assessed by assaying malondialdehyde (MDA), reactive oxygen species (ROS), and Fe2+ levels, glutathione (GSH) activity, as well as ferroptosis-related proteins GPX4 and SLC3A2 by using matched kits and performing western blot. Ferritinophagy was assessed by Lyso tracker staining and FerroOrange staining, immunofluorescence analysis of Ferritin and LC-3, and western blot analysis of LC-3II/I, p62, FTH1, and NCOA4. The bind of YTH domain containing 1 (YTHDC1) to Mir22hg or angiopoietin-like-4 (Angptl4) was verified by RNA pull-down and/or immunoprecipitation (RIP) assays. RESULTS: Mir22hg silencing lightened ferroptosis and ferritinophagy in LPS-induced MLE-12 cells and sepsis mouse models, as presented by the downregulated MDA, ROS, Fe2+, NCOA4, and SLC3A2 levels, upregulated GPX4, GSH, and FTH1 levels, along with a decrease in autophagy. Mir22hg could bind to the m6A reader YTHDC1 without affecting its expression. Mechanistically, Mir22hg enhanced Angptl4 mRNA stability through recruiting the m6A reader YTHDC1. Furthermore, Angptl4 overexpression partly overturned Mir22hg inhibition-mediated effects on ferroptosis and ferritinophagy in LPS-induced MLE-12 cells. CONCLUSION: Mir22hg contributed to in ferritinophagy-mediated ferroptosis in sepsis via recruiting the m6A reader YTHDC1 and strengthening Angptl4 mRNA stability, highlighting that Mir22hg may be a potential target for sepsis treatment based on ferroptosis.

Hsa_circ_0049472 contributed to amyloid-beta peptide-induced neurotoxicity, apoptosis and inflammation via regulating PI3K-AKT signaling pathway by interacting with miR-22-3p/ZNF217 axis.

BACKGROUND: Circular RNAs (circRNAs) exhibited important roles in Alzheimer's disease (AD). Here, we focused on the dysregulation of hsa_circ_0049472 (circ_0049472) and potential functions in SK-N-SH cells with amyloid-beta peptide (Aß) treatment in AD. METHODS: RNA expression was detected by real-time quantitative PCR. Cell viability and proliferation were measured by MTS and Edu assays. Flow cytometry was used for apoptosis detection, and cell inflammation was assessed using enzyme-linked immunosorbent assay. Target interaction was validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. Protein expression and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) pathway were examined by Immunoblotting. RESULTS: Aß treatment inhibited cell viability and proliferation of SK-N-SH cells, but enhanced apoptosis rate, apoptosis protein levels (Bcl2-associated X protein and cleaved-caspase-3) and inflammatory cytokines (interleukin -6, IL-1ß, tumor necrosis factor-α). Then, circ_0049472 expression was shown to be upregulated in response to Aß stimulation and knockdown of circ_0049472 has ameliorated Aß-induced cell injury. Circ_0049472 was identified as a sponge for miR-22-3p, and miR-22-3p inhibition reversed the regulation of circ_0049472 knockdown in Aß-treated cells. Furthermore, ZNF217 acted as a target of miR-22-3p and circ_0049472 could regulate ZNF217 expression via binding to miR-22-3p. Overexpression of miR-22-3p abated Aß-induced apoptosis and inflammation via downregulating ZNF217. Furthermore, Aß reduced proteins levels of p-PI3K and p-AKT, and this inhibition of PI3K-AKT pathway was restored by the regulation of circ_0049472/miR-22-3p/ZNF217 axis. CONCLUSION: Circ_0049472 was involved in Aß-induced neural injury by regulating miR-22-3p/ZNF217 axis to affect PI3K-AKT pathway. This study has discovered an innovative mechanism for AD.

CircPRELID2 functions as a promoter of renal cell carcinoma through the miR-22-3p/ETV1 cascade.

