Overexpression of miR-96 leads to retinal degeneration in mice.

Double knockout of miR-183 and miR-96 results in retinal degeneration in mice; however, single knockout of miR-96 leads to developmental delay but not substantial retinal degeneration. To further explore the role of miR-96, we overexpressed this miRNA in mouse retinas. Interestingly, we found that overexpression of miR-96 at a safe dose results in retinal degeneration in the mouse retina. The retinal photoreceptors dramatically degenerated in the miR-96-overexpressing group, as shown by OCT, ERG and cryosectioning at one month after subretinal injection. Degenerative features such as TUNEL signals and reactive gliosis were observed in the miR-96-overexpressing retina. RNA-seq data revealed that immune responses and microglial activation occurred in the degenerating retina. Further qRT‒PCR and immunostaining experiments verified the microglial activation. Moreover, the number of microglia in the miR-96-overexpressing retinas was significantly increased. Our findings demonstrate that appropriate miR-96 expression is required for mouse retinal homeostasis.

LncRNA ZFAS1 Alleviated NLRP3 Inflammasome-Mediated Pyroptosis through Regulating miR-96-5p/Smad7 Signaling in Allergic Rhinitis.

INTRODUCTION: The NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptosis was positively correlated with the allergic rhinitis progression and was reported to be regulated by SMAD family member 7 (Smad7). Bioinformatics analysis revealed that Smad7 might be targeted by miR-96-5p, and miR-96-5p might be targeted by long noncoding RNA zinc finger antisense 1 (ZFAS1). However, the effects and regulatory mechanisms of the ZFAS1/miR-96-5p/Smad7 functional axis in allergic rhinitis have not been investigated. METHODS: Human nasal mucosa epithelial cell line RPMI 2650 and C57BL/6 mice were obtained for in vitro and in vivo studies. Dual-luciferase reporter assay and RNA immunoprecipitation were implemented for detecting molecular interactions. Cell counting kit-8 and flow cytometry were used for measuring cell viability and pyroptosis. ELISA was obtained for monitoring cytokine secretion. RT-qPCR and Western blot were examined for determining RNA and protein expression. RESULTS: In vitro studies revealed that ZFAS1 was downregulated in interleukin (IL)-13-treated RPMI 2650 cells, while overexpression of ZFAS1 enhanced cell viability and inhibited NLRP3-mediated pyroptosis and inflammatory response. ZFAS1 directly inhibited miR-96-5p to suppress NLRP3-mediated pyroptosis in IL-13-treated RPMI 2650 cells. MiR-96-5p bound to the 3'-untranslated region of Smad7 and knockdown of Smad7 significantly reversed the effects of miR-96-5p depletion. Moreover, in vivo experiments further confirmed the findings of in vitro studies and showed ZFAS1 overexpression or miR-96-5p inhibition alleviated allergic rhinitis in vivo. CONCLUSION: ZFAS1 downregulated the expression of miR-96-5p to upregulate Smad7 level, which subsequently inhibited NLRP3-mediated pyroptosis and inflammatory response to ameliorate allergic rhinitis.

CircItgb5 promotes synthetic phenotype of pulmonary artery smooth muscle cells via interacting with miR-96-5p and Uba1 in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal cardiopulmonary disease mainly characterized by pulmonary vascular remodeling. Aberrant expression of circRNAs has been reported to play a crucial role in pulmonary vascular remodeling. The existing literature predominantly centers on studies that examined the sponge mechanism of circRNAs. However, the mechanism of circRNAs in regulating PAH-related protein remains largely unknown. This study aimed to investigate the effect of circItgb5 on pulmonary vascular remodeling and the underlying functional mechanism. MATERIALS AND METHODS: High-throughput circRNAs sequencing was used to detect circItgb5 expression in control and PDGF-BB-treated pulmonary arterial smooth muscle cells (PASMCs). Localization of circItgb5 in PASMCs was determined via the fluorescence in situ hybridization assay. Sanger sequencing was applied to analyze the circularization of Itgb5. The identification of proteins interacting with circItgb5 was achieved through a RNA pull-down assay. To assess the impact of circItgb5 on PASMCs proliferation, an EdU assay was employed. Additionally, the cell cycle of PASMCs was examined using a flow cytometry assay. Western blotting was used to detect biomarkers associated with the phenotypic switch of PASMCs. Furthermore, a monocrotaline (MCT)-induced PAH rat model was established to explore the effect of silencing circItgb5 on pulmonary vascular remodeling. RESULTS: CircItgb5 was significantly upregulated in PDGF-BB-treated PASMCs and was predominately localized in the cytoplasm of PASMCs. In vivo experiments revealed that the knockdown of circItgb5 attenuated MCT-induced pulmonary vascular remodeling and right ventricular hypertrophy. In vitro experiments revealed that circItgb5 promoted the transition of PASMCs to synthetic phenotype. Mechanistically, circItgb5 sponged miR-96-5p to increase mTOR level and interacted with Uba1 protein to activate the Ube2n/Mdm2/ACE2 pathway. CONCLUSIONS: CircItgb5 promoted the transition of PASMCs to synthetic phenotype by interacting with miR-96-5p and Uba1 protein. Knockdown of circItgb5 mitigated pulmonary arterial pressure, pulmonary vascular remodeling and right ventricular hypertrophy. Overall, circItgb5 has the potential for application as a therapeutic target for PAH.

Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96-3p.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. METHODS: Specific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96-3p to CPA6 3'UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96-3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. RESULTS: Knockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96-3p could directly regulate CPA6 expression by targeting its 3'UTR, and miR-96-3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96-3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. CONCLUSION: CPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96-3p negatively regulates the expression of CPA6.

Transcription Factor Sox9 Exacerbates Kidney Injury through Inhibition of MicroRNA-96-5p and Activation of the Trib3/IL-6 Axis.

INTRODUCTION: Our study investigated the possible mechanisms of the role of the transcription factor Sox9 in the development and progression of kidney injury through regulation of the miR-96-5p/Trib3/IL-6 axis. METHODS: Bioinformatics analysis was performed to identify differentially expressed genes in kidney injury and normal tissues. An in vivo animal model of kidney injury and an in vitro cellular model of kidney injury were constructed using LPS induction in 8-week-old female C57BL/6 mice and human normal renal tubular epithelial cells HK-2 for studying the possible roles of Sox9, miR-96-5p, Trib3, and IL-6 in kidney injury. RESULTS: Sox9 was highly expressed in both mouse and cellular models of kidney injury. Sox9 was significantly enriched in the promoter region of miR-96-5p and repressed miR-96-5p expression. Trib3 was highly expressed in both mouse and cellular models of kidney injury and promoted inflammatory responses and kidney injury. In addition, Trib3 promoted IL-6 expression, which was highly expressed in kidney injury, and promoted the inflammatory response and extent of injury in kidney tissue. In vivo and in vitro experiments confirmed that the knockdown of Sox9 improved the inflammatory response and fibrosis of mouse kidney tissues and HK-2 cells, while the ameliorative effect of silencing Sox9 was inhibited by overexpression of IL-6. CONCLUSION: Collectively, Sox9 up-regulates miR-96-5p-mediated Trib3 and activates the IL-6 signaling pathway to exacerbate the inflammatory response, ultimately promoting the development and progression of kidney injury.

Molecular characterization of cattle and sheep isolates of Echinococcus granulosus from Elazig province in Turkey and expression analysis of the non-coding RNAs, egr-miR-7, egr-miR-71 and egr-miR-96.

Cystic Echinococcosis (CE) is a common zoonotic disease seen in human and animals worldwide, caused by the larval form of Echinococcus granulosus. In this study, E. granulosus s.l. species and haplotypes were determined in hydatid cysts isolated from cattle and sheep, and the expression levels of egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were compared in different cyst structures. A total of 82 (cattle, n = 41; sheep, n = 41) hydatid cyst isolates (germinal membranes and/or protoscoleces) were collected from a slaughterhouse in Elazig province of Turkey. After mt-CO1 gene sequences were made, 81 out of 82 hydatid cyst isolates were determined as E. granulosus s.s. (G1 and G3), while an isolate of cattle origin was determined as Echinococcus canadensis (G6/7). A total of 26 nucleotide polymorphisms and 29 haplotype groups were identified in the samples. miRNA expressions in germinal membranes of sterile cysts and germinal membrane and protoscoleces of fertile cysts were investigated by qRT-PCR and Real Time PCR analyses. It was determined that miRNAs were expressed at high levels in 79.31% of the 29 haplotype groups and at low levels in the remaining 10.34%. In 10 fertile samples of sheep origin, egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were found to be 44, 168, and 351-fold higher in expression, respectively, in the germinal membrane compared to the protoscoleces. Especially egr-miR-96 may have the potential to be used as biomarkers in the diagnosis of active CE.

