Function Of miR-34a Research Articles

Myeloid Mir34a suppresses colitis-associated colon cancer: characterization of mediators by single-cell RNA sequencing.

We have previously shown that general deletion of the gene encoding the p53-inducible Mir34a microRNA enhances the number and invasion of colitis-associated colorectal cancers (CACs) in mice. Since the p53-pathway has been implicated in tumor-suppression mediated by cells in the tumor microenvironment (TME) we deleted Mir34a in myeloid cells and characterized CACs in these with scRNA-Seq (single cell RNA sequencing). This revealed an increase inspecificmacrophage subtypes, such as Cdk8+ macrophagesand Mrc1+,M2-like macrophages. The latterdisplayed elevated expression of 21 known Mir34a target mRNAs, including Csf1r, Axl, Foxp1, Ccr1, Nampt, and Tgfbr2, and 32 predicted Mir34a target mRNAs. Furthermore, Mir34a-deficient BMDMs showed enhanced migration, elevated expression of Csf1r and a shift towards M2-like polarization when compared to Mir34a-proficient BMDMs. Concomitant deletion of Csf1r or treatment with a Csf1r inhibitor reduced the CAC burden and invasion in these mice. Notably, loss of myeloid Mir34a function resulted in a prominent, inflammatory CAC cell subtype, which displayed epithelial and macrophage markers. These cells displayed high levels of the EMT transcription factor Zeb2 and may therefore enhance the invasiveness of CACs. Taken together, our results provide in vivo evidence for a tumor suppressive role of myeloid Mir34a in CACs which is, at least in part, mediated by maintaining macrophages in an M1-like state via repression of Mir34a targets, such as Csf1r. Collectively, these findings may serve to identify new therapeutic targets and approaches for treatment of CAC.

MiR-34a is a tumor suppressor in zebrafish and its expression levels impact metabolism, hematopoiesis and DNA damage.

Li-Fraumeni syndrome is caused by inherited TP53 tumor suppressor gene mutations. MicroRNA miR-34a is a p53 target and modifier gene. Interestingly, miR-34 triple-null mice exhibit normal p53 responses and no overt cancer development, but the lack of miR-34 promotes tumorigenesis in cancer-susceptible backgrounds. miR-34 genes are highly conserved and syntenic between zebrafish and humans. Zebrafish miR-34a and miR-34b/c have similar expression timing in development, but miR-34a is more abundant. DNA damage by camptothecin led to p53-dependent induction of miR-34 genes, while miR-34a mutants were adult-viable and had normal DNA damage-induced apoptosis. Nevertheless, miR-34a-/- compound mutants with a gain-of-function tp53R217H/ R217H or tp53-/- mutants were more cancer-prone than tp53 mutants alone, confirming the tumor-suppressive function of miR-34a. Through transcriptomic comparisons at 28 hours post-fertilization (hpf), we characterized DNA damage-induced transcription, and at 8, 28 and 72 hpf we determined potential miR-34a-regulated genes. At 72 hpf, loss of miR-34a enhanced erythrocyte levels and up-regulated myb-positive hematopoietic stem cells. Overexpression of miR-34a suppressed its reporter mRNA, but not p53 target induction, and sensitized injected embryos to camptothecin but not to γ-irradiation.

Effect of miR-34a on the expression of clock and clock-controlled genes in DLD1 and Lovo human cancer cells with different backgrounds with respect to p53 functionality and 17β-estradiol-mediated regulation.

