GRg1 inhibits the TLR4/NF-kB signaling pathway by upregulating miR-216a-5p to reduce growth factors and inflammatory cytokines in DR.

BACKGROUND: Diabetic retinopathy (DR) is a common diabetic neurodegenerative disease that affects vision in severe cases. Current therapeutic drugs are ineffective for some patients with severe side effects, and ginsenoside-Rg1 (GRg1) has been shown to protect against DR and may serve as a new potential drug for DR. This study aimed to confirm the protective effect of GRg1 against DR and its molecular mechanism. METHODS: Human retinal microvascular endothelial cells (hRMECs) and rats were used to construct DR models in vitro and in vivo. Cell proliferation was detected by BrdU assays, the cell cycle was detected by flow cytometry, and TNF-α, IL-6 and IL-1ß levels were detected by ELISA. qRT‒PCR, Western blotting and immunohistochemistry were used to detect the expression of related genes and proteins, and angiogenesis assays were used to assess angiogenesis. RIP and RNA pull down assays were used to determine the relationship between miR-216a-5p and TLR4; retinal structure and changes were observed by HE staining and retinal digestive spread assays. RESULTS: GRg1 effectively inhibited HG-induced hRMEC proliferation, cell cycle progression and angiogenesis and reduced the levels of intracellular inflammatory cytokines and growth factors. HG downregulated the expression of miR-216a-5p and upregulated the expression of TLR4/NF-kB signaling pathway-related proteins. Importantly, GRg1 inhibited TLR4/NF-kB signaling pathway activation by upregulating miR-216a-5p, thereby inhibiting HG-induced cell proliferation, cell cycle progression, angiogenesis, and the production of inflammatory cytokines and growth factors. In addition, animal experiments confirmed the results of the cell experiments. CONCLUSIONS: GRg1 inhibits TLR4/NF-kB signaling by upregulating miR-216a-5p to reduce growth factors and inflammatory cytokines in DR, providing a potential therapeutic strategy for DR.

Ginsenoside Rb2 improves heart failure by down-regulating miR-216a-5p to promote autophagy and inhibit apoptosis and oxidative stress.

BACKGROUND: Ginsenoside Rb2 is beneficial in cardiovascular disease treatment, yet its role in heart failure (HF) is obscure. This study aimed to investigate the effect and mechanism of ginsenoside Rb2 on HF. METHODS: The left anterior descending branch-ligated HF rat model and oxygen-glucose deprivation/reoxygenation (OGD/R) H9c2 cell model were constructed. Ginsenoside Rb2 were applied for intervention. Heart function indexes, miR-216a-5p expression, autophagy, oxidative stress, apoptosis, cell morphology, and proliferation were detected to explore the effect of ginsenoside Rb2 on HF. Overexpression of miR-216a-5p was employed to explore the specific mechanism of ginsenoside Rb2 on HF. RESULTS: Ginsenoside Rb2 improved the heart function of HF rats, including the reduction of heart rate, LVEDP, and heart weight/body weight ratio, and the increase of LVSP, +dP/dtmax, -dP/dtmax, LVEF, and LVFS. It also down-regulated miR-216a-5p expression and enhanced OGD/R-induced cardiomyocyte viability. Ginsenoside Rb2 up-regulated Bcl2, LC3B II/I, and Beclin1, and down-regulated Bax, Caspase-3, and p62 in the myocardium of HF rats and OGD/R-induced H9c2 cells. Moreover, ginsenoside Rb2 increased the levels of SOD and CAT, but decreased the levels of MDA and ROS in the myocardium of HF rats and OGD/R-induced H9c2 cells. However, overexpression of miR-216a-5p promoted the apoptosis and oxidative stress of cardiomyocytes and inhibited autophagy, thus reversing the therapeutic effect of ginsenoside Rb2 on HF in vivo and in vitro. CONCLUSION: Ginsenoside Rb2 demonstrated potential as a therapeutic intervention for HF by enhancing autophagy and reducing apoptosis and oxidative stress through miR-216a-5p downregulation. Further research could explore its application in clinical trials and investigate the complex mechanism networks underlying its effects.