CircRIMS promotes cerebral ischemia-reperfusion injury through increasing apoptosis and targeting the miR-96-5p/JAK/STAT1 axis.

OBJECTIVE: This study aims to explore the function of circRIMS in cerebral ischemia/reperfusion (CIR) and its regulatory mechanism. METHOD: The expression of the circRIMS was examined in GEO chip data and validated by qRT-PCR analysis. A middle cerebral artery occlusion/repression (MCAO/R) model was developed using C57BL/6J mice. Starbase and circinteractome were employed to identify the target miRNA and mRNA. The result was confirmed by dual-luciferase reporter assay, and biotinylated RNA-pulldown assay. The cell viability and apoptosis were confirmed through CCK-8 and flow cytometry assay. RESULTS: This study revealed that circRIMS expression was upregulated in MCAO mice model and OGD/RX-simulated cell model. Knockdown circRIMS demonstrated the functional of circRIMS in increasing cell viability, reducing apoptosis, LDH activity and inflammatory factors secretion in OGD/RX-simulated CIR injury in vitro. Additionally, miR-96-5p was identified as a target of circRIMS, while the STAT1 gene is a downstream gene of miR-96-5p, and JAK was also considered to be a downstream gene of the JAK-STAT pathway. Furthermore, inhibition of miR-96-5p or overexpression of STAT1 promoted the progression of CIR injury by elevating apoptosis, reducing cell viability, and increasing the secretion of inflammatory cytokines. CONCLUSION: CircRIMS contributes to the progression of CIR injury via regulating miR-96-5p/JAK/STAT1 axis.

[Effect of miR-96-5p targeting IRS1 on apoptosis of PC12 cells induced by aluminum maltol].

Objective: To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. Methods: In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 µmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3ß (p-GSK3ß) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 µmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3ß were measured. Results: After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3ß proteins were significantly decreased (P<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3ß proteins were significantly increased (P<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3ß proteins were significantly decreased (P<0.05) . Conclusion: The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.

circFBXO7/miR-96-5p/MTSS1 axis is an important regulator in the Wnt signaling pathway in ovarian cancer.

BACKGROUND: CircRNAs are a novel class of evolutionarily conserved noncoding RNA molecules that form covalently closed continuous loop structures without 5' caps and 3' poly(A) tails. Accumulating evidence suggests that circRNAs play important regulatory roles in cancer and are promising biomarkers for cancer diagnosis and prognosis, as well as targets for cancer therapy. In this study, we identify and explore the role of a novel circRNA, circFBXO7, in ovarian cancer. METHODS: rRNA-depleted RNA-sequencing was performed to identify differentially expressed circRNAs between ovarian cancerous and normal tissues. qRT-PCR and single-molecule RNA in-situ hybridization was used to quantify circFBXO7 expression in tumor tissues. The association of circFBXO7 expression with patient prognosis was evaluated by Kaplan-Meier survival analysis. The biological function of circFBXO7 was also investigated using loss-of-function and gain-of-function assays in vivo and in vitro. Luciferase reporter and TOP/FOP-Flash reporter assays were then conducted together with RNA immunoprecipitation and western blot to assess the circFBXO7/miR-96-5p/MTSS1/Wnt/ß-catenin axis. RESULTS: circFBXO7 was downregulated in ovarian cancer which was associated with poor prognosis. Biologically, circFBXO7 overexpression significantly suppressed ovarian cancer cell proliferation, migration, and invasion in vitro, and inhibited tumor growth and metastasis in vivo, whereas its knockdown exerted an opposite role. Mechanistically, circFBXO7 functioned as a competing endogenous RNA for miR-96-5p to regulate the expression of MTSS1. Consequently, downregulation of MTSS1 led to excessive accumulation of ß-catenin and increased phosphorylation of GSK3ß, leading to the translocation of ß-catenin to the nucleus, thereby activating the Wnt/ß-catenin signaling pathway and ultimately promoting ovarian cancer progression. CONCLUSIONS: Our findings indicate that circFBXO7 acts as a bone fide tumor suppressor in ovarian cancer and that the circFBXO7/miR-96-5p/MTSS1 axis is an important regulator in the Wnt/ß-catenin signaling pathway which may provide a promising target for ovarian cancer therapy.