The small non-coding RNA miR-34a is a p53-regulated miRNA that acts as a tumour suppressor of colorectal cancer (CRC). Oncogenesis is also negatively influenced by deregulation of the circadian system in many types of tumours with various genetic backgrounds. As the clock gene per2 was recently recognized as one of the target genes of miR-34a, we focused on the miR-34a-mediated influence on the circadian oscillator in CRC cell lines DLD1 and LoVo, which differ in their p53 status. Previously, a sex-dependent association between the expression of per2 and that of miR-34a was demonstrated in CRC patients. Therefore, we also investigated the effect of 17β-estradiol (E2) on miR-34a oncostatic functions. miR-34a mimic caused a pronounced inhibition of per2 expression in both cell lines. Moreover, miR-34a mimic significantly inhibited bmal1 expression in LoVo and rev-erbα expression in DLD1 cells and induced clock gene expression in both cell lines. miR-34a mimic caused a pronounced decrease in sirt1 and cyclin D1 expression, which may be related to the inhibition of proliferation observed after mir-34a administration in DLD1 cells. E2 administration inhibited the migration and proliferation of DLD1 cells. E2 and miR-34a, when administered simultaneously, did not potentiate each other's effects. To conclude, miR-34a strongly influences the expression of components of the circadian oscillator without respect to p53 status and exerts its oncostatic effects via inhibition of sirt1 and cyclin D1 mRNA expression. E2 administration inhibits the growth of DLD1 cells; however, this effect seems to be independent of miR-34a-mediated action. With respect to the possible use of miR-34a in cancer treatment, clock genes can be considered as off-target genes, as changes in their expression induced by miR-34a treatment do not contribute to the oncostatic functions of miR-34a. Possible ambiguous oncogenic characteristics should be taken into consideration in future clinical studies focused on miR-34a.

MiR-26a Improves Microglial Activation and Neuronal Apoptosis in a Rat Model of Cerebral Infarction by Regulating the TREM1-TLR4/MyD88/NF-κB Axis

Emerging studies have indicated that abnormally expressed microRNAs (miRNAs) are related to the pathogenesis of cerebral ischemia. Nevertheless, the function of miR-26a in neuronal damage and microglial activation during cerebral infarction remains elusive. It was revealed that miR-26a was downregulated in oxygen-glucose deprivation (OGD)-treated microglia and neurons. Overexpressing miR-26a reduced the inflammatory reaction in BV2 cells and decreased neuronal apoptosis following OGD stimulation. miR-26a upregulation inactivated the TLR4/MyD88/NF-κB pathway and inhibited TREM1 expression. Repressing NF-κB phosphorylation inhibited the miR-26a level. As supported by the dual-luciferase reporter assay, TREM1 was directly targeted by miR-26a. Furthermore, a rat model of middle cerebral artery occlusion (MCAO) was built. We discovered that miR-26a improved cognitive, learning, and motor functions and reduced cerebral edema in MCAO rats. Mechanistically, upregulating miR-26a reduced inflammation and neuronal apoptosis by mitigating the TREM1-TLR4/MyD88/NF-κB pathway in the MCAO rat model. Collectively, this study verified that the miR-26a-TREM1-TLR4/MyD88/NF-κB axis contributes to modulating OGD-mediated microglial activation and neuronal injury.

MiR-34a targets SIRT1 to reduce p53 deacetylation and promote sevoflurane inhalation anesthesia-induced neuronal autophagy and apoptosis in neonatal mice

This study is to investigate the function of miR-34a and interactions between miR-34a, SIRT1, and p53 in sevoflurane-induced neuronal apoptosis and autophagy in neonatal mice. A mouse model was established by inhalation anesthesia with sevoflurane and injected with genetic reagents, followed by tests of learning and memory abilities and histological staining of the hippocampus. CCK-8 and AnnexinV/PI staining respectively measured the survival and apoptosis rates of primary hippocampal neurons cultured with sevoflurane. The expression levels of miR-34a, SIRT1, p53, Ac-p53, and autophagy- or apoptosis-related proteins were measured. Sevoflurane impaired the learning and memory abilities of mice, increased TUNEL-positive cells in their hippocampus, and hindered the survival of hippocampal neurons. Sevoflurane increased miR-34a, Bax, cleaved caspase-3, and the ratio of LC3-II/LC3-I and reduced SIRT1 and p62. MiR-34a overexpression promoted sevoflurane-induced neural damage, whereas SIRT1 inhibition or p53 upregulation counteracted the neuroprotection of miR-34a knockdown. SIRT1 was a target of miR-34a and promoted p53 deacetylation. MiR-34a promotes sevoflurane-stimulated neuronal apoptosis and autophagy in neonatal mice by inhibiting SIRT1 expression and subsequent p53 deacetylation.

