miR-34a Inhibitor Research Articles

Comprehensive transcriptome analysis of hypothalamus reveals genes associated with disorders of sex development in pigs

XX sex reversal, also called XX disorders of sex development (XX-DSD), is a condition affecting the development of the gonads or genitalia, and is relatively common in pigs. However, its genetic etiology and transcriptional regulation mechanism in the hypothalamic-pituitary-gonadal axis (HPGA) remain mostly unknown. XX-DSD (SRY-negative) pigs and normal sows were selected by external genitalia observation. The hypothalamus, which is the integrated center of the HPGA was sampled for whole-transcriptome RNA-seq. The role of DEmiRNA was validated by its overexpression and knockdown in vitro. A total of 1,258 lncRNAs, 1,086 mRNAs, and 61 microRNAs differentially expressed in XX-DSD pigs compared with normal female pigs. Genes in the hormone biosynthesis and secretion pathway significantly up-regulated, and the up-regulation of GNRH1, KISS1 and AVP may associate with the abnormal secretion of GnRH. We also predicted the lncRNA-miRNA-mRNA co-expression triplets and constructed three competing endogenous RNA (ceRNA) potentially associated with XX-DSD. Functional enrichment studies suggested that TCONS_00340886, TCONS_00000204 and miR-181a related to GnRH secretion. Further, miR-181a inhibitor up-regulated GNRH1, PAK6, and CAMK4 in the GT1-7 cells. Conversely, transfection of miR-181a mimics obtained the opposite trends. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly higher in XX-DSD gondas than those in normal sows. Taken together, we proposed that the balance of endocrine had broken in XX-DSD pigs. The current study is the first to examine the transcriptomic profile in the hypothalamus of XX-DSD pigs. It provides new insight into coding and non-coding RNAs that may be associated with DSD in pigs.

Could miR-34a Inhibition be Used as a Tool to Overcome Drug Resistance in MCF-7 Cells Treated with Synthesized Steroidal Heterocycles?

Background:Progesterone derivatives have explored an improved effect on human cancer cells through combination of the explored heterocycles with progesterone moiety.miRNAs have an important role in moderating cancer cell survival, proliferation and drug resistance. The current study tested the hypothesis “whether miR-34a inhibitor has a negative impact on apoptosis and angiogenesis in MCF-7 cells treated with newly synthesized progesterone derivatives”. Methods:MCF-7 cells were treated with progesterone derivatives individually and in combination with miR-34a inhibitor. miR-34a expression levels were measured in MCF-7 cells treated with progesterone derivatives using QRT-PCR. MCF-7 cells treated with progesterone derivatives individually showed increased miR-34a expression levels. miR-34a deficient cells were treated with the newly synthesized progesterone derivatives, after that, apoptotic and angiogenic gene expression levels were determined using QRT-PCR. The studied genes were as follows: apoptotic (Bcl-2, survivin, CCND1, CDC2, P53 and P21) and angiogenic (VEGF, Hif-1α, MMP-2, Ang-1, Ang-2, and FGF-1). Results:The results showed that miR-34a deficient MCF-7 cells treated with the newly progesterone derivatives still have promising effects on apoptotic and angiogenic genes. Besides, results revealed that miRNA-34a deficient MCF-7 cells exhibited improved effect of tested compounds in some apoptotic and angiogenic genes such as CDC-2, MMP-2. Conclusion:These results revealed that miR-34a inhibitor did not have remarkable negative effect on apoptosis and angiogenesis. On contrary, it showed an improved effect on some genes. And consequently, miR-34a inhibitor could be used safely as a tool to tackle drug resistance in breast cancer cells.

Glucagon-like peptide-1 (GLP-1) improved diabetic lung fibrosis via AMPK and microRNA-27a (miR-27a).