Propofol rescues LPS-induced toxicity in HRT-8/SVneo cells via miR-216a-5p/TLR4 axis.

OBJECTIVE: The purpose of this study was to evaluate the effect of propofol on lipopolysaccharide (LPS)-induced toxicity in HTR-8/SVneo cells. METHODS: In this study, HTR-8/SVneo cells were induced by LPS. The cells were treated with different concentrations of propofol. Cell proliferation, apoptosis, invasion, and wound healing rate were measured by MTT, flow cytometry, Transwell, and wound-healing assay. The relative mRNA expression levels of miR-216a-5p, TLR, MyD88, and NF-κB(p65) were measured by qRT-PCR. The protein expression levels of TLR, MyD88, and p-NF-κB(p65) were detected by western blot. The p-NF-κB(p65) nuclear volume was evaluated by cell immunofluorescence. RESULTS: Compared with control group, the cell proliferation, invasion, and wound healing rate were significantly decreased and the cell apoptosis rate was significantly increased in LPS- induced HTR-8/SVneo cells (P < 0.01). With propofol supplement, the cell proliferation, migration, and invasion abilities were significantly recovered and apoptosis rate was significantly inhibited (P < 0.05). The expression levels of miR-216a-5p, TLR4, MyD88, NF-κB(p65), and p-NF-κB(p65), and p-NF-κB(p65) nuclear volume were significantly different between propofol group and model group (P < 0.05). However, after knockdown of miR-216a-5p expression by si-miR-216a-5p transfection, the cell proliferation, migration, and invasion abilities were significantly inhibited and apoptosis rate was notably increased (P < 0.05). CONCLUSION: Propofol improves LPS-induced toxicity in HTR-8/SVneo cells via regulation miR-216a-5p/TLR4 axis.

Glucose starvation suppresses gastric cancer through targeting miR-216a-5p/Farnesyl-Diphosphate Farnesyltransferase 1 axis.

BACKGROUND: Fasting mimic diet is an effect approach for gastric cancer (GC) treatment. Exploring mechanisms of glucose deprivation-mediated GC suppression is required to develop novel therapeutic regimens. Farnesyltransferase 1 (FDFT1), as a novel target in basic research, has been reported to regulate malignant progression in some types of cancer. However, biological functions of FDFT1 in GC are still unclear. This study focused on biological functions of FDFT1 in GC and the association between glucose starvation (GS) and FDFT1. METHODS: The data derived from the Kaplan-Meier Plotter database were collected to identify the relationship between survival time and FDFT1 expression levels of GC patients. Bioinformatic analysis was performed to explore the biological functions of FDFT1. The expression levels of targeted genes and microRNAs (miRNAs) were detected with immunohistochemistry, quantitative real-time PCR and western blot. Malignant behaviors were measured using cell counting, cell counting kit-8, 5-ethynyl-2-deoxyuridine, wound healing, invasion transwell assays in vitro and constructions of subcutaneous and lung-metastatic tumors in vivo. The glycolysis of GC cells was determined by a series of metabolites, including lactate acid, pyruvic acid, ATP production, rates of glucose uptake, extracellular acidification rate and oxygen consumption rate. RESULTS: FDFT1 was downregulated in GC and negatively correlated with pathological T stage, pathological TNM stage and cancer differentiation. High expression of FDFT1 also indicated better prognosis of GC patients. FDFT1 upregulation attenuated proliferation, migration and invasion of GC. miR-216a-5p was identified as a critical suppressor of FDFT1 expression and miR-216a-5p/FDFT1 axis regulated malignant behaviors and glycolysis of GC cells. GS suppressed malignant behaviors of GC by targeting miR-216a-5p/FDFT1 axis both in vitro and in vivo. CONCLUSION: This study illustrated novel mechanisms by which GS effectively suppresses GC. FDFT1 may become a potential prognostic indicator and novel target of GC therapy.

Knockdown of circ_0011946 targets miR-216a-5p/BCL2L2 axis to regulate proliferation, migration, invasion and apoptosis of oral squamous cell carcinoma cells.