LncRNA NDRG1 aggravates osteosarcoma progression and regulates the PI3K/AKT pathway by sponging miR-96-5p.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumors in children and adolescents. Large numbers of studies have focused on the long non-coding RNA (lncRNA) that plays essential roles in the progression of osteosarcoma. Nevertheless, the functions and underlying mechanisms of LncRNA NDRG1 in osteosarcoma remain unknown. METHODS: Differentially expressed lncRNAs between osteosarcoma and adjacent normal tissues were identified through RNA sequencing. The role of LncRNA NDRG1 in osteosarcoma proliferation and metastasis were investigated through in vitro and in vivo functional experiments. The interaction between LncRNA NDRG1 and miR-96-5p was verified through bioinformatic analysis and luciferase reporter assay. Regulation relationship between LncRNA NDRG1 and miR-96-5p was further evaluated by the rescue experiments. Additionally, the changes in the expression of epithelial-mesenchymal transition (EMT) and the PI3K/AKT pathway were verified by Western blot. RESULTS: LncRNA NDRG1 was up-regulated in osteosarcoma cell lines and tissues and the expression of LncRNA NDRG1 was correlated with the overall survival of osteosarcoma patients. Functional experiments exhibited that LncRNA NDRG1 aggravated osteosarcoma proliferation and migration in vitro; meanwhile, animals experiments showed that LncRNA NDRG1 promoted osteosarcoma growth and metastasis in vivo. Mechanistically, LncRNA NDRG1 was found to aggravate osteosarcoma progression and regulate the PI3K/AKT pathway by sponging miR-96-5p. CONCLUSIONS: LncRNA NDRG1 aggravates osteosarcoma progression and regulates the PI3K/AKT pathway by sponging miR-96-5p. Therefore, LncRNA NDRG1 could act as a prognostic marker and a therapeutic target for osteosarcoma in the future.

Identification of microRNA-96-5p as a postoperative, prognostic microRNA predictor in nonviral hepatocellular carcinoma.

AIM: The microRNA (miR) clusters miR-183/96/182 and miR-217/216a/216b are significantly upregulated in nonviral hepatocellular carcinoma (NBNC-HCC). Here, we investigate the impact of each member of these clusters on the clinical outcome of NBNC-HCC and analyze the antitumor effects of miR-96-5p. METHODS: The association between recurrence-free survival of 111 NBNC-HCC patients and the levels of miR-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, miR-216a-5p, and miR-216b-5p in tumor and adjacent tissues was investigated. The impact of miR-96-5p on apoptosis and invasion of a hepatoma cell line, HepG2, was investigated by cell counting, Transwell assay, and flow cytometry, respectively. RESULTS: MicroRNA-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, and miR-216b-5p were significantly upregulated in tumor tissues compared to the adjacent tissues (p = 0.0005, p = 0.0030, p = 0.0002, p = 0.0011, and p = 0.0288, respectively). By multivariate Cox regression analysis, high tumor/adjacent ratios of miR-182-5p (p = 0.007) and miR-217-5p (p = 0.008) were associated with poor recurrence-free survival. In contrast, a low tumor/adjacent ratio of miR-96-5p (p < 0.001) was associated with poor recurrence-free survival. It suggested that further upregulation of miR-96-5p in tumors might have an inhibitory effect on recurrence. Transfection of miR-96-5p mimic significantly induced apoptosis of HepG2 cells, in association with downregulation of Nucleophosmin 1 (NPM1) and a decrease of phosphorylated AKT protein. Interestingly, simultaneous knockdown of the NPM1 and AKT genes induced apoptosis. MicroRNA-96-5p also suppressed proliferation and invasion, which inhibited epithelial-to-mesenchymal transition of HCC cells. CONCLUSION: MicroRNA-96-5p as a tumor suppressor would be valuable to stratify NBNC-HCC patients at high risk of recurrence.

miR-96-5p regulates myocardial infarction-induced cardiac fibrosis via Smad7/Smad3 pathway.