BackgroundExtracellular matrix proliferation is an issue which leads to lung tissue damage in diabetes mellitus. Glucagon-like peptide-1 (GLP-1) analogues can improve the proliferation of extracellular matrix in diabetic pulmonary disease. In this study, we investigated the effect of GLP-1 on pulmonary fibrosis through the AMPK/microRNA-27a (miR-27a) pathway.MethodsHuman embryonic lung fibroblast (MRC-5) cells were cultured with a high-glucose medium, and were treated with miR-27a inhibitor, GLP-1 analogues, and AMPK inhibitor. Cell Counting Kit-8 (CCK-8) detected the proliferation of MRC-5 cells. The fibrosis-related genes were analyzed, including Col-IV, fibronectin, NF-κB p65, α-SMA, and TGF-β1. Bioinformatics and dual-luciferase reporter assays were used to identify the targets for miR-27a.ResultsCompared with the control group, the expression of miR-27a in the hyperglycemic group was significantly up-regulated (P<0.01) and the expression of peroxisome proliferator-activated receptor γ (PPARγ) significantly down-regulated (P<0.01). The expression of Col-IV, fibronectin, NF-κB p65, α-SMA and TGF-β1 increased significantly (P<0.01). The expression level of apoptosis factor caspase-3 decreased significantly (P<0.01). MiR-27a inhibitor could reverse the expression of these proteins. The effect of GLP-1 on miR-27a was time- and concentration-dependent. After pretreating MRC-5 cells via GLP-1, with or without compound C (AMPK inhibitor), the expression of miR-27a in the GLP-1 treated group was significantly lower than that in Vehicle group. The expression of miR-27a was increased after inhibition of the AMPK pathway. A predictive TargetScan algorithm showed that the PPARγ gene was a potential target of miR-27a. MiR-27a was also shown to target 3'-UTR of PPARγ.ConclusionsMiR-27a plays an important regulatory role in diabetic pulmonary fibrosis. GLP-1 could down-regulate the expression level of miR-27a by activating AMPK. Furthermore, the target gene PPARγ was up-regulated, consequently improving extracellular matrix proliferation in MRC-5 cells.

MiR-181a, delivered by hypoxic PTC-secreted exosomes, inhibits DACT2 by downregulating MLL3, leading to YAP-VEGF-mediated angiogenesis

Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, and angiogenesis plays critical roles in its recurrence and metastasis. In this study, we investigated the effects of hypoxia-induced exosomal microRNA-181 (miR-181a) from PTC on tumor growth and angiogenesis. Thyroid-cancer-related differentially expressed miR-181a was identified by microarray-based analysis in the Gene Expression Omnibus (GEO) database. We validated that miR-181a was highly expressed in PTC cells and even more so in cells cultured under hypoxic conditions, which also augmented exosome secretion from PTC cells. Exosomes extracted from PTC cells with manipulated miR-181a and mixed-lineage leukemia 3 (MLL3) were subjected to normoxic or hypoxic conditions. Human umbilical vein endothelial cells (HUVECs) were transfected with miR-181a inhibitor/mimic or small interfering RNA (siRNA)-MLL3 or treated with exosomes from hypoxic PTC cells. Hypoxic exosomal miR-181a delivery promoted proliferation and capillary-like network formation in HUVECs. Mechanistically, miR-181a targeted and inhibited MLL3. Furthermore, miR-181a downregulated DACT2 and upregulated YAP and vascular endothelial growth factor (VEGF). Further, hypoxic exosomal miR-181a induced angiogenesis and tumor growth in vivo, which was reversed by hypoxic exosomal miR-181a inhibitor. In conclusion, exosomal miR-181a from hypoxic PTC cells promotes tumor angiogenesis and growth through MLL3 and DACT2 downregulation, as well as VEGF upregulation.

RETRACTED ARTICLE: MiR-34a inhibitor protects mesenchymal stem cells from hyperglycaemic injury through the activation of the SIRT1/FoxO3a autophagy pathway