BACKGROUND: Circular RNAs (circRNAs) are implicated in the development of oral squamous cell carcinoma (OSCC). The aim of current research is to elucidate the role and mechanism of circ_0011946 in the functional behaviors of OSCC cells. METHODS: Circ_0011946, microRNA (miR)-216a-5p, B cell lymphoma-2-like 2 protein (BCL2L2) abundances were exposed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blot. Cell proliferation, migration, invasion and apoptosis were detected by MTT, colony formation assay, transwell, wound-healing and flow cytometry assays, respectively. Target correlation was tested by dual-luciferase reporter and RNA pull-down assays. An in vivo xenograft experiment was employed to investigate the function of circ_0011946 on tumor growth in vivo. RESULTS: Circ_0011946 and BCL2L2 levels were increased, while miR-216a-5p level was decreased in OSCC tissues and cells. Circ_0011946 knockdown impeded proliferation, migration, and invasion, but promoted apoptosis in OSCC cells. Circ_0011946 functioned as a sponge for miR-216a-5p, and BCL2L2 was targeted by miR-216a-5p. Besides, miR-216a-5p or BCL2L2 knockdown partly attenuated the inhibitory influences of circ_0011946 silence or miR-216a-5p overexpression on OSCC cell progression. Furthermore, circ_0011946 post-transcriptionally regulated BCL2L2 through sponging miR-216a-5p. Moreover, circ_0011946 knockdown constrained OSCC tumor growth in vivo. CONCLUSION: Circ_0011946 silence repressed OSCC cell proliferation, migration, and invasion, but promoted apoptosis through the regulation of the miR-216a-5p/BCL2L2 axis.

LncRNA OIP5-AS1 Promotes Breast Cancer Progression by Regulating miR-216a-5p/GLO1.

BACKGROUND: Breast cancer is a familiar malignant tumor, which is a great threat to women's life. Long noncoding RNA Opa interacting protein 5-antisense RNA 1 (OIP5-AS1) has been reported to be associated with numerous cancers. This study aimed to explore the role of OIP5-AS1 and the mechanism of its action in the progression of breast cancer. METHODS: The expression of OIP5-AS1 and miR-216a-5p was detected by quantitative real-time polymerase chain reaction. Cell proliferation, apoptosis, migration, or invasion was assessed by 4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, flow cytometry, or transwell assay, respectively. The binding sites were predicted by bioinformatics tool starBase2.0 (http://starbase.sysu.edu.cn/starbase2/index.php). The interaction between miR-216a-5p and OIP5-AS1 or glyoxalase 1 (GLO1) was confirmed by dual-luciferase reporter assay. The expression of GLO1 was quantified by Western blot. Nude mouse tumorigenicity assays were conducted to verify the role of OIP5-AS1 in vivo. RESULTS: OIP5-AS1 and GLO1 were highly expressed in both clinical tumor tissues and cell lines, whereas miR-216a-5p was downregulated. Knockdown of OIP5-AS1 suppressed proliferation, migration, and invasion but promoted apoptosis of breast cancer cells. MiR-216a-5p was a target of OIP5-AS1 and interacted with GLO1. MiR-216a-5p inhibition or GLO1 overexpression reversed the effects of OIP5-AS1 knockdown on the development of breast cancer cells. OIP5-AS1 knockdown depleted tumor growth in vivo. CONCLUSIONS: OIP5-AS1 knockdown suppressed the progression of breast cancer by inducing GLO1 expression via competitively binding to miR-216a-5p, suggesting that OIP5-AS1 was a hopeful biomarker for the therapy of breast cancer.

MiR-216a-5p alleviates LPS-induced inflammation in the human bronchial epithelial cell by inhibition of TGF-ß1 signaling via down-regulating TGFBR2.

