A cell type-specific approach to elucidate the role of miR-96 in inner ear hair cells.

Introduction: Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Methods: Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes. Results: We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs. Conclusion: Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

Prostate cancer invasion is promoted by the miR-96-5p-induced NDRG1 deficiency through NF-κB regulation.

BACKGROUND: The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). METHODS: The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. RESULTS: Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. CONCLUSIONS: MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

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.

miR-96-5p alleviates cerebral ischemia-reperfusion injury in mice by inhibiting pyroptosis via downregulating caspase 1.

Ischemic stroke is one of the leading causes of global mortality and disability. Nevertheless, successful treatment remains limited. In this study, we investigated the efficacy and the mechanism of miR-96-5p in protecting acute ischemic brain injury in adult mice. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in adult male C57BL/6 mice. MiR-96-5p or the negative control was administered via intracerebroventricular injection. The expression of pyroptosis-related genes and activation of various resident cells in the brain was assessed by RT-qPCR, western blot, immunohistochemistry, and immunofluorescence. Modified neurological severity score, rotarod test, cylinder test, brain water content, and cerebral infarction volume were used to evaluate the behavioral deficits and the severity of brain injury after MCAO. Flow cytometry, TUNEL staining, and Nissl staining were employed to assess the neuron damage. MiR-96-5p decreased markedly in the ischemic stroke model in vivo and in vitro. MiR-96-5p mimics suppressed the expression of caspase 1 and alleviated the apoptosis rate in OGD/R treatment N2a cells, however, the miR-96-5p inhibitor caused the opposite results. Intracerebroventricular delivery of miR-96-5p agomir significantly mitigated behavioral deficits, brain water content, and cerebral infarction volume after MCAO. In addition, treatment with miR-96-5p agomir downregulated the expression of caspase 1/cleaved caspase 1 and Gsdmd/Gsdmd-N, while alleviating the neuron damage. In summary, overexpression of miR-96-5p suppresses pyroptosis and reduces brain damage in the acute phase of ischemic stroke, providing new insight into the treatment of acute ischemic stroke.

LncRNA urothelial cancer associated 1 promotes the osteogenic differentiation of human periodontal ligament stem cells by regulating the miR-96-5p/Osx axis.

OBJECTIVES: To investigate the expression of long non-coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) in human periodontal ligament stem cells (hPDLSCs), its effect on osteogenic differentiation of hPDLSCs and its mechanism. DESIGN: The expression of osteogenic genes Osx, Runx2, Ocn and Opn was explored by qPCR. Protein expression in hPDLSCs was estimated by Western blot. The osteogenic differentiation of hPDLSCs was detected by Alizarin red staining assays. The interaction between UCA1 and miR-96-5p was explored by RNA pulldown assay and dual luciferase assay. The interaction between miR-96-5p and Osx 3'-UTR was measured by dual luciferase assay. RESULTS: The expression of UCA1 and miR-96-5p was negatively correlated in hPDLSCs. During the osteogenic differentiation of hPDLSCs, the expression of UCA1 was increased, while the expression of miR-96-5p was decreased. Knockdown of UCA1 in hPDLSCs inhibited osteogenic differentiation but induced upregulation of miR-96-5p expression, and vice versa. In addition, miR-96-5p partially reversed the positive effect of UCA1 on osteogenic differentiation of hPDLSCs. Notably, UCA1 was identified as a miR-96-5p sponge, and miR-96-5p targeted Osx. CONCLUSIONS: Our results demonstrated that the novel UCA1/miR-96-5p/Osx pathway regulates osteogenic differentiation of hPDLSCs and sheds new insights and targets for periodontitis therapeutics.

LncRNA DLEU1 promotes angiogenesis in diabetic foot ulcer wound healing by regulating miR-96-5p.