Fibrotic remodelling contributes to heart failure in myocardial infarction. MicroRNAs (miRNAs) play a crucial role in myocardial fibrosis. However, current antifibrotic therapeutic strategies using miRNAs are far from effective. In this study, we aim to investigate the effect of miR-96-5p on cardiac fibrosis. Our work reveals a significant upregulation of miR-96-5p level in the ventricular tissues of myocardial infarction mice, as well as in neonatal rat cardiac fibroblasts stimulated with TGF-ß or Ang II as shown by qPCR assay. In myocardial infarction mice, miR-96-5p knockdown using antagomir alleviates the aggravated cardiac fibrosis and exacerbated myocardial function caused by myocardial infarction surgery as shown by the echocardiography and Masson's staining analysis. In contrast, immunofluorescence staining results reveal that miR-96-5p overexpression in neonatal rat cardiac fibroblasts contributes to an increase in the expressions of fibrosis-associated genes and promotes the proliferation and differentiation of cardiac fibroblasts. Conversely, miR-96-5p downregulation using inhibitor presents adverse consequences. Furthermore, Smad7 expression is downregulated in fibrotic cardiac tissues, and the Smad7 gene is identified as a direct target of miR-96-5p by dual luciferase assay. Indeed, Smad7 knockdown weakens the anti-fibrotic effect of the miR-96-5p inhibitor on cardiac fibroblasts. Moreover, Smad3 phosphorylation is elevated in fibrotic cardiac tissues, and interestingly, the Smad3 inhibitor suppresses the profibrotic effect of the miR-96-5p mimic. Taken together, our findings demonstrate that the Smad7/Smad3 signaling pathway mediates the profibrotic effect of miR-96-5p in cardiac fibrosis.

AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR-96-5p-AIMP3-p53 axis.

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a tumour suppressor, however, the roles of AIMP3 in non-small cell lung cancer (NSCLC) are not explored yet. Here, we reported that AIMP3 significantly inhibited the cell growth and metastasis of NSCLC (lung adenocarcinoma) in vitro and in vivo. We have firstly identified that AIMP3 was down-regulated in human NSCLC tissues compared with adjacent normal lung tissues using immunohistochemistry and western blot assays. Overexpression of AIMP3 markedly suppressed the proliferation and migration of cancer cells in a p53-dependent manner. Furthermore, we observed that AIMP3 significantly suppressed tumour growth and metastasis of A549 cells in xenograft nude mice. Mechanically, we identified that AIMP3 was a direct target of miR-96-5p, and we also observed that there was a negative correlation between AIMP3 and miR-96-5p expression in paired NSCLC clinic samples. Ectopic miR-96-5p expression promoted the proliferation and migration of cancer cells in vitro and tumour growth and metastasis in vivo which partially depended on AIMP3. Taken together, our results demonstrated that the axis of miR-96-5p-AIMP3-p53 played an important role in lung adenocarcinoma, which may provide a new strategy for the diagnosis and treatment of NSCLC.

miR-96-5p is the tumor suppressor in osteosarcoma via targeting SYK.

BACKGROUND: Osteosarcoma is a multiple malignant tumor in adolescents. MicroRNAs (MiRNAs) have been found to express abnormally in OS tissues and are considered as potential targets for OS prognosis and treatment. METHODS: MiR-96-5p and SYK expression in clinical samples, osteoblast and OS cell lines were detected. The changes of cell proliferation, apoptosis, adhesion and metastasis of OS cells were detected by CCK-8, BrdU, caspase-3 activity and transwell assay. Dual luciferase report analysis and RNA pull-down were used to confirm binding relation of miR-96-5p and SYK. RESULTS: MiR-96-5p was increased in OS tissue and cells. Moreover, miR-96-5p inhibits proliferation, adhesion and migration of HOS and Saos-2 cells, and promotes cell apoptosis. SYK has been identified to be targeted by miR-96-5p. Overexpressed SYK inhibits the suppressive impact of miR-96-5 on OS cells. CONCLUSION: MiR-96-5p may function as an effective target in OS treatment.

miRNA96 expression level within red blood cells is probably associated with RSL indicators during the storage of red blood cell units.