OBJECTIVE: Bronchopneumonia is a common respiratory infection disease and is the leading cause of hospitalization in children under 5 years of age. Inflammation is the primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation induced by bronchopneumonia and investigate the potential mechanism underlying it. METHODS: Human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccha-rides (LPS) to trigger bronchopneumonia in vitro. The production of interleukin (IL)-1ß, IL-6, and Tumor necrosis factor (TNF)-α was measured using the enzyme-linked immunosorbent assay. The luciferase assay was conducted to explore the relationship between miR-216a-5p and TGFBR2. Quantitative real-time polymerase chain reaction and western blot were used to detect the gene expression. RESULTS: miR-216a-5p gene expression decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of IL-1ß, IL-6, and TNF-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor (TGF)-ß1, and phosphorylation of SMAD family member 2 (smad2),. This ectopic expression of miR-216a-5p was restored by overexpressed TGFBR2. CONCLUSION: miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-ß1 signaling via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia.Bronchopneumonia is a common respiratory infection disease and is the main cause of hospitalization in children under 5 years of age. Inflammation is a primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation caused by bronchopneumonia and investigate the potential mechanism underlying it. In this study, human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccharides (LPS) to trigger bronchopneumonia in vitro. miR-216a-5p was decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of interleukin (IL)-1ß, IL-6, and Tumor necrosis factor (TNF)-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor-beta 1 (TGF-ß1), and phosphorylation of SMAD family member 2 (smad2. This ectopic overexpression of miR-216a-5p was restored by overexpressed TGFBR2. In conclusion, miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-ß1 signaling via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia.

Lucanthone, Autophagy Inhibitor, Enhances the Apoptotic Effects of TRAIL through miR-216a-5p-Mediated DR5 Upregulation and DUB3-Mediated Mcl-1 Downregulation.

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization.

BACKGROUND: Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. METHODS: Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS: Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. CONCLUSION: Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

DANCR contributed to hepatocellular carcinoma malignancy via sponging miR-216a-5p and modulating KLF12.

Long noncoding RNA (lncRNA) differentiation antagonizing nonprotein coding RNA (DANCR) has been identified as an oncogene in several cancers. However, the biological function and role of DANCR in hepatocellular carcinoma (HCC) remain unclear. Our current study aimed to investigate the detailed mechanism of DANCR in HCC. We found that DANCR was significantly upregulated in HCC cell lines in comparison to LO2 cells. Then, we observed that knockdown of DANCR could greatly inhibit Huh7 and HepG2 cell proliferation. In addition, HCC cell apoptosis was increased by silence of DANCR and meanwhile, cell cycle progression was blocked in G1 phase. Apart from these, downregulation of DANCR repressed HCC cell migration and invasion ability obviously. As predicted by the bioinformatics analysis, microRNA-216a-5p (miR-216a-5p) could serve as a direct target of DANCR. MiR-216a-5p has been reported to be involved in many cancers. Here, the correlation between miR-216a-5p and DANCR was confirmed using dual-luciferase reporter assay and radioimmunoprecipitation assay. Subsequently, Kruppel-like factor 12 (KLF12) exerts an important role in different tumor types. KLF12 can function as a downstream target of miR-216a-5p. Finally, the in vivo experiments were used and the data proved that DANCR also strongly suppressed HCC tumor growth in vivo via targeting miR-216a-5p and KLF12. In conclusion, our study indicated that DANCR might provide a new perspective for HCC treatment.

lncRNA A1BG-AS1 suppresses proliferation and invasion of hepatocellular carcinoma cells by targeting miR-216a-5p.

Extensive evidence indicate that long noncoding RNAs (lncRNAs) regulates the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the expression and biological function of lncRNA A1BG antisense RNA 1 (A1BG-AS1) were poorly known in HCC. Here, we found the underexpression of A1BG-AS1 in HCC via analysis of The Cancer Genome Atlas database. Further analyses confirmed that A1BG-AS1 expression in HCC was markedly lower than that in noncancerous tissues based on our HCC cohort. Clinical association analysis revealed that low A1BG-AS1 expression correlated with poor prognostic features, such as microvascular invasion, high tumor grade, and advanced tumor stage. Follow-up data indicated that low A1BG-AS1 level evidently correlated with poor clinical outcomes of HCC patients. Moreover, forced expression of A1BG-AS1 repressed proliferation, migration, and invasion of HCC cells in vitro. Conversely, A1BG-AS1 knockdown promoted these malignant behaviors in HepG2 cells. Mechanistically, A1BG-AS1 functioned as a competing endogenous RNA by directly sponging miR-216a-5p in HCC cells. Notably, miR-216a-5p restoration rescued A1BG-AS1 attenuated proliferation, migration and invasion of HCCLM3 cells. A1BG-AS1 positively regulated the levels of phosphatase and tensin homolog and SMAD family member 7, which were reduced by miR-216a-5p in HCC cells. Altogether, we conclude that A1BG-AS1 exerts a tumor suppressive role in HCC progression.