BACKGROUND: Emerging evidence has indicated that a number of circular RNAs (circRNAs) participate in renal cell carcinoma (RCC) carcinogenesis. Nevertheless, the activity and molecular process of circPRELID2 (hsa_circ_0006528) in RCC progression remain unknown. METHODS: CircPRELID2, miR-22-3p and ETS variant 1 (ETV1) levels were gauged by qRT-PCR. Effect of the circPRELID2/miR-22-3p/ETV1 axis was evaluated by detecting cell growth, motility, and invasion. Immunoblotting assessed related protein levels. The relationships of circPRELID2/miR-22-3p and miR-22-3p/ETV1 were confirmed by RNA immunoprecipitation (RIP), luciferase reporter or RNA pull-down assay. RESULTS: CircPRELID2 was up-regulated in RCC. CircPRELID2 silencing suppressed RCC cell growth, motility and invasion. Moreover, circPRELID2 silencing weakened M2-type macrophage polarization in THP1-induced macrophage cells. CircPRELID2 sequestered miR-22-3p, and circPRELID2 increased ETV1 expression through miR-22-3p. Moreover, the inhibitory impact of circPRELID2 silencing on RCC cell malignant behaviors was mediated by the miR-22-3p/ETV1 axis. Furthermore, circPRELID2 knockdown in vivo hampered growth of xenograft tumors. CONCLUSION: Our study demonstrates that circPRELID2 silencing can mitigate RCC malignant development through the circPRELID2/miR-22-3p/ETV1 axis, highlighting new therapeutic targets for RCC treatment.

Albumen exosomes alleviate LPS-induced inflammation of intestinal epithelial cells via miR-22/ATM/p53/NF-κB axis.

Biological macromolecules identified in albumen were found benefit to intestinal health, whether albumen contains exosomes and function of their cargos in intestinal inflammation remain unknown. This study aimed to investigate characteristics and cargos of albumen exosomes, as well as their potential roles in alleviating inflammation in intestinal epithelial cells. Our results demonstrated that albumen contains exosomes that are cup-shaped morphology vesicles with diameter ranging from 50 to 200 nm. There were 278 miRNAs and 45 proteins with higher expression levels in albumen exosomes, and they were mainly involved in immune responses and programmed cell death pathways, including apoptosis and p53 signaling pathway. LPS induced overexpression of pro-inflammatory cytokines IL-1ß and TNF-α and excessive apoptosis, which could be reversed by albumen exosomes. The beneficial effects of exosomes could be mainly attributed to miRNA cargos and their inhibition on inflammatory response signaling pathways (p53 and NF-κB pathways). Mechanically, exosome miR-22 targeted ATM and inhibited p53/NF-κB pathway, alleviating LPS-induced overexpression of Caspase-3 and Bax, and inflammatory response. Collectively, albumen exosomes alleviate inflammation of intestinal epithelial cells via miR-22/ATM/p53/NF-κB axis and these findings may provide theoretical basis to the potential application of albumen exosomes for intestinal inflammation.

Small nucleolar RNA host gene 18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

AIMS: Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. METHODS AND RESULTS: Analysis of single-cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFß1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering with the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. CONCLUSION: We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

Knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney.

BACKGROUND: Sepsis is a life-threatening, systemic inflammatory disease that can lead to a variety of conditions, including septic acute kidney injury (AKI). Recently, multiple circular Rnas (circRNAs) have been implicated in the development of this disease. METHODS: In this study, we aimed to elucidate the role of circ-Gatad1 in sepsis induced AKI and its potential mechanism of action. High-throughput sequencing was used to investigate abnormal expression of circRNA in AKI and healthy volunteer. Bioinformatics analysis and luciferase reporting analysis were used to clarify the interacted relationship among circRNA, miRNA and mRNA. HK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. HK2 cells were employ to analysis the ROS, inflammatory cytokines expression, proliferation and apoptosis under LPS condition. RESULTS: The result show that the expression of circ-Gatad1 was increased in septic acute kidney patients. Downregulation circ-Gatad1 suppressed LPS-treated induced HK2 cells injury including apoptosis, proliferation ability, ROS and inflammatory cytokines level. Bioinformatics and luciferase report analysis confirmed that both miR-22-3p and TRPM7 were downstream targets of circ-Gatad1. Overexpression of TRPM7 or downregulation of miR-22-3p reversed the protective effect of si-circ-Gatad1 to HK2 after exposure to LPS (5 µg/ml) microenvironment. CONCLUSION: In conclusion, knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney.