BackgroundMesenchymal stem cells (MSCs) are favourable treatments for ischaemic diseases; however, MSCs from diabetic patients are not useful for this purpose. Recent studies have shown that the expression of miR-34a is significantly increased in patients with hyperglycaemia; the precise role of miR-34a in MSCs in diabetes needs to be clarified.ObjectiveThe aim of this study is to determine the precise role of miR-34a in MSCs exposed to hyperglycaemia and in recovery heart function after myocardial infarction (MI) in diabetes mellitus (DM) rats.MethodsDM rat models were established by high-fat diet combined with streptozotocin (STZ) injection. MSCs were isolated from the bone marrow of donor rats. Chronic culture of MSCs under high glucose was used to mimic the DM micro-environment. The role of miR-34a in regulating cell viability, senescence and paracrine effects were investigated using a cell counting kit-8 (CCK-8) assay, senescence-associated β-galactosidase (SA-β-gal) staining and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) ELISA, respectively. The expression of autophagy- and senescence-associated proteins in MSCs and silent information regulator 1 (SIRT1) and forkhead box class O 3a (FoxO3a) were analysed by western blotting. Autophagic bodies were analysed by transmission electron microscopy (TEM). The MI model was established by left anterior descending coronary artery (LAD) ligation, and then, the rats were transplanted with differentially treated MSCs intramuscularly at sites around the border zone of the infarcted heart. Thereafter, cardiac function in rats in each group was detected via cardiac ultrasonography at 1 week and 3 weeks after surgery. The infarct size was determined through a 2,3,5-triphenyltetrazolium chloride (TTC) staining assay, while myocardial fibrosis was assessed by Masson staining.ResultsThe results of the current study showed that miR-34a was significantly increased under chronic hyperglycaemia exposure. Overexpression of miR-34a was significantly associated with impaired cell viability, exacerbated senescence and disrupted cell paracrine capacity. Moreover, we found that the mechanism underlying miR-34a-mediated deterioration of MSCs exposed to high glucose involved the activation of the SIRT1/FoxO3a autophagy pathway. Further analysis showed that miR-34a inhibitor-treated MSC transplantation could improve cardiac function and decrease the scar area in DM rats.ConclusionsOur study demonstrates for the first time that miR-34a mediates the deterioration of MSCs’ functions under hyperglycaemia. The underlying mechanism may involve the SIRT1/FoxO3a autophagy signalling pathway. Thus, inhibition of miR-34a might have important therapeutic implications in MSC-based therapies for myocardial infarction in DM patients.

MicroRNA-133a exerts tumor suppressive role in oral squamous cell carcinoma through the Notch signaling pathway via downregulation of CTBP2.

microRNAs (miRNAs) have been revealed to participate in some oral cancers and are proved to be effective. In the present study, we tried to explore the biological function of miR-133a in oral squamous cell carcinoma (OSCC) cells. The relationship that C-terminal-binding proteins 2 (CTBP2) was the putative target gene of miR-133a revealed from bioinformatics analysis was further was further validated by dual-luciferase reporter gene assay. In total, 40 patients with OSCC were enrolled for characterization of miR-133a, CTBP2, and Notch signaling pathway-related gene expression in clinical OSCC tissues. Low expression of miR-133a and high expression of CTBP2, Hes1, Notch-1, and Notch-3 were determined in OSCC tissues. OSCC cell lines were transfected with miR-133a inhibitor, miR-133a mimic, or shRNA targeting CTBP2, in response to which cell proliferation, migration, invasion, cell cycle, and apoptosis were evaluated. Transfection of miR-133a mimic induced apoptosis and inhibited OSCC cell proliferation, migration, and invasion and this was demonstrated to be attributable to decreased CTBP2 expression and suppression of the Notch signaling pathway. Taken together, we concluded that miR-133a acted as a tumor suppressor in OSCC through inhibition of the Notch signaling pathway via binding to CTBP2.

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide.

This study aimed to investigate the effects of microRNA-146a (miR-146a) on the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS). Lymphocytes were harvested from mouse spleen and cultured in vitro. The cells were treated with P. gingivalis LPS, miR-146a mimic, or miR-146a inhibitor. Scramble RNA served as the negative control of mimic and inhibitor. The production of inflammatory cytokines was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Compared with non-LPS-stimulated group, P. gingivalis LPS could increase the levels of interleukin (IL)-1β, IL-6, receptor activator NF-κB ligand (RANKL), and IL-10 (P<0.05) and decrease the mRNA level of osteoprotectin (OPG) (P<0.05). However, it did not significantly change the secretion of OPG. Compared with the negative control group, miR-146a mimic upregulated the levels of IL-10 and OPG (P<0.05), downregulated IL-1β, IL-6, and RANKL (P<0.05). Meanwhile, miR-146a inhibitor had a reverse effect on these cytokines (P<0.05) in P.gingivalis LPS-treated-lymphocytes. MiR-146a can provide a suitable microenvironment for bone formation by preventing the inflammatory effects of P.gingivalis LPS through the inhibition of IL-1β, IL-6, and RNAKL, thereby enhancing IL-10 and OPG.

MiR-146a mediates TLR-4 signaling pathway to affect myocardial fibrosis in rat constrictive pericarditis model.