The estrogen-regulated lncRNA H19/miR-216a-5p axis alters stromal cell invasion and migration via ACTA2 in endometriosis.

Fibrotic tissue may contribute to the origin of some endometriosis-related symptoms, such as chronic pelvic pain and infertility. Alterations in the H19/miR-216a-5p/ACTA2 pathway may mediate the regulation of eutopic endometrial stromal cell (euESC) invasion and migration and may represent a potential mechanism underlying fibrous tissue formation or fibrosis in women with endometriosis. In this study, we aimed to determine the expression of H19 and ACTA2 in endometrial tissues of women with endometriosis. Two groups of 23 infertile women with endometriosis and 23 matched infertile women without endometriosis were investigated. Primary cultured cells of endometrial tissues were analyzed using RT-PCR and western blotting (WB) to determine expression of H19 and ACTA2. 5-Ethyl-2'-deoxyuridine, CCK8 and Transwell assays were used to study the functions of H19 and ACTA2. Human embryonic kidney 293 cells were used for luciferase assays to study miR-216a-5p binding sites with H19 and ACTA2. We found that H19 and ACTA2 levels were significantly higher in endometriosis euESCs than in control euESCs (P < 0.05) and were positively correlated in endometriosis euESCs. Luciferase assays indicated that H19 regulates ACTA2 expression via competition for inhibitory miR-216a-5p binding sites. Our results indicate that alterations in the estrogen/H19/miR-216a-5p/ACTA2 pathway regulated endometriosis euESC invasion and migration. Downregulation of H19 or ACTA2 inhibited endometriosis euESC invasion and migration; however, estrogen promoted endometriosis euESC invasion and migration via H19. The main limitation of our study was that experiments were conducted in vitro and further in vivo studies are required in the future. However, our study showed that primary cultured cells represented endometriosis cells more clearly than cell lines.

MiR-216a-5p targets TCTN1 to inhibit cell proliferation and induce apoptosis in esophageal squamous cell carcinoma.

BACKGROUND: MiR-216a-5p has been reported to be associated with several tumors, including prostate cancer and melanoma. However, its expression level and potential role in esophageal squamous cell carcinoma (ESCC) remain uncertain. RESULTS: Here, we found that miR-216a-5p expression was significantly down-regulated in clinical ESCC tissues and cells. Functional assays were performed to evaluate the biological effects of miR-216a-5p on cell proliferation and cell apoptosis by CCK-8 assay and flow cytometry in ESCC cell lines, EC9706 and TE-9. The results showed that miR-216a-5p overexpression repressed cell proliferation and induced cell apoptosis. Through bioinformatics prediction and luciferase reporter assay, we revealed that miR-216a-5p could directly target tectonic family member 1 (TCTN1). Moreover, TCTN1 was obviously suppressed by miR-216a-5p overexpression. In addition, TCTN1 expression was significantly increased and inversely correlated with the levels of miR-216a-5p in ESCC tissues. More importantly, down-regulation of TCTN1 imitated, while restoration of TCTN reversed the effects of miR-216a-5p on cell proliferation and apoptosis. At the molecular level, we further found that TCTN1 overexpression reversed the effects of miR-216a-5p transfection on the expression of PCNA, Bcl-2 and Bad. CONCLUSIONS: Our results demonstrate that miR-216a-5p might serve as a tumor suppressor in ESCC cells through negatively regulating TCTN1 expression, indicating the possibility that miR-216a-5p and TCTN1 might be attractive targets for ESCC therapeutic intervention.