Dynamical modeling of miR-34a, miR-449a, and miR-16 reveals numerous DDR signaling pathways regulating senescence, autophagy, and apoptosis in HeLa cells

Transfection of tumor suppressor miRNAs such as miR-34a, miR-449a, and miR-16 with DNA damage can regulate apoptosis and senescence in cancer cells. miR-16 has been shown to influence autophagy in cervical cancer. However, the function of miR-34a and miR-449a in autophagy remains unknown. The functional and persistent G1/S checkpoint signaling pathways in HeLa cells via these three miRNAs, either synergistically or separately, remain a mystery. As a result, we present a synthetic Boolean network of the functional G1/S checkpoint regulation, illustrating the regulatory effects of these three miRNAs. To our knowledge, this is the first synthetic Boolean network that demonstrates the advanced role of these miRNAs in cervical cancer signaling pathways reliant on or independent of p53, such as MAPK or AMPK. We compared our estimated probability to the experimental data and found reasonable agreement. Our findings indicate that miR-34a or miR-16 may control senescence, autophagy, apoptosis, and the functional G1/S checkpoint. Additionally, miR-449a can regulate just senescence and apoptosis on an individual basis. MiR-449a can coordinate autophagy in HeLa cells in a synergistic manner with miR-16 and/or miR-34a.

MicroRNA-26a represses pancreatic cancer cell malignant behaviors by targeting E2F7

Dysregulation of microRNAs (miRNAs) exerts key roles in the development of pancreatic cancer (PCa). miR-26a is reportedly a tumor suppressor in cancers. However, whether miR-26a modulates PCa progression is poorly understood. Here, we found that miR-26a was down-regulated in PCa. Overexpressed miR-26a suppressed PCa cell proliferation, colony formation, and tumor stem cell properties. Mechanically, the transcription factor E2F7 is a downstream target of miR-26a. miR-26a decreased E2F7 expression through binding to the 3’-untranslated region (UTR) of E2F7. Decreased miR-26a in PCa tissues was inversely correlated with E2F7. The inhibitory effects of miR-26a in PCa were reversed by E2F7 overexpression. Consistently, the knockout of E2F7 further significantly inhibited the growth of PCa cells combined with miR-26a overexpression. Further study revealed that E2F7 bound the promoter of vascular endothelial growth factor A (VEGFA), a key factor in angiogenesis, and transcriptionally activated the expression of VEGFA. miR-26a overexpression attenuated the effects of E2F7 on VEGFA promotion. Our results uncovered the novel function of miR-26a/E2F7/VEGFA in PCa, making miR-26a a possible target for PCa treatment.

MiR-34a promotes fibrosis of hepatic stellate cells via the TGF-β pathway.

BackgroundPrevious studies have confirmed that MicroRNA (miRNA) is a key regulator exhibiting different effects in human liver fibrosis. However, the function of miR-34a in liver fibrosis has not been reported. Hence, this study aimed to investigate the regulatory mechanism of miR-34a in liver fibrosis.MethodsThe expression of miR-34a was measured in fibrosis tissues via the quantitative real-time PCR (qRT-PCR) assay. Subsequently, 30 male C57BL/6J mice were divided into control and treatment groups and used to establish animal models of liver fibrosis to explore the expression level of miR-34a. Moreover, Cell Counting Kit 8 (CCK-8) and transwell assays were preformed to identify the regulatory mechanism of miR-34a in cells. The effect of miR-34a on the activity of transforming growth factor-β (TGF-β) pathway was observed by western blot.ResultsUp-regulation of miR-34a was detected in fibrosis cells. Moreover, the cellular phenotype was suppressed by miR-34a down-regulation in a primary culture of hepatic stellate cells (HSCs). Besides, it was found that increased miR-34a could significantly promote the invasion and migration of HSCs. Moreover, miR-34a activates HSCs through transforming TGF-β, α-smooth muscle actin (α-SMA), and Monocyte chemoattractant protein-1 (MCP-1), which further affects liver fibrosis.ConclusionsMiR-34a promotes the fibrosis of HSCs as well as cell proliferation, migration, and invasion.