BACKGROUND: Diabetic foot ulcer (DFU) carries high rates of major amputation and mortality. AIMS: The goals of this study were to identify expression of circulating lncRNA DLEU1 and miR-96-5p in patients with diabetic foot ulcer (DFU) and to explore the function of lncRNA DLEU1/miR-96-5p axis in DFU. METHODS: Matched patients with DFU and healthy individuals were randomly selected. Serum samples from all subjects were used for circulating lncRNA DLEU1 and miR-96-5p assessment by RT-qPCR. Receiver operating characteristic (ROC) curve was plotted to assess the discriminative capacity of lncRNA DLEU1 and miR-96-5p in identifying DFU. Cell proliferation was detected by CCK-8 assay. Cell apoptosis was assayed by Annexin V-FITC/PI staining method. Bioinformatics, luciferase reporter activity assay, and in vitro cell experiments were used to explore the relationship between lncRNA DLEU1 and miR-96-5p. RESULTS: LncRNA DLEU1 and miR-96-5p were significantly up- and downregulated in patients with DFU, respectively, compared with controls. After ROC assessment, lncRNA DLEU1 and miR-96-5p were found to discriminate DFU from miR-96-5p. Furthermore, lncRNA DLEU1 inhibited human umbilical vein endothelial cells (HUVECs) cell proliferation and increased HUVECs apoptosis and oxidative stress through sponging miR-96-5p. CONCLUSION: Our findings suggest lncRNA DLEU1 and miR-96-5p as circulating biomarkers for DFU. Also, we provide the clue for the pathogenic significance of lncRNA DLEU1/miR-96-5p in DFU, as well as insights for new potential targets.

Inhibition of miR-96-5p alleviates intervertebral disc degeneration by regulating the peroxisome proliferator-activated receptor γ/nuclear factor-kappaB pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is the main pathogenesis of low back pain. MicroRNAs (miRNAs) have been found to exert regulatory function in IDD. This study aimed to investigate the effect and potential mechanism of miR-96-5p in IDD. METHODS: In vitro cell model of IDD was established by treating human nucleus pulposus cells (HNPCs) with interleukin-1ß (IL-1ß). The level of peroxisome proliferator-activated receptor γ (PPARγ) was examined in the IDD cell model by Western blot and quantification real-time reverse transcription-polymerase chain reaction (qRT-PCR). The expression level of miR-96-5p was detected by RT-qPCR. Effects of PPARγ or/and PPARγ agonist on inflammatory factors, extracellular matrix (ECM), apoptosis, and nuclear factor-kappaB (NF-κB) nuclear translocation were examined through enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry assay, and immunofluorescence staining. The Starbase database and dual luciferase reporter assay were used to predict and validate the targeting relationship between miR-96-5p and PPARγ, and rescue assay was performed to gain insight into the role of miR-96-5p on IDD through PPARγ/NF-κB signaling. RESULTS: PPARγ expression reduced with concentration and time under IL-1ß stimulation, while miR-96-5p expression showed the reverse trend (P < 0.05). Upregulation or/and activation of PPARγ inhibited IL-1ß-induced the increase in inflammatory factor levels, apoptosis, degradation of the ECM, and the nuclear translocation of NF-κB (P < 0.05). MiR-96-5p was highly expressed but PPARγ was lowly expressed in IDD, while knockdown of PPARγ partially reversed remission of IDD induced by miR-96-5p downregulation (P < 0.05). MiR-96-5p promoted NF-κB entry into the nucleus but PPARγ inhibited this process. CONCLUSION: Inhibition of miR-96-5p suppressed IDD progression by regulating the PPARγ/NF-κB pathway. MiR-96-5p may be a promising target for IDD treatment clinically.

MiR-96-5p Suppresses Progression of Arsenite-Induced Human Keratinocyte Proliferation and Malignant Transformation by Targeting Denticleless E3 Ubiquitin Protein Ligase Homolog.

Long-term exposure to arsenic has been linked to a variety of cancers, among which skin cancer is the most prevalent form. However, the mechanism underlying arsenic carcinogenesis is unclear, and there is still limited information on the role of miRNAs in arsenic-induced skin cancer. This study aims to explore the role of miR-96-5p in the arsenite-induced proliferation and malignant transformation of human HaCaT keratinocytes. The GEO database (accession numbers GSE97303, GSE97305, and GSE97306) was used to extract mRNA and miRNA expression profiles of HaCaT cells treated with or without 0.1 µmol/L sodium arsenite for 3 and 7 weeks. In this paper, according to the CCK8 assay result, HaCaT cells exposed to 0.1 µmol/L sodium arsenite for 48 h were finalized. CCK8, MTT, EdU incorporation, and colony formation assays were used to determine the viability and proliferation of HaCaT cells and transformed HaCaT (T-HaCaT) cells. The subcellular localization and relative expression levels of DTL, as well as miR-96-5p in HaCaT cells induced by arsenite, were determined via immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was performed to identify miR-96-5p bound directly to DTL. Transfection of miR-96-5p mimics or DTL siRNA was conducted to verify the arsenite-induced viability of HaCaT cells and T-HaCaT cells. T-HaCaT cells and nude mice were used to construct arsenite-induced malignant transformation and an in vivo xenograft model to demonstrate the over-expressed effect of miR-96-5p. The results showed that DTL was the target gene of miR-96-5p. Meanwhile, we also found that 0.1 µmol/L sodium arsenite upregulated DTL by decreasing the miR-96-5p level, leading to the proliferation and malignant transformation of HaCaT cells. MiR-96-5p agomir treatment slowed the growth of transplanted HaCaT cells transformed by arsenite in a manner associated with DTL downregulation in the nude mice xenograft model. Taken together, we confirmed that miR-96-5p, as a potent regulator of DTL, suppressed arsenite-induced HaCaT cell proliferation and malignant transformation, which might provide a novel therapeutic target for the treatment of arsenic-induced skin cancer.