MiR-216a-5p/Hexokinase 2 axis regulates uveal melanoma growth through modulation of Warburg effect.

Hexokinase-2 (HK2), the initial as well as the rate-limiting step in glycolysis, is overexpressed in many human cancers, and correlates with poor clinical outcomes. Aerobic glycolysis is a hallmark of cancer, and drugs targeting its enzymes, including HK2, are being developed. However, the mechanisms of HK2 inhibition and the physiological significance of the HK2 inhibitors in cancer cells are rarely reported. Here, we show that microRNA-216a-5p (miR-216a-5p) inhibits HK2 expression by directly targeting its 3'-UTR in uveal melanoma cells. Through inhibition of HK2, miR-216a-5p dampens glycolysis by reducing HK activity, glucose uptake, lactate production, ATP generation, extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in uveal melanoma cells. Importantly, glycolysis regulated by miR-216a-5p is critical for its regulating uveal melanoma tumor growth both in vitro and in vivo. miR-216a-5p expression is negatively correlated with HK2 expression and predicts better outcome in uveal melanoma patients. Our findings provide clues regarding the role of miR-216a-5p as a tumor suppressor in uveal melanoma through the inhibition of HK2. Targeting HK2 through miR-216a-5p could be a promising therapeutic strategy in uveal melanoma.

Ginsenoside Rg3 induces mesangial cells proliferation and attenuates apoptosis by miR-216a-5p/MAPK pathway in diabetic kidney disease.

BACKGROUND: Ginsenoside Rg3 is an active saponin isolated from ginseng, which can reduce renal inflammation. However, the role and mechanism of Rg3 in diabetic kidney disease (DKD) are far from being studied. METHODS: The effects of Rg3 and miR-216a-5p on the proliferation, apoptosis, and MAPK pathway in high glucose (HG)-induced SV40 MES 13 were monitored by CCK-8, TUNEL staining, and western blot. RESULTS: Rg3 treatment could accelerate proliferation and suppress apoptosis in HG-induced SV40 MES. Moreover, miR-216a-5p inhibition also could alleviate renal injury, prevent apoptosis, and activate the MAPK pathway in kidney tissues of diabetic model mice. CONCLUSION: Rg3 could attenuate DKD progression by downregulating miR-216a-5p, suggesting Rg3 and miR-216a-5p might be the potential drug and molecular targets for DKD therapy.

MALAT1 promotes FOXA1 degradation by competitively binding to miR-216a-5p and enhancing neuroendocrine differentiation in prostate cancer.

OBJECTIVES: Prostate cancer (PC) is a leading cause of cancer-related death in males worldwide. Neuroendocrine differentiation (NED) is a feature of PC that often goes undetected and is associated with poor patient outcomes. Long non-coding RNAs (lncRNAs), microRNAs (miRNAs/miRs), and messenger RNAs (mRNAs) play important roles in the development and progression of PC. METHODS: In this study, we used transcriptome sequencing and bioinformatics analysis to identify key regulators of NED in PC. Specifically, we examined the expression of PC-related lncRNAs, miRNAs, and mRNAs in PC cells and correlated these findings with NED phenotypes. RESULTS: Our data revealed that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and zinc finger protein 91 (ZFP91) were upregulated in PC, while miR-216a-5p was down-regulated. Ectopic expression of MALAT1 induced NED and promoted malignant phenotypes of PC cells. Furthermore, we found that MALAT1 competitively bound to miR-216a-5p, upregulated ZFP91, and promoted the degradation of forkhead box A1 (FOXA1), a key gene involved in NED of PC. CONCLUSION: Taken together, these results suggest that MALAT1 plays an oncogenic role in NED and metastasis of PC via the miR-216a-5p/ZFP91/FOXA1 pathway. Our study highlights the potential of targeting this pathway as a novel therapeutic strategy for PC.

Evaluation of circulating miR-216a and miR-217 as biomarkers of pancreatic damage in the L-arginine-induced acute pancreatitis mouse model.