BACKGROUND AND OBJECTIVES: Many biochemical and hematological changes occur during the storage of RBC units. Collectively, these changes are known as RSLs. Previous studies found miRNA96 as non-coding RNA that its expression level changed during RBC storage. However, its correlation with mechanical and biochemical RSL indicators is not yet determined. Therefore, this study aimed to assess possible correlations between miRNA96a and some RSLs indicators to clarify its biomarker capability for evaluating the storage quality of RBC units. MATERIALS AND METHODS: Samples were collected from ten leuko-reduced RBC units on days 0, 14, 28, and 42 of storage. miRNA96 gene expression level and RSLs indicators including hemolysis, mechanical fragility index (MFI), total antioxidant capacity (TAC), lipid peroxidation (TBARs), thiol groups, and RBC indices were measured on the days mentioned above. RESULTS: Significant correlations were found between the changes in miRNA96 expression level and the levels of hemolysis, TAC, TBARs, and MFI indices (p values < 0.05). The donors were classified into the high risk group and low risk group, according to four important characteristics and lifestyle habits (smoking, physical activity, age, and BMI). The high risk group had a significantly lower rate of hemolysis, free hemoglobin, MFI, TAC, and a higher rate of lipid peroxidation compared to low risk group (p values < 0.05). CONCLUSION: The finding suggested that upregulation of miRNA96 could prevent hemolysis of RBCs, despite the accumulation of oxidative injuries in them. The miRNA96 expression level was probably a potential predictor for mechanical and biochemical RSL indicators.

LncRNA FGF14-AS2 represses growth of prostate carcinoma cells via modulating miR-96-5p/AJAP1 axis.

OBJECTIVE: This investigation devoted to lncRNA FGF14 antisense RNA 2 (FGF14-AS2) in prostate carcinoma progression. METHODS: The levels of lncRNA FGF14-AS2, miR-96-5p, and Adherens junction-associated protein-1 (AJAP1) in prostate carcinoma were tested by Western blot and qRT-PCR. How these two genes interacted was confirmed by RNA immunoprecipitation and dualluciferase gene methods. The effect of FGF14-AS2/miR-96-5p/AJAP1 axis in prostate carcinoma progression was determined by MTT, Transwell, and nude mice tumor model. RESULTS: FGF14-AS2 was a downregulated lncRNA in prostate carcinoma tissue and cells. FGF14-AS2 could restrain miR-96-5p expression while miR-96-5p hampered AJAP1. FGF14-AS2 could effectively decrease the biological behaviors of prostate carcinoma cells, while knock-down of FGF14-AS2 triggered opposite results. Moreover, miR-96-5p mimic presented a cancer promoter role in prostate carcinoma cells. AJAP1 expression level could affect levels of proteins related to epithelial-mesenchymal transition. In vivo experiment suggested that overexpressing FGF14-AS2 could reverse the promotion of silenced AJAP1 on prostate carcinoma cell metastasis, thus to inhibit tumor growth. CONCLUSION: lncRNA FGF14-AS2 was a downregulated lncRNA in prostate carcinoma and influenced cell proliferation and metastasis. The influence relied on modulating miR-96-5p and its target gene AJAP1.

Circ-KIAA0907 inhibits the progression of oral squamous cell carcinoma by regulating the miR-96-5p/UNC13C axis.

BACKGROUND: Circular RNA (circRNA) plays an important role in regulating cell biological function and has been shown to be involved in cancer progression, including oral squamous cell carcinoma (OSCC). Circ-KIAA0907 has been found to play an anti-cancer role in OSCC, so it is worth exploring more functions and new mechanisms of circ-KIAA0907 in OSCC progression. METHODS: Quantitative real-time PCR (qRT-PCR) was used to detect the expression of circ-KIAA0907, microRNA (miR)-96-5p, and unc-13 homolog C (UNC13C). Transwell assay, flow cytometry, and colony formation assay were employed to measure the migration, invasion, apoptosis, and radiosensitivity of cells. Besides, glucose uptake, lactate production, and extracellular acidification rate (ECAR) were determined to evaluate the glycolysis ability of cells. Dual-luciferase reporter assay and RIP assay were performed to confirm the interactions among circ-KIAA0907, miR-96-5p, and UNC13C. And RNA pull-down assay was used to verify the binding degree of miR-96-5p to its targets. Moreover, UNC13C protein level was examined using western blot (WB) analysis. OSCC xenograft models were constructed to perform in vivo experiments. RESULTS: Circ-KIAA0907 was a stability circRNA with lowly expression in OSCC. Overexpressed circ-KIAA0907 could inhibit migration, invasion, and glycolysis, while promoting apoptosis and radiosensitivity in OSCC cells. In the terms of mechanism, circ-KIAA0907 could sponge miR-96-5p to regulate UNC13C expression. MiR-96-5p overexpression could reverse the inhibitory effect of circ-KIAA0907 on OSCC progression, and UNC13C knockdown also could overturn the suppressive effect of miR-96-5p inhibitor on OSCC progression. Animal experiments revealed that circ-KIAA0907 could reduce the tumor growth of OSCC by regulating the miR-96-5p/UNC13C axis. CONCLUSION: Our study suggests that circ-KIAA0907 restrains OSCC progression via the miR-96-5p/UNC13C axis, indicating that it may be a potential target for OSCC treatment.