MiR-34a/SIRT1 Axis Modulating Therapeutic Effect of Olaparib on Pancreatic Cancer through Suppressing PARP1 and EMT

ABSTRACT This study analyzed functions of miR-34a and SIRT1 in modulating efficacy of Olaparib in pancreatic cancer in vitro. RNA expressions of miR-34a and SIRT1 were evaluated through RT-qPCR in HPDE6-C7, SW1990, PANC-1 and MIA PaCa-2 cells. MiR-34a was promoted in cancer cell lines while SIRT1 was decreased. Luciferase reporter assay verified bindings between miR-34a and SIRT1. Moreover, E-cadherin was promoted by miR-34a mimics and SIRT1 suppression while N-cadherin and Vimentin were both downregulated. Additionally, PARP1 protein expression was increased after miR- 34a promotion and SIRT1 downregulation. Besides that, PARP1 protein levels and EMT were significantly inhibited after treated by Olaparib (0, 0.5, 1 and 1.5ìM). In Olaparib-treated cancer cells, SIRT1, PARP1 and EMT were all distinctly decreased after SIRT1 suppression and overexpression of miR-34a induced much lower level of SIRT1.

AP2-microRNA-26a overexpression reduces visceral fat mass and blood lipids

BackgroundMicroRNA-26a (miR-26a) is a key player in tumor suppression and plays important roles in glucose and lipid metabolism. However, its function in adipose tissue is not well defined. ObjectiveThe study aimed to examine the effect on fat expansion and function of miR-26a in adipose tissue. MethodsAdipose-specific miR-26a transgenic mice (Ap2-miR-26a) were firstly generated by breeding miR-26a floxed (Mir26aloxP/loxP) mice with Ap2-Cre recombinase transgenic mice. The effects of miR-26a adipose-specific overexpression on body weight, body fat composition, fat pad weight, adipocyte size, blood lipid levels, glucose metabolism, and adipogenesis were investigated in mice on a chow diet and a high fat diet. White adipose tissue browning was evaluated by energy expenditure, adipocyte morphology and browning related genes expression levels both at room temperature and after cold exposure. Gene expression was determined by Real-Time quantitative PCR and western blotting. ResultsMiR-26a was specifically overexpressed in adipose by ~4 folds. Ap2-miR-26a mice had a moderate decrease in body weight, body fat composition, epididymal white adipose (eWAT) weight and blood lipid levels, along with smaller adipocytes in eWAT. The favorable phenotype was not due to white adipose tissue browning (even after cold exposure) or adipogenesis or lipolysis. Ap2-miR-26a mice exhibited no significant metabolic phenotype under high-fat-diet feeding. ConclusionThis study suggests that adipose-specific overexpression of miR-26a could moderately reduce visceral fat pad mass and lipid levels independent of white adipose tissue browning, adipogenesis and adipose lipolysis based on the gene expression level.

Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion

BackgroundMicroRNAs (miRs) have been shown to play an important role in tumorigenesis, including in head and neck squamous cell carcinoma (HNSCC). The miR-34 family is thought to play a role in tumor suppression, but the exact mechanism of their action in HNSCC is not well understood. Moreover, the impact of chromosomal changes and mutation status on miR-34a expression remains unknown.MethodsDifferential expression of miR-34a, MET, and genomic alterations were assessed in the Cancer Genome Atlas (TCGA) datasets as well as in primary HNSCC and adjacent normal tissue. The biological functions of miR-34a in HNSCC were investigated in samples derived from primary human tumors and HNSCC cell lines. The expression of MET was evaluated using immunohistochemistry, and the molecular interaction of miR-34a and MET were demonstrated by RNA pulldown, RNA immunoprecipitation, luciferase reporter assay, and rescue experiments. Lastly, locked nucleic acid (LNA) miRs in mouse xenograft models were used to evaluate the clinical relevance of miR-34a in HNSCC tumor growth and modulation of the tumor microenvironment in vivo.ResultsChromosome arm 1p loss and P53 mutations are both associated with lower levels of miR-34a. In HNSCC, miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET. Our studies found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Consistent with these findings, administration of LNA-miR-34a in an in vivo model of HNSCC leads to diminished HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Consistently, LNA-miR-34a induced a decreased number of immunosuppressive PDL1-expressing tumor-associated macrophages in the tumor microenvironment. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells and CD8 naïve T cells.ConclusionsOur results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity. We propose miR-34a as a potential new therapeutic approach for HNSCC.