BackgroundMyocardial fibrosis (MF) is thought to be associated with constrictive pericarditis (CP). miR-146a has been reported to be related to the survival of myocardial fibroblasts and related signal transduction pathways. The aim of this study was to investigate the expression of miR-146a in CP with MF and the activation of the Toll-like receptor 4 (TLR-4) signaling pathway, to understand the molecular mechanism of MF involvement in CP.MethodsThirty rats with different disease duration were randomly divided into three groups: an 8-week model group (CP-8W group), a 16-week model group (CP-16W group) model, and a normal control group (N group). After the CP model was established in the rats, the myocardial tissues were collected. The expression of miR-146a, the key factors of TLR-4 signaling pathway, including IL-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor-κB (NF-κB) and p-NF-κB, and the MF indicator α-SMA in myocardial tissue were detected. After treatment with lipopolysaccharide (LPS), primary cultured rat cardiac fibroblasts (CFs) were transfected with miR-146a. RT-PCR and western blot were used to detect the expression of downstream effectors to further verify the function of miRNA-146a in regulating MF via the TLR-4 signaling pathway.ResultsmiR-146a was increased in the CP-8W group but not in the CP-16W group. IRAK1 and TRAF6 in the CP-16W group were found to be higher than in the N group and CP-8W group. α-SMA in the model groups was higher than in the N group. Compared with the CP-8W group, α-SMA in the CP-16W model group was further increased. In the experiments using CFs, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA increased in the LPS-treated group compared with the N group. After transfection of CFs with the miR-146a mimics, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA decreased compared with the LPS-treated group. Following transfection of CFs with miR-146a inhibitors, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA increased compared with the LPS-treated group.ConclusionsThe expression of miR-146a demonstrated a dynamic change in the CP model; it was increased at the early time point (CP-8W) and then decreased at the 16W time point. miR-146a suppressed MF by inhibiting the target genes TRAF6 and IRAK1 via the TLR-4 signaling pathway.

Metformin attenuates ischemia/reperfusion-induced apoptosis of cardiac cells by downregulation of p53/microRNA-34a via activation of SIRT1.

Metformin has been demonstrated to be beneficial for the treatment of an impaired myocardium as a result of ischemia/reperfusion (I/R) injury, and miR-34a may be involved in this process. The aim of the present study was to determine the mechanisms by which metformin attenuated myocardial I/R injury-induced apoptosis. In the in vivo I/R model using Sprague-Dawley rats, metformin reduced the area of damaged myocardium and serum creatine MB isoform (CKMB) activity resulting in protection of the myocardium. Metformin also reduced apoptosis and the expression of apoptosis associated proteins, including caspase 3 and cleaved caspase, and decreased the expression of miR-34a, which is upregulated during I/R injury, which in turn resulted in corresponding changes in expression of Bcl-2, a direct target of miR-34a both in vitro and in vivo. To further examine the role of miR-34a in this process, H9C2 cells were transfected by a miR-34a mimic and inhibitor. Overexpression of miR-34a increased apoptosis in H9C2 cells induced by oxygen-glucose deprivation/recovery and knockdown of miR-34a expression-reduced apoptosis under the same conditions. Therefore, the effect of metformin on miR-34a in vitro were assessed. Metformin decreased the deacetylation activity of silent information regulator 1 resulting in reduced Ac-p53 levels, which reduced the levels of pri-miR-34a, and thus in turn reduced miR-34a levels. To confirm these results clinically, 90 patients with ST-segment elevation myocardial infarction following percutaneous coronary intervention were recruited. Patients who took metformin regularly before infarction had lower miR-34a levels and lower serum CKMB activity. Metformin also improved the sum ST-segment recovery following I/R injury. In conclusion, metformin may be helpful in the treatment of myocardial I/R.

Icariin stimulates osteogenesis and suppresses adipogenesis of human bone mesenchymal stem cells via miR-23a-mediated activation of the Wnt/β-catenin signaling pathway