Sex-specific regulation of miR-22 and ERα in white adipose tissue of obese dam's female offspring impairs the early postnatal development of functional beige adipocytes in mice.

During inguinal adipose tissue (iWAT) ontogenesis, beige adipocytes spontaneously appear between postnatal 10 (P10) and P20 and their ablation impairs iWAT browning capacity in adulthood. Since maternal obesity has deleterious effects on offspring iWAT function, we aimed to investigate its effect in spontaneous iWAT browning in offspring. Female C57BL/6 J mice were fed a control or obesogenic diet six weeks before mating. Male and female offspring were euthanized at P10 and P20 or weaned at P21 and fed chow diet until P60. At P50, mice were treated with saline or CL316,243, a ß3-adrenoceptor agonist, for ten days. Maternal obesity induced insulin resistance at P60, and CL316,243 treatment effectively restored insulin sensitivity in male but not female offspring. This discrepancy occurred due to female offspring severe browning impairment. During development, the spontaneous iWAT browning and sympathetic nerve branching at P20 were severely impaired in female obese dam's offspring but occurred normally in males. Additionally, maternal obesity increased miR-22 expression in the iWAT of male and female offspring during development. ERα, a target and regulator of miR-22, was concomitantly upregulated in the male's iWAT. Next, we evaluated miR-22 knockout (KO) offspring at P10 and P20. The miR-22 deficiency does not affect spontaneous iWAT browning in females and, surprisingly, anticipates iWAT browning in males. In conclusion, maternal obesity impairs functional iWAT development in the offspring in a sex-specific way that seems to be driven by miR-22 levels and ERα signaling. This impacts adult browning capacity and glucose homeostasis, especially in female offspring.

Translation regulatory long non-coding RNA 1 negatively regulates cell radiosensitivity via the miR-22-3p/SP1 axis in non-small cell lung cancer.

OBJECTIVE: Non-small cell lung cancer (NSCLC) occupies 85% of lung cancer. Long non-coding RNAs (LncRNAs) can regulate the radiosensitivity of cancers. This study explored the mechanism of lncRNA TRERNA1 in the radiosensitivity of NSCLC cells. METHODS: LncRNA TRERNA1 level in NSCLC cell lines was determined. NSCLC cell radiation tolerance was measured. TRERNA1 expression was silenced or overexpressed in A549/HCC827 cells with the highest/lowest radiation tolerance, respectively. The contents of γ-H2AX and SA-ß-gal in NSCLC cells after radiation induction were detected. The targeted binding of TRERNA1 to miR-22-3p and miR-22-3p to SP1 were verified by dual-luciferase assay. SP1 expression were detected. Functional rescue experiments were implemented to confirm the roles of miR-22-3p and SP1 in the regulatory mechanism of TRERNA1. RESULTS: TRERNA1 was upregulated in NSCLC cells. TRERNA1 silencing enhanced radiosensitivity of NSCLC cells. TRERNA1 silencing elevated the contents of γ-H2AX and SA-ß-gal in A549 cells after radiation induction, while TRERNA1 overexpression showed an opposite trend in HCC827 cells. There were targeting relationships between TRERNA1 and miR-22-3p, and miR-22-3p and SP1. miR-22-3p repression or SP1 overexpression abolished the effects of TRERNA1 silencing. CONCLUSION: TRERNA1 silencing enhanced radiosensitivity of NSCLC cells via the miR-22-3p/SP1 axis. This study may offer new targets for NSCLC treatment.

Expression of miR-22 profiling in colorectal normal-adenoma-carcinoma sequence.