We investigated the usefulness of circulating miR-216a-5p and miR-217-5p that are pancreas-enriched micro RNAs (miRNAs) as biomarkers of acute pancreatic damage, and compared them with conventional pancreatic biomarkers in L-arginine-induced acute pancreatitis mouse model. As the results, amylase and lipase levels apparently increased and peaked on Day 3 when acute pancreatitis including acinar cell degeneration/necrosis and inflammatory cell infiltration reached its peak. In contrast, miR-216a-5p and miR-217-5p increased from Day 1 when histopathological findings in the acinar cells were limited to decreased zymogen granules, and the increases in ratios were much higher than those of amylase and lipase. The miRNAs remained at high levels until Day 5 when the pseudo-tubular complex and replacement of inflammatory cells and fibrotic cells were apparent instead of necrosis, whereas amylase and lipase levels decreased to the control levels. Furthermore, we examined the relationship between biomarker levels and histopathological degeneration/necrosis scores in the acinar cells. miR-216a-5p and miR-217-5p levels increased depending on the score of degeneration/necrosis, and all individual miRNAs exceeded the control levels from a score of 2 (focal necrosis), whereas all individual amylase and lipase levels exceeded the control levels at scores of 4 (lobular necrosis) and 3 (sublobular necrosis), respectively. In conclusion, we demonstrated that circulating miR-216a-5p and miR-217-5p could detect pancreatic damage earlier with greater magnitude, and the sensitivity to detect acinar cell degeneration/necrosis was superior to that of conventional biomarkers in the L-arginine-induced acute pancreatitis mouse model.

Hypoxic ASCs-derived Exosomes Attenuate Colitis by Regulating Macrophage Polarization via miR-216a-5p/HMGB1 Axis.

BACKGROUND: Exosomes derived from mesenchymal stem cells have shown therapeutic effects for colitis. As a more clinically accessible resource, the therapeutic potential of exosomes from adipose-derived stem cells (ASCs) has not been fully elucidated, and whether hypoxia precondition could improve the therapeutic effect of ASC-derived exosomes in colitis remains elusive. METHODS: In this study, exosomes were derived from ASCs under normoxia (NExos) and hypoxia (HExos) and were identified by detecting their morphology, size distribution, and exosome surface markers. The concentration of inflammation-related cytokines was detected by ELISA, and macrophage phenotype-related genes were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and immunofluorescence. A miRNA microarray sequencing analysis was conducted to confirm the differentially expressed miRNAs. Dextran sulfate sodium-induced colitis was employed as an in vivo assay. RESULTS: Administration of NExos alleviated inflammation by modulating the balance of macrophages both in cellular assays and in vivo experiments, and HExos showed higher therapeutic efficiency than NExos. The miR-216a-5p in HExos was significantly enriched and promoted macrophage M2 polarization through transfer to macrophages by exosomes. The miR-216a-5p was confirmed to target the 3'-UTR of HMGB1. Mechanistically, hypoxia-induced ASCs release miR-216a-5p in an exosomal way that induced macrophage M2 polarization by regulating the HMGB1/TLR4/NF-κB signaling pathway. CONCLUSIONS: Exosomal miR-216a-5p released from hypoxia-prime ASCs showed higher therapeutic efficiency than NExos in experimental colitis by promoting the M2 macrophage phenotype, which indicated that hypoxia prime may represent a promising approach to optimizing the function of ASC-derived exosomes.

Exosomal miR-216a-5p released from hypoxia-prime ASCs showed higher therapeutic efficiency than NExos in experimental colitis by promoting the M2 macrophage phenotype, which indicated that hypoxia prime may represent a promising approach to optimize the function of ASC-derived exosomes.

[Moxibustion relieves colonic inflammation by up-regulating expression of miR-345-3p/miR-216a-5p and down-regulating NF-κB p65 in colonic tissue of rats with diarrhea-predominant irritable bowel syndrome].