BackgroundIcariin (ICA) is a bioactive compound isolated from epimedium-derived flavonoids that modulates bone mesenchymal stem cell osteogenesis and adipogenesis. However, its precise mechanism in this process is unknown. PurposeThe purpose of this study was to elucidate the role of ICA on human bone mesenchymal stem cell (hBMSC) osteogenesis and adipogenesis by focusing on miR-23a mediated activation of the Wnt/β-catenin signaling pathway. MethodsAfter ICA treatment, hBMSC osteogenesis and adipogenesis were evaluated using alkaline phosphatase staining, an alkaline phosphatase activity assay, Oil Red O staining, and cellular triglyceride levels. Moreover, the mRNA and protein expression levels of osteogenic and adipogenic markers as well as key factors of the Wnt/β-catenin signaling pathway were measured using quantitative reverse transcription polymerase chain reaction and western blotting. Lithium chloride, an activator of the Wnt/β-catenin signaling pathway, was used as a positive control. Finally, to investigate the role of miR-23a in ICA-induced activation of the Wnt/β-catenin signaling pathway, hBMSCs were transfected with miR-23a mimics or a miR-23a inhibitor. ResultsICA significantly promoted hBMSC osteogenic differentiation by upregulating alkaline phosphatase activity and the expression of bone sialoprotein II (BSPII) and runt-related transcription factor-2 (Runx-2). In contrast, ICA inhibited hBMSC adipogenic differentiation by reducing lipid droplet formation and cellular triglyceride levels as well as by downregulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT enhancer-binding protein-α (C/EBP-α). ICA mediated its effects on hBMSCs by activating the Wnt/β-catenin signaling pathway. It did so by upregulating β-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and T cell factor 1 (TCF1). Notably, the up-regulation of these proteins was blocked by Dickkopf-related protein 1 (DKK1). Critically, the effects of ICA on hBMSCs were similar to that of the positive control, lithium chloride. Notably, ICA-induced activation of the Wnt/β-catenin signaling pathway was significantly attenuated following miR-23a up-regulation. Conversely, miR-23a downregulation affected hBMSCs in the same manner as ICA; i.e., it activated the Wnt/β-catenin signaling pathway. ConclusionICA promotes and inhibits, respectively, hBMSC osteogenesis and adipogenesis via miR-23a-mediated activation of the Wnt/β-catenin signaling pathway.

The antagonistic effects and mechanisms of microRNA‐26a action in hypertensive vascular remodelling

Hypertensive vascular remodelling is responsible for end-organ damage and is the result of increased extracellular matrix accumulation and excessive vascular smooth muscle cell (VSMC) proliferation. MicroRNA-26a (miR-26a), a non-coding small RNA, is involved in several cardiovascular diseases. We aimed to validate the effect and mechanisms of miR-26a in hypertensive vascular remodelling. Male spontaneously hypertensive rats (SHRs) were injected intravenously with recombinant adeno-associated virus-miR-26a. Samples of thoracic aorta were examined histologically with H&E staining. In vitro, angiotensin II (AngII)-induced VSMCs cultured from thoracic aortae of female Sprague-Dawley rats, were transfected with miR-26a mimic or inhibitor. Western blots, qRT-PCR and immunohistological methods were used, along with chromatin-immunoprecipitation and luciferase reporter assays. Specific siRNAs were used to silence Smad production in VSMCs KEY RESULTS: Levels of miR-26a were lower in the thoracic aorta and plasma of SHRs than in WKY rats. Overexpression of miR-26a inhibited extracellular matrix deposition by targeting connective tissue growth factor (CTGF) and decreased VSMC proliferation by regulating the enhancer of zeste homologue 2 (EZH2)/p21 pathway both in vitro and in vivo. AngII-mediated Smad3 activation suppressed miR-26a expression, which in turn promoted Smad3 activation via targeted regulation of Smad4, leading to further down-regulation of miR-26a. Our data show that AngII stimulated a Smads/miR-26a positive feedback loop, which further reduced expression of miR-26a, leading to collagen production and VSMC proliferation and consequently vascular remodelling. MiR-26a has an antagonistic effect on hypertensive vascular remodelling and can be a strategy for treating hypertensive vascular remodelling.

BCL-6 promotes the methylation of miR-34a by recruiting EZH2 and upregulating CTRP9 to protect ischemic myocardial injury.