Colorectal cancer (CRC) is the third most common cancer in the world. MiRNA-22 has emerged as a potential candidate with diagnostic significance; however, its expression profile across the normal-adenoma-carcinoma transition in colorectal remains unexplored. In this study, we evaluated serum miRNA-22 levels in patients with varying stages of CRC. The study cohort comprised 49 healthy controls, 50 patients with polyps, 51 individuals with colorectal adenoma (CRA), and 50 cases of CRC, confirmed through proctocolonoscopy and pathological biopsy. Real-time quantitative polymerase chain reaction was employed to validate the significantly differential expression of serum miRNA-22 among different stages of CRC progression. The 2-ΔΔCT method was utilized to assess the relative changes in serum miRNA-22 expression levels. Our results revealed no significant differences in gender, adenoma grade, location, or TNM classification stage in terms of serum miR-22 expression across the four groups. Notably, both the CRC and CRA groups exhibited higher miR-22 expression levels compared to the control group (p = 0.0001, p = 0.0004), with the CRA and CRC groups displaying higher expression levels than the polyp group (p = 0.02, p = 0.043). Ordered multicategorical logistic regression analysis model revealed the utilization of age, gender, smoking status, and miR-22 expression collectively exhibited the highest value for the area under the curve (AUC = 0.748) in the discrimination between individuals CRC and healthy. The independent factor of expression of miR-22 demonstrated the most notable predictive capacity (AUC = 0.753) when distinguishing between CRA and healthy individuals. Furthermore, the independent expression of miR-22 exhibited discernible potential (AUC = 0.654, 0.636) differentiation between polyps and CRA/ CRC. Notably, the factor of age displayed the most substantial discriminatory power (AUC = 0.741) when distinguishing between polyps and healthy individuals. Our findings provide supportive evidence for considering miR-22 as a potential biomarker for CRC early screening. Nonetheless, the molecular mechanisms of miR-22 regulation in colorectal lesions still need to be investigated.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

CircMAP3K5 promotes cardiomyocyte apoptosis in diabetic cardiomyopathy by regulating miR-22-3p/DAPK2 Axis.

BACKGROUND: Diabetic cardiomyopathy (DCM) is one of the serious complications of the accumulated cardiovascular system in the long course of diabetes. To date, there is no effective treatment available for DCM. Circular RNA (circRNA) is a novel r2egulatory RNA that participates in a variety of cardiac pathological processes. However, the regulatory role of circular RNA MAP3K5 (circMAP3K5) in DCM is largely unclear. METHODS AND RESULTS: Microarray analysis of DCM rats' heart circular RNAs was performed and the highly species-conserved circRNA mitogen-activated protein kinase kinase kinase 5 (circMAP3K5) was identified, which participates in DCM processes. High glucose-provoked cardiotoxicity leads to the up-regulation of circMAP3K5, which mechanistically contributes to cardiomyocyte cell death. Also, in high glucose-induced H9c2 cardiomyocytes, the level of apoptosis was significantly increased, as well as the expression of circMAP3K5. In contrast, the depletion of circMAP3K5 could reduce high glucose-induced apoptosis in cardiomyocytes. In terms of mechanism, circMAP3K5 acts as a miR-22-3p sponge and miR-22-3p directly target death-associated protein kinase 2 (DAPK2) in H9c2 cardiomyocytes, where in circMAP3K5 upregulates DAPK2 expression by targeting miR-22-3p. Moreover, we also found that miR-22-3p inhibitor and pcDNA DAPK2 could antagonize the protective effects brought by the depletion of circMAP3K5. CONCLUSION: CircMAP3K5 is a highly conserved noncoding RNA that is upregulated during DCM process. We concluded that circMAP3K5 promotes high glucose-induced cardiomyocyte apoptosis by regulating the miR-22-3p/DAPK2 axis. The results of this study highlight a novel and translationally important circMAP3K5-based therapeutic approach for DCM.