MicroRNA-96-5p is negatively regulating GPC3 in the metastasis of papillary thyroid cancer.

Backgrounds: Papillary thyroid cancer is the most common pathological type of thyroid cancer. miR-96-5p, a member of the miR-183 family, constitute a polycistronic miRNA cluster. In breast cancer, miR-96-5p promotes cell invasion, migration, and proliferation in vitro by inhibiting PTPN9. Moreover, miR-96-5p was reported to function as an oncogene in many cancers. However, whether miR-96-5p is involved in the development of papillary thyroid cancers and its potential mechanism is still unknown. The present study aims to explore the relationship between miR-96-5p and GPC3 expression in the development of papillary thyroid cancers. Methods: Transcriptomic sequencing was carried out using six pairs of papillary thyroid cancer and adjacent normal tissues. Quantitative real-time polymerase chain reaction (PCR) experiments were performed to examine the expression of genes. Results: In total, there were 1588 up-regulated and 1803 down-regulated differentially expressed genes between papillary thyroid cancer and normal tissues. Gene ontology and Kyoto encyclopedia of genes and genomes analysis revealed that extracellular matrix structure and proteoglycans were mainly involved in papillary thyroid cancer. Among the cluster of proteoglycans, GPC3 was significantly down-regulated in papillary thyroid cancer and is a target of miR-96. Conclusion: miR-96-5p participates in the development of papillary thyroid cancer by regulating the expression of GPC3. Thus, targeting miR-96-5p may be a potential therapeutic approach for preventing and treating papillary thyroid cancer.

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.

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.

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.

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.

microRNA-96 targets the INS/AKT/GLUT4 signaling axis: Association with and effect on diabetic retinopathy.

Background: miR-96-5p is a highly expressed microRNA in the retina of subjects with diabetes. The INS/AKT/GLUT4 signaling axis is the main cell signaling pathway of glucose uptake in cells. Here, we investigated the role of miR-96-5p in this signaling pathway. Methods: Expression levels of miR-96-5p and its target genes were measured under high glucose conditions, in the retina of streptozotocin-induced diabetic mice, in the retina of AAV-2-eGFP-miR-96 or GFP intravitreal injected mice and in the retina of human donors with diabetic retinopathy (DR). MTT, wound healing, tube formation, Western blot, TUNEL, angiogenesis assays and hematoxylin-eosin staining of the retinal sections were performed. Results: miR-96-5p expression was increased under high glucose conditions in mouse retinal pigment epithelial (mRPE) cells, in the retina of mice receiving AAV-2 carrying miR-96 and STZ-treated mice. Expression of the miR-96-5p target genes related to the INS/AKT/GLUT4 signaling pathway was reduced following miR-96-5p overexpression. mmu-miR-96-5p expression decreased cell proliferation and thicknesses of retinal layers. Cell migration, tube formation, vascular length, angiogenesis, and TUNEL-positive cells were increased. Conclusions: In in vitro and in vivo studies and in human retinal tissues, miR-96-5p regulated the expression of the PIK3R1, PRKCE, AKT1, AKT2, and AKT3 genes in the INS/AKT axis and some genes involved in GLUT4 trafficking, such as Pak1, Snap23, RAB2a, and Ehd1. Because disruption of the INS/AKT/GLUT4 signaling axis causes advanced glycation end product accumulation and inflammatory responses, the inhibition of miR-96-5p expression could ameliorate DR.

[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.

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.

Anti-Liver Fibrosis Role of miRNA-96-5p via Targeting FN1 and Inhibiting ECM-Receptor Interaction Pathway.