Acute myocardial infarction (AMI) and the following heart failure are public health problems faced all over the globe. The current study set out to investigate the role of B-cell lymphoma 6 (BCL-6) in cardiac protection after AMI. Initially, AMI mouse models and H9c2 cell oxygen-glucose deprivation (OGD) models were established. The cell models were transfected with the vectors containing oe-BCL-6, oe-EZH2, sh-EZH2, miR-34a mimic, and miR-34a inhibitor. RT-qPCR and Western blot analysis were applied to detect the expression patterns of microRNA-34a (miR-34a), BCL-6, enhancer of zeste homolog 2 (EZH2), and C1q tumor necrosis factor-related protein 9 (CTRP9) in the treated cell models. ChIP-qPCR and co-immunoprecipitation assay were performed to detect EZH2 enrichment and H3K27me3 levels in the miR-34a promoter region and the interaction between BCL-2 and EZH2, respectively. EdU staining, TUNEL staining, and flow cytometry were performed to detect cell proliferation and apoptosis, while ELISA was conducted to detect the oxidative stress levels. It was found that miR-34a was highly expressed in heart tissues of AMI models, while BCL-6 and EZH2 were poorly expressed. BCL-2 overexpression increased the recruitment of EZH2, upregulated H3K27me3 level in the miR-34a promoter region, and inhibited the miR-34a expression. Ctrp9, the downstream negative-regulatory molecule of miR-34a, was upregulated. Besides, miR-34a/CTRP9 expression changes were found to affect cardiomyocyte apoptosis, oxidation stress, and proliferation, and prevent myocardial injury in AMI mice. Our findings indicate that BCL-6 increases the level of H3K27me3 in the promoter region of miR-34a via EZH2 recruitment and CTRP9 upregulation, which inhibits the apoptosis of myocardial cells.

Resveratrol prevents steroid-induced osteonecrosis of the femoral head via miR-146a modulation.

The purpose of this study was to investigate the possible use of resveratrol (Res) to reverse abnormal osteogenesis/osteoclastogenesis activity that occurs during femoral head osteonecrosis and to explore the detailed mechanisms. Application of Res to bone marrow-derived mesenchymal stem cells in vitro promoted survival, inhibited apoptosis, and downregulated expression of reactive oxygen species expression. Moreover, Res application was associated with elevated microRNA-146a (miR-146a) expression, osteogenic differentiation, and suppressed osteoclastic differentiation, which were markedly reversed by miR-146a inhibitor. Histopathological observations and micro-computed tomography scanning results indicated that the Res-treated group had lower incidence of osteonecrosis and better bone microstructure than the untreated group. Res inhibited osteoclastogenesis through altering the levels of sirtuin1 (Sirt1), nuclear transcription factor-κB (NF-κB), and receptor activator of NF-κB ligand (RANKL). Simultaneously, Res treatment improved bone formation and increased β-catenin and runt-related transcription factor 2 (Runt2) expression levels, while reducing forkhead box class O (FOXO) family protein levels. The results of our study suggest that Res prevents steroid-induced osteonecrosis by upregulating miR-146a, and thereby stabilizes osteogenesis/osteoclastogenesis homeostasis via Wnt/FOXO and Sirt1/NF-κB pathways.

Prediction and verification of target of helenalin against hepatic stellate cell activation based on miR-200a-mediated PI3K/Akt and NF-κB pathways

Hepatic stellate cell (HSC) activation is a crucial event in the progress of liver fibrosis. In this study, the target of helenalin was firstly predicted by bioinformatics analysis, and then the prediction was verified by various experiments. HSC‐T6 cells were activated by interleukin-1 beta (IL-1β) and then treated with helenalin. Moreover, HSC‐T6 cells were transfected with miR-200a mimic or inhibitor, and the effect of helenalin on the miR-200a-mediated PI3K/Akt and NF-κB signaling pathways was investigated. The bioinformatics analysis indicated that miR-200a might regulate the PI3K/Akt pathway, NF-κB activation, Bcl-2 family and Caspases, ultimately affecting cell survival and apoptosis. Interestingly, the molecular docking demonstrated that the target of helenalin might be miR-200a-mediated the PI3K/Akt and NF-κB pathways. Moreover, the experiments showed that helenalin administration led to the inactivation of HSC-T6 cells, as evidenced by the inhibition of cell proliferation, α-SMA expression and collagen production. The mechanism studies showed that helenalin reduced collagen accumulation by restoring the balance of MMPs/TIMPs. Moreover, helenalin markedly suppressed HSC activation by inhibiting the PI3K/Akt pathway and alleviated inflammatory response by blocking the NF-κB signal transduction. Further study indicated that helenalin up-regulated miR-200a expression, thus leading to the inhibition of the PI3K/Akt and NF-κB signaling pathways. In conclusion, helenalin inhibits HSC activation via inhibiting the miR-200a-mediated PI3K/Akt and NF-κB pathways, and it may be developed as a potential medicine for the treatment of liver fibrosis.