LncRNA MIR22HG/microRNA-9-3p/IGF1 in nonalcoholic steatohepatitis, the ceRNA network increases fibrosis by inhibiting autophagy and promoting pyroptosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is known to progress due to the impact of long non-coding RNAs (lncRNAs), which have been linked to autophagy, pyroptosis, and fibrosis in NASH cells. However, the exact mechanisms underpinning these processes remain unclear. This study focuses on the role of lncRNA MIR22HG (MIR22HG) in NASH. METHODS: The expression of differentially expressed lncRNA was analyzed by RNA sequencing. Mouse models of NASH induced by MCD and HFD were validated. The expression of MIR22HG in HFD and MCD mouse liver tissue samples, FFA cells constructed with HepG2 and Huh7, and human liver tissue samples were detected by QRT-PCR. In addition, We used RNA immunoprecipitation, luciferase reporting, miRNA transfection, plasmid construction, immunofluorescence, Western blot, qRT-PCR, ELISA, and hybridization techniques to elucidate the relationship between MIR22HG, microRNA-9-3p (miR-9-3p), and IGF1. In addition, the mechanism of MIR22HG and PTEN/AKT was explored by Western blot analysis. RESULTS: RNA-seq found that 3751 mRNAs and 23 lncRNAs were differentially expressed, which constituted a lncRNA-miRNA-mRNA regulatory network. Studies demonstrated the downregulation of MIR22HG in HFD and MCD mouse liver tissue samples (p = 1.00E-04 and p = 4.6E-03). Our results showed that overexpression of MIR22HG promoted autophagy and inhibited pyroptosis and fibrosis through the miR-9-3p/IGF1 pathway, thus slowing the occurrence and development of NASH. Further, we observed a low expression of MIR22HG and IGF1, but a high expression of miR-9-3p in NASH patients, a finding in alignment with our in vivo and in vitro results. CONCLUSION: Using MIR22HG as a biomarker and therapeutic target for NASH patients, we found that it plays a pivotal role in detecting autophagy, pyroptosis, and fibrosis through the ceRNA pathway.

miR-22-3p in the rostral ventrolateral medulla promotes hypertension through inhibiting ß-arrestin-1.

It has been documented that increased sympathetic activity contributes to the development of cardiovascular diseases, such as hypertension. We previously reported that ß-arrestin-1, a multifunctional cytoskeletal protein, was downregulated in the rostral ventrolateral medulla (RVLM) of the spontaneously hypertensive rat (SHR), and its overexpression elicited an inhibitory effect on sympathetic activity in hypertension. microRNA (miR)-22-3p has been reported to be associated with the pathological progress of hypertension. The purpose of this study was to determine the role of miR-22-3p in ß-arrestin-1-mediated central cardiovascular regulation in hypertension. It was observed that miR-22-3p was upregulated in the RVLM of SHRs compared with normotensive Wistar-Kyoto (WKY) rats, and it was subsequently confirmed to target the ß-arrestin-1 gene using a dual-luciferase reporter assay. miR-22-3p was downregulated in the RVLM using adeno-associated virus with 'tough decoys', which caused a significant increase of ß-arrestin-1 expression and decrease of noradrenaline and blood pressure (BP) in SHRs. However, upregulation of miR-22-3p using lentivirus in the RVLM of WKY rats significantly increased BP. In in vitro PC12 cells, enhanced oxidative stress activity induced by angiotensin II was counteracted by pretreatment with miR-22-3p inhibitor, and this effect could be abolished by ß-arrestin-1 gene knockdown. Furthermore, microglia exhaustion significantly diminished miR-22-3p expression, and enhanced ß-arrestin-1 expression in the RVLM of SHRs. Activation of BV2 cells in vitro evoked a significant increase of miR-22-3p expression, and this BV2 cell culture medium was also able to facilitate miR-22-3p expression in PC12 cells. Collectively, our findings support a critical role for microglia-derived miR-22-3p in inhibiting ß-arrestin-1 in the RVLM, which is involved in central cardiovascular regulation in hypertension. KEY POINTS: Impairment of ß-arrestin-1 function in the rostral ventrolateral medulla (RVLM) has been reported to be associated with the development of sympathetic overactivity in hypertension. However, little is known about the potential mechanisms of ß-arrestin-1 dysfunction in hypertension. miR-22-3p is implicated in multiple biological processes, but the role of miR-22-3p in central regulation of cardiovascular activity in hypertension remains unknown. We predicted that miR-22-3p could directly bind to the ß-arrestin-1 gene (Arrb1), and this hypothesis was confirmed by using a dual-luciferase reporter assay. Inhibition of ß-arrestin-1 by miR-22-3p was further verified in both in vivo and in vitro experiments. Furthermore, our results suggested miR-22-3p as a risk factor for oxidative stress in the RVLM, thus contributing to sympatho-excitation and hypertension. Our present study provides evidence that microglia-derived miR-22-3p may underlie the pathogenesis and progression of neuronal hypertension by inhibiting ß-arrestin-1 in the RVLM.