The aberrant expression of mRNAs participates in the pathogenesis of hepatic fibrosis. However, the precise mechanisms regulated by microRNAs (miRNAs) remain unclear. This study aims to investigate the functions about differentially expressed mRNAs (DEMs) in liver fibrosis and their regulatory mechanisms. The DEMs datasets about hepatic stellate cells (HSCs) obtained from hepatic fibrosis mice versus HSCs obtained from normal mice were downloaded from the GEO database (GSE120281). According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the GSE120281 datasets, ECM-receptor interaction was the most significant enrichment pathway that was correlated with hepatic fibrosis, and the fibronectin 1 (FN1) gene was upregulated most significantly in the signaling pathway. Downregulation of the expression of the FN1 gene by transfecting with FN1-siRNA alleviated the activity of HSCs. Four different bioinformatics web-based tools were used to predict that microRNA-96-5p (miR-96-5p) would directly target FN1, and a luciferase assay further confirmed this. Moreover, miR-96-5p was declined in activated HSCs and FN1, whereas laminin γ1 (LAMC1), collagen 1α1 (COL1A1) in the ECM-receptor interaction pathway, and the fibrosis marker α-smooth muscle actin (α-SMA) could be reduced by upregulation of the miRNA. Additionally, miR-96-5p expression was low in CCl4-induced liver fibrosis mice. Increased miR-96-5p expression alleviated liver fibrosis, improved liver function, and inhibited the expression of α-SMA, FN1, COL1A1, and LAMC1. In conclusion, this study indicated that upregulation of miR-96-5p could reduce HSC activation and relieve hepatic fibrosis by restraining the FN1/ECM-receptor interaction pathway.

miR96- and miR182-driven regulation of cytoskeleton results in inhibition of glioblastoma motility.

Glioblastoma multiforme (GBM) is one of the most common forms of brain tumor. As an excessively invasive tumor type, GBM cannot be fully cured due to its invasion ability into healthy brain tissues. Therefore, molecular mechanisms behind GBM migration and invasion need to be deeply investigated for the development of effective GBM treatments. Cellular motility and invasion are strictly associated with the cytoskeleton, especially with actins and tubulins. Palladin, an actin-binding protein, tightly bundles actins during initial invadopodia and contraction fiber formations, which are essential for cellular motility. Spastin, a microtubule-binding protein, cuts microtubules into small pieces and acts on invadopodia elongation and cellular trafficking of invadopodia-associated proteins. Regulation of proteins such as spastin and palladin involved in dynamic reorganization of the cytoskeleton, are rapidly carried out by microRNAs at the posttranscriptional level. Therefore, determining possible regulatory miRNAs of spastin and palladin is critical to elucidate GBM motility. miR96 and miR182 down-regulate SPAST and PALLD at both transcript and protein levels. Over-expression of miR96 and miR182 resulted in inhibition of the motility. However, over-expression of spastin and palladin induced the motility. Spastin and palladin rescue of miR96- or miR182-transfected U251 MG cells resulted in diminished effects of the miRNAs and rescued the motility. Our results demonstrate that miR96 and miR182 over-expressions inhibit GBM motility by regulating cytoskeleton through spastin and palladin. These findings suggest that miR96 and miR182 should be investigated in more detail for their potential use in GBM therapy.

Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice.

Alcohol-associated liver disease (ALD) and its complications are significant health problems worldwide. Several pathways in ALD are influenced by alcohol that drives inflammation, fatty acid metabolism, and fibrosis. Although miR-96 has become a key regulator in several liver diseases, its function in ALD remains unclear. In contrast, sonic hedgehog (SHH) signaling has a well-defined role in liver disease through influencing the activation of hepatic stellate cells (HSCs) and the inducement of liver fibrosis. In this study, we investigated the expression patterns of miR-96 and Hh molecules in mouse and human liver samples. We showed that miR-96 and Shh were upregulated in ethanol-fed mice. Furthermore, alcoholic hepatitis (AH) patient specimens also showed upregulated FOXO3a, TGF-ß1, SHH, and GLI2 proteins. We then examined the effects of Hh inhibitor MDB5 and anti-miR-96 on inflammatory and extracellular matrix (ECM)-related genes. We identified FOXO3 and SMAD7 as direct target genes of miR-96. Inhibition of miR-96 decreased the expression of these genes in vitro in AML12 cells, HSC-T6 cells, and in vivo in ALD mice. Furthermore, MDB5 decreased HSCs activation and the expression of ECM-related genes, such as Gli1, Tgf-ß1, and collagen. Lipid nanoparticles (LNPs) loaded with the combination of MDB5, and anti-miR-96 ameliorated ALD in mice. Our study demonstrated that this combination therapy could serve as a new therapeutic target for ALD.