MiR-26a regulated adipogenic differentiation of ADSCs induced by insulin through CDK5/FOXC2 pathway.

Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes, since insulin can induce adipogenic differentiation of human adipose-derived stem cells (ADSCs). MiR-26a was reported to be highly expressed in ADSCs under induction and Forkhead box C2 (FOXC2), as a key substrate of cyclin-dependent kinase 5 (CDK5) could inhibit white adipocyte differentiation, which was mediated by miR-26a. However, the relationship between miR-26a and CDK5/FOXC2 during ADSCs differentiation remains unknown. We want to verify the regulated mechanism of miR-26a/CDK5/FOXC2 axis participating in the adipogenic differentiation of ADSCS. ADSCs were isolated and verified by flow cytometry. Oil Red O staining was performed to assess the capacity for adipogenic differentiation of ADSCs. The proliferation ability of ADSCs was verified by MTT assay. The expression of miR-26a, peroxisome proliferator-activated receptors γ (PPARγ), CDK5, and FOXC2 were tested by qRT-PCR and Western blot, and the relationship between miR-26a and CDK5 was verified by dual-luciferase reporter gene assay. MiR-26a and PPARγ were upregulated and CDK5 and FOXC2 were downregulated during adipogenic differentiation of ADSCs. Knockdown of miR-26a or overexpression of CDK5 could inhibit adipogenic differentiation of ADSCs induced by insulin. MiR-26a could directly target CDK5 and the effect of miR-26a inhibitor on adipogenic differentiation of ADSCs could be blocked by si-CDK5. We demonstrated that miR-26a regulated insulin-induced adipogenic differentiation of ADSCs by regulating CDK5/FOXC2 pathway, which could provide the key to a comprehensive mechanistic understanding of obesity and type 2 diabetes.

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models.

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

LncRNA SNHG7 Mediates the Chemoresistance and Stemness of Breast Cancer by Sponging miR-34a.

Chemoresistance is considered to be a major cause of the recurrence and metastasis of breast cancer (BC). LncRNA SNHG7 has been reported to be upregulated in breast cancer and to promote tumor progression and metastasis. Nevertheless, the function and potential regulatory mechanism of SNHG7 in BC drug resistance are still largely unclear. This study indicated that SNHG7 was highly expressed in chemoresistant BC tissues and cells. Upregulated SNHG7 might predict a low pCR rate and poor clinical outcome in BC patients. Knockdown of SNHG7 enhanced drug sensitivity and drug-induced apoptosis in chemoresistant BC cells. In terms of the mechanism, miR-34a was found to be a target of SNHG7 and its expression in breast cancer tissues and chemoresistant cell lines was negatively correlated with SNHG7 expression. Importantly, sh-SNHG7 upregulated miR-34a expression, reduced the percentages of CD44+/CD24−cells, and inhibited sphere-formation and stem cell factor (Oct4, Nanog, SOX2) expression. Functional loss experiments showed that the repressive effect of SNHG7 knockdown on BC cell stemness was partially reversed by transfection with miR-34a inhibitors. In summary, this study indicated that SNHG7 contributed to the chemoresistance of BC and mediated chemoresistance and cancer stemness by sponging miR-34a.

DNMT3B silencing suppresses migration and invasion by epigenetically promoting miR-34a in bladder cancer.

The role of DNA methyltransferase 3B (DNMT3B) in tumorigenesis and development has been widely recognized; however, the mechanism underlying its action remains unclear. Considering its function in de novo methylation, we aimed to investigate whether DNMT3B plays its role via microRNA (miR)-34a promoter methylation in bladder cancer. We found that DNMT3B expression was low in 10 bladder cancer tissues and high in 20 bladder cancer tissues. miR-34a expression was higher in bladder cancer tissues with low expression of DNMT3B than that in bladder cancer tissues with high expression of DNMT3B. The level of miR-34a was negatively correlated with the level of DNMT3B. The methylation ratio of the miR-34a promoter was positively correlated with the level of DNMT3B and negatively correlated with the level of miR-34a. DNMT3B knockdown increased the expression of miR-34a and the transcriptional activity of the miR-34a promoter, while decreasing miR-34a promoter methylation. DNMT3B knockdown inhibited migration and invasion, while decreasing the protein levels of hepatocyte nuclear factor 4 gamma and Notch1 which are downstream targets of miR-34a. These inhibitory effects of DNMT3B were mitigated by the miR-34a inhibitor. In conclusion, DNMT3B silencing suppresses migration and invasion by epigenetically promoting miR-34a in bladder cancer.