MiR-22-3p facilitates bone marrow mesenchymal stem cell osteogenesis and fracture healing through the SOSTDC1-PI3K/AKT pathway.

Bone fractures are the most common form of musculoskeletal trauma worldwide. Numerous microRNAs (miRNAs) have been suggested to be participants in regulating bone-related diseases. Recent studies revealed the regulatory role of miR-22-3p in osteogenic differentiation, but its role in fracture healing has not been investigated previously. Here, a rat femoral fracture model was established, Bone marrow mesenchymal stem cells (BMSCs) were isolated to detect the specific function and underlying mechanisms of miR-22-3p. MiR-22-3p and sclerostin domain-containing 1 (SOSTDC1) expression was determined by RT-qPCR and immunohistochemistry staining. The levels of proteins associated with osteogenic differentiation were assessed by western blotting. Flow cytometry was conducted to identify the isolated rat BMSCs. Alizarin red staining, alkaline phosphatase staining and Oil Red O staining were used to evaluate the osteogenic and adipogenic differentiation of rat BMSCs. The interaction between miR-22-3p and SOSTDC1 was verified using a luciferase reporter assay. Haematoxylin and Eosin (H&E) staining of the bone tissues was performed to analyse the effect of miR-22-3p on histopathological changes in vivo. MiR-22-3p was downregulated in the callus tissues of rat femoral fracture, while the expression of SOSTDC1 was upregulated. The isolated rat BMSCs had the capacity for both osteogenic and adipogenic differentiation. The differentiation capacity of BMSCs into osteoblasts was increased by miR-22-3p overexpression. MiR-22-3p activated the PI3K/AKT pathway by targeting SOSTDC1. SOSTDC1 overexpression and PI3K/AKT signalling inhibitor LY294002 abolished the enhancing effect of miR-22-3p overexpression on the osteogenesis of BMSCs. Thus MiR-22-3p facilitated the femoral fracture healing in rats. MiR-22-3p overexpression promoted fracture healing via the activation of PI3K/AKT pathway by targeting SOSTDC1.

Mechanism Study of LncRNA LINC01137 Promoting the Progression of Non-small Cell Lung Cancer by Inducing CD8+T Cell Exhaustion / 现代检验医学杂志