Glutathione in the Brain.

Glutathione (GSH) is the most abundant non-protein thiol, and plays crucial roles in the antioxidant defense system and the maintenance of redox homeostasis in neurons. GSH depletion in the brain is a common finding in patients with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and can cause neurodegeneration prior to disease onset. Excitatory amino acid carrier 1 (EAAC1), a sodium-dependent glutamate/cysteine transporter that is selectively present in neurons, plays a central role in the regulation of neuronal GSH production. The expression of EAAC1 is posttranslationally controlled by the glutamate transporter-associated protein 3-18 (GTRAP3-18) or miR-96-5p in neurons. The regulatory mechanism of neuronal GSH production mediated by EAAC1 may be a new target in therapeutic strategies for these neurodegenerative diseases. This review describes the regulatory mechanism of neuronal GSH production and its potential therapeutic application in the treatment of neurodegenerative diseases.

MicroRNA regulation of K-Ras in pancreatic cancer and opportunities for therapeutic intervention.

The Ras family of GTPases is involved in cell proliferation, cell survival, and angiogenesis. It is upregulated in several cancers, including pancreatic cancer (PC) and leads to uncontrolled growth and aggressiveness. PC is well known to be a lethal disease with poor prognosis, plagued by limited therapeutic modalities. MicroRNAs (miRNAs), which are short non-coding RNA molecules, have recently emerged as regulators of signaling networks and have shown potential to target pathway components for therapeutic use in several malignancies. K-Ras mutations are widespread in PC cases (90%), with mutations detectable as early as pancreatic intraepithelial neoplasias and in later metastatic stages alike; therefore, these mutations in K-Ras are obvious drivers and potential targets for PC therapy. Several K-Ras targeting miRNAs have lately been discovered, and many of them have shown promise in combating pancreatic tumor growth in vitro and in mouse models. However, the field of miRNA therapy is still in its infancy, and miRNA mimics or anti-miRNA oligonucleotides that target Ras pathway have thus far not been evaluated in PC patients. In this review, we summarize the role of several miRNAs that regulate oncogenic K-Ras signaling in PC, with their prospective roles as therapeutic agents for targeting K-Ras pathway.

MiR-96-5p promotes breast cancer migration by activating MEK/ERK signaling.

BACKGROUND: Breast cancer is the leading cause of cancer deaths in women worldwide. The purpose of the current study was to investigate the potential role of miR-96-5p in breast cancer. METHODS: Breast cancer tissues and matched para-cancerous tissues were collected. The expression of microRNA-96-5p (miR-96-5p) and arginine kinase 3 (AK3) was detected by quantitative real-time PCR (qRT-PCR). The correlation between miR-96-5p and AK3 was calculated by Pearson's Chi-square test. Moreover, mimics or inhibitors of miR-96-5p were applied to explore whether miR-96-5p influences the migration capacity in Transwell and wound healing assays. Bioinformatics analysis was performed to identify the target genes of miR-96-5p through the TargetScan, miRDB and miRanda databases. A luciferase reporter assay was performed to verify AK3 as a downstream target gene of miR-96-5p. RESULTS: The expression of miR-96-5p was significantly increased in breast cancer tissue and breast cancer cell lines compared with para-cancerous tissue and a breast cell line, respectively. The expression of miR-96-5p negatively correlated with AK3 gene expression. AK3 was demonstrated to be a direct mRNA target of miR-96-5p. AK3 was positively associated with the overall survival of breast cancer patients. Kaplan-Meier curve and log rank test analyses revealed that decreased AK3 levels were significantly associated with reduced overall survival. miR-96-5p was shown to promote the migration of breast cancer cells through the MEK/ERK signaling pathway. CONCLUSION: Our results identify a role for miR-96-5p in promoting breast cancer cell migration through activation of MEK/ERK signaling by targeting AK3.