MiR-146a Overexpression in Oral Squamous Cell Carcinoma Potentiates Cancer Cell Migration and Invasion Possibly via Targeting HTT

Huntingtin (HTT) is one of the target genes of miR-146-a and regulates various cancer cell activities. This study aims to explore the miR-146a expression pattern in oral squamous cell carcinoma (OSCC) and its role and mechanism in OSCC progression and metastasis via targeting the HTT gene. OSCC tissue and non-cancerous matched tissue (NCMT) were obtained from 14 patients. OSCC cell lines and normal HOK cells were used to analyze migration and invasion assay. OSCC-induced miR-146a knockout mice (B6.Cg-Mir146tm1.1Bal) model was developed. Transwell cell migration/invasion and scratch wound assays were used to investigate the OSCC cell migration and invasion in vitro. Kaplan-Meier survival analysis was used to investigate the association of HTT expression patterns in cancer tissue with patient survival percentage and duration. Pearson’s correlation analysis tested the association between miR-146a and HTT expression in OSCC tissues. miR-146a mimic and inhibitor transfection were performed to overexpress and knockdown the miR-146a in OSCC cells, respectively. miR-146a expression was highly upregulated in OSCC tissues and OSCC cell lines. Cancer cell migration/invasion was enhanced in miR-146a overexpressed cells and reduced in mi-R146a knockdowned cells. HTT expression was reduced in OSCC tissues and cell lines compared to NCMT and HOK cells, respectively. HTT expression was downregulated in miR-146a overexpressed OSCC cells and upregulated in miR-146a knockdowned OSCC cells. The expression pattern of miR-146a in OSCC cell lines and tissues was inversely correlated with HTT expression. Prediction of miRNA target analysis showed that HTT possesses the binding sites for miR-146a. HTT overexpression in OSCC tissues was associated with patients’ higher survival percentage and duration. HTT knockdown in OSCC cells enhanced miR-146a expression and cell migration/invasion. Inducing OSCC in miR-146a knockout mice increased the HTT expression in tongue tissue and alleviated the cancer aggressiveness and epithelial damage. Overexpressed miR-146a in OSCC targets the HTT gene and enhances cancer cell migration/invasion unraveling the possible role of HTT in miR146a-mediated OSCC cell migration and invasion.

Downregulation of miR-34a ameliorates inflammatory response and apoptosis induced by renal ischemia-reperfusion by promoting Kruppel-like factor 4 expression.

To explore the influence of micro ribonucleic acid (miR)-34a on renal ischemia-reperfusion by regulating Kruppel-like factor 4 (KLF4). A total of 36 Sprague-Dawley (SD) rats weighing 180-200 g were randomly divided into sham operation group (n=12), model group (n=12), and miR-34a inhibitor group (n=12). Renal ischemia-reperfusion modeling was performed in rats of model group and miR-34a inhibitor group. Those in the sham operation group received the same procedures without ligation. 200 μL of miR-34a inhibitor was pre-injected before modeling in rats of miR-34a inhibitor group. An automatic biochemical analyzer was used to detect serum creatinine and urea nitrogen levels in each group of rats, thus reflecting renal functions. The expressions of B-cell lymphoma 2 (Bcl-2), an apoptotic protein, and KLF4, a transcription factor, were detected via Western blotting. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was conducted to measure the expression levels of miR-34a and KLF4 in rat renal tissues in each group. Immunohistochemistry was employed to determine the expressions of inflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10). Additionally, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was utilized to determine the apoptotic rate in rat kidney tissues in each group. Compared with those in the sham operation group, serum creatinine level, urea nitrogen level, expressions of miR-34a, TNF-α and Bcl-2 (p<0.05) increased, while the levels of KLF4 and IL-10 (p<0.05) decreased in the model group. Apoptosis rate was also higher in the model group than the controls. In comparison with the model group, miR-34a inhibitor group had lowered serum creatinine level, urea nitrogen level, expressions of miR-34a, TNF-α and Bcl-2, and apoptotic rate (p<0.05), but raised levels of KLF4 and IL-10 (p<0.05), showing statistically significant differences. Downregulation of miR-34a ameliorates inflammatory response and apoptosis induced by renal ischemia-reperfusion by promoting KLF4 level, thus improving renal functions in rats.