Objective To investigate the biological function of long non-coding RNA(LncRNA)LINC01137 in immune escape of non-small cell lung cancer(NSCLC)cells and its potential regulatory mechanisms.Methods The blood samples of 24 healthy volunteers and 24 NSCLC patients were collected.The tumor tissues and paracancerous tissues of 24 NSCLC patients were collected,and the levels of LINC01137 were detected.The binding sites of LINC01137 and miR-22-3p were predicted by Starbase database and verified by the luciferase reporter gene analysis.A549 cells were transfected with exosomes derived from A549 cells and/or sh-LINC01137 interference sequence to detect cell proliferation and invasion.The supernatant of A549 cells were collected to culture CD8+T cells,and the levels of CD8+T cell exhaustion markers,including interfereron-γ(IFN-γ),tumor necrosis factor-α(TNF-α),granzyme B and interleukin-2(IL-2),and the percentage of PD-1+Tim3+CD8+T cells were detected.CD8+T cells were transfected with exosomes and/or miR-22-3p mimics to detect the protein level of PD-1.Results The expression of LINC01137 in tumor tissues of patients with NSCLC was increased compared with paracancerous tissues(3.357±0.548 vs 1.011±0.371),while the expression of LINC01137 in peripheral blood of patients with NSCLC was increased compared with healthy volunteers(3.216±0.342 vs 1.007±0.313),with statistically significant differences(t=-17.367,-17.147,all P<0.001).There was a positive correlation between the expression of LINC01137 in tumor tissue and peripheral blood(r=0.755,P<0.05).LINC01137 was significantly enriched in exosomes derived from A549 cells.Compared with Exo+sh-NC group,the cell viability(65.85%±4.71%vs 100.15%±11.93%)and cell invasion(21.46%±3.48%vs 43.12%±1.44%)in Exo+sh-LINC01137 group were decreased,and the differences were statistically significant(t=4.630,9.953,all P<0.01).The expression of LINC01137 in peripheral blood of NSCLC patients was negatively correlated with the percentage of CD8+T cells(r=-0.520,P<0.05).Compared with Exo+sh-NC group,the IFN-γ(3 865.31±543.85 pg/ml vs 1 786±105.98 pg/ml),TNF-α(4 631.93±510.71 pg/ml vs 1 973.24±379.62 pg/ml),Granzyme B(3 876.49±312.43 pg/ml vs 1 879.43±287.58 pg/ml),and IL-2 mRNA levels(3.286±0.437 vs 1.015±0.314)were increased,and the percentage of PD-1+Tim3+CD8+T cells(7.68%±2.18%vs 18.95%±3.21%)was decreased in Exo+sh-LINC01137 group,with statistical significances(t=-6.497,-7.237,-8.146,-7.310,5.021,all P<0.01).Our results showed that miR-22-3p was the target gene of LINC01137.Compared with Exo+NC mimic group,the level of PD-1 protein in Exo+miR-22-3p group(0.384±0.087 vs 1.003±0.147)was significantly decreased,and the difference was statistically significant(t=6.277,P<0.01).Conclusion The expression of LINC01137 was significantly up-regulated in tumor tissues and plasma of NSCLC patients.Exosomes LINC01137 derived NSCLC cell induces CD8+T cell exhaustion by targeting miR-22-3p and inhibiting its expression,and thus promoting NSCLC cell immune escape.

[Effect of miR-22 Targeting FMNL2 on Cell Migration and Apoptosis in Childhood Acute Myeloid Leukemia].

OBJECTIVE: To investigate the effect of miR-22 targeting formin-like protein 2 (FMNL2) on the migration and apoptosis of childhood acute myeloid leukemia (AML) cells. METHOD: Peripheral blood samples from 11 children with AML, 10 children with immune thrombocytopenia, human AML cell lines TF-1a, HL-60, THP-1 and human bone marrow stromal cells HS-5 were used as the research objects. UniCel DxH 800 automatic hematology analyzer detected platelet count, hemoglobin, and white blood cell count in peripheral blood samples, and RT-qPCR detected miR-22 expression in peripheral blood samples and AML cells. HL-60 cells were transfected with LipofectamineTM 2000 kit, the experiments were divided into seven groups: blank (no cells transfected), miR-NC, miR-22 mimics, si-NC, si-FMNL2 , miR-22 mimics+OE-NC and miR-22 mimics+OE-FMNL2 . RT-qPCR was used to detect the expression of miR-22 in each group. Transwell was used to detect cell migration. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter gene detection experiments verified the targeting relationship between miR-22 and FMNL2 . Western blot was used to detect the expression of FMNL2 protein. RESULTS: Compared with the control group, the number of leukocytes in the peripheral blood of children with AML was significantly increased (P <0.001), while the concentration of hemoglobin and the number of platelets were significantly decreased P <0.001). The expression level of miR-22 in peripheral blood of children with AML was significantly lower than that in control group (P <0.001). Compared with HS-5 cells, the expression levels of miR-22 in TF-1a, HL-60, and THP-1 cells were significantly decreased (P <0.05), and in HL-60 cells was the lowest. Therefore, HL-60 cells were selected for subsequent experiments. Up-regulation of miR-22 or silencing of FMNL2 could reduce the number of migrating cells and increase apoptosis rate (P <0.05). MiR-22 targeted and negatively regulated the expression of FMNL2 . FMNL2 overexpression reversed the effects of up-regulated miR-22 on migration and apoptosis of HL-60 cells. CONCLUSION: MiR-22 can inhibit the migration and promote apoptosis of HL-60 cells by down regulating the expression of FMNL2 .