OBJECTIVE: To observe the effect of moxibustion on the expression of miR-345-3p, miR-216a-5p and nuclear factor-κB p65(NF-κB p65) in colonic tissue of rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its anti-inflammatory mechanism in relieving IBS-D. METHODS: SD rats were randomly divided into normal control (n=12), model (n=12), moxibustion (n=12) and ammonium pyrrolidine dithiocarbamate (PDTC,n=12) groups. The IBS-D model was established by neonatal mother-child separation combined with acetic acid enema stimulation and chronic binding methods. The rats in the moxibustion group received moxibustion stimulation of "Tianshu"(ST25) and "Shangjuxu"(ST37) for 20 min, once a day, for 7 days, and those of the PDTC group received intraperitoneal injection of PDTC (50 mg·kg-1·d-1) once daily for 7 days. After the intervention, the body weight, loose stool rate and the minimum volume threshold of abdominal withdrawal reflex (AWR) were observed, and histopathological changes of colonic mucosa were observed by HE staining. The contents of interleukin-1ß (IL-1ß), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF- α) in serum were measured by ELISA. The expression of miR-345-3p, miR-216a-5p and NF-κB p65 mRNA in the colon tissue were detected by quantitative real-time PCR, and the immunoactivities of IL-1ß, IL-6, TNF-α and NF-κB p65 in the colon tissue were determined by immunofluorescence histochemistry. RESULTS: Compared with the normal control group, the loose stool rate, contents of IL-1ß, IL-6 and TNF-α, experssion of NF-κB p65 mRNA and the immunoactivities of IL-1ß, IL-6, TNF-α and NF-κB p65 were significantly increased (P<0.01), whereas the body weight, minimum volume threshold of AWR, content of IL-4, and the relative expression of miR-345-3p and miR-216a-5p were remarkably decreased in the model group (P<0.01). In comparison with the model group, the loose stool rate, contents of IL-1ß, IL-6, TNF-α, expression of NF-κB p65 mRNA and the immunoactivities of IL-1ß, IL-6, TNF-α and NF-κB p65 were considerably down-regulated (P<0.01), while the content of IL-4 and the relative expressions of miR-345-3p and miR-216a-5p were obviously up-regulated in both moxibustion and PDTC groups (P<0.01, P<0.05). The content of IL-6 in serum was significantly lower in the PDTC group than in the moxibustion group (P<0.01). CONCLUSION: Moxibustion can reduce the level of intestinal inflammation and visceral hypersensitivity in IBS-D rats, which may be related to its functions in increasing the expression levels of miR-345-3p and miR-216a-5p and in inhibiting the expression of NF-κB p65, thus reducing the levels of inflammatory factors.

circPLOD2 knockdown suppresses endometriosis progression via the miR-216a-5p/ZEB1 axis.

The present study aimed to identify the role of circPLOD2 in endometriosis and its underlying mechanisms. We determined circPLOD2 and miR-216a-5p expression in ectopic endometrial (EC) and eutopic endometrial (EU) samples as well as in endometrial samples from uterine fibroids of ectopic patients (EN) and embryonic stem cells (ESCs) using qRT-PCR. The association between circPLOD2 and miR-216a-5p or miR-216a-5p and zinc finger E-box binding homeobox 1 (ZEB1) expression was analyzed using Starbase, TargetScan, and dual-luciferase reporter gene assays. Cell viability, apoptosis, and migration and invasion were assessed using MTT, flow cytometry, and transwell assays, respectively. In addition, qRT-PCR and western blotting was used to measure circPLOD2, miR-216a-5p, E-cadherin, N-cadherin, and ZEB1 expression. circPLOD2 was upregulated and miR-216a-5p was downregulated in EC samples compared with that in EU samples. Similar trends were observed in ESCs. circPLOD2 interacted and negatively regulated miR-216a-5p expression in EC-ESCs. circPLOD2-siRNA significantly inhibited EC-ESC growth; promoted cellular apoptosis; and inhibited EC-ESC migration, invasion, and epithelial-mesenchymal transition; these effects could be reversed following miR-216a-5p inhibitor transfection. miR-216a-5p directly targeted and negatively regulated ZEB1 expression in EC-ESCs. In conclusion, circPLOD2 promotes the proliferation, migration, and invasion of EC-ESCs and inhibits their apoptosis by targeting miR-216a-5p. These findings indicate potential therapeutic targets for endometriosis.