MiR-34a reverses radiation resistance on ECA-109 cells by inhibiting PI3K/AKT/mTOR signal pathway through downregulating the expression of SIRT1

Background Radiotherapy is an effective treatment for esophageal squamous cell carcinoma (ESCC). However, many ESCC patients relapsed after receiving radiotherapy due to the inherent resistance. The function of miR-34a and SIRT1, as well as the correlation between miR-34a and SIRT1 has been widely claimed in multiple types of malignant tumors. This study aimed to investigate the effects of miR-34a on radiation resistance against ESCC and the underlying mechanism. Methods In this study, CCK8, flow cytometry, wounding healing assays, and cell clone formation assay were used to determine the in vitro anti-tumor effects of radiation on radiation-resistant ESCC cell line (rECA-109). The luciferase activity and Western Blot assays were used to investigate the relationship among miR-34a, SIRT1, and the anti-radiation resistant effects. The xenograft experiments were used to verify the important function of miR-34a and SIRT1 in radiation resistance against ESCC. The apoptosis state of tumor tissues was evaluated by TUNEL assay. Results The introduction of miR-34a significantly induced the cell death and apoptosis of rECA-109 and inhibit the migration of rECA-109 treated by radiation. The anti-tumor effect was accompanied by the downregulation of SIRT1 and the inhibition of PI3K/AKT/mTOR signal pathway. The radiation resistance on rECA-109 cells was reversed by silencing SIRT1, accompanied by the PI3K/AKT/mTOR signal pathway inhibited. In vivo experiments revealed that the radiation resistance on ESCC was reversed by the introduction of miR-34a, the effect of which was promoted by the activation of SIRT1. Conclusion Our results showed that miR-34a could reverse the radiation resistance on rECA-109 cells by downregulating the expression of SIRT1through inhibiting the PI3K-AKT-mTOR signal pathway.

Sevoflurane induction alleviates the progression of gastric cancer by upregulating the miR-34a/TGIF2 axis.

This study aims to elucidate how sevoflurane affects the malignant progression of gastric cancer (GC) and its pharmacological mechanism. Dose-dependent and time-dependent regulations of sevoflurane on proliferation inhibition rate in AGS and BGC-823 cells were examined, and thus the optimal dose and treatment time of sevoflurane on GC cells were selected. Subsequently, proliferative and migratory abilities in sevoflurane-induced AGS and BGC-823 cells (3.4% sevoflurane induction for 6 h) were detected by CCK-8 and transwell assay, respectively. After sevoflurane induction, relative levels of miR-34a and TGIF2 in GC cells were determined by qRT-PCR and Western blot. Regulatory effects of miR-34a on GC cell phenotypes were also assessed. Furthermore, the in vivo function of miR-34a in GC growth was explored by generating xenografted GC in nude mice. Sevoflurane induction time-dependently and dose-dependently enhanced proliferation inhibition rate in AGS and BGC-823 cells. The proliferative and migratory abilities in GC cells induced with 3.4% sevoflurane for 6 h were markedly attenuated. sevoflurane induction upregulated miR-34a, but downregulated TGIF2 in GC cells. TGIF2 was negatively regulated by miR-34a. Notably, overexpression of miR-34a inhibited proliferative and migratory abilities in sevoflurane-induced GC cells, and knockdown of miR-34a yielded the opposite results. In nude mice with xenografted GC tissues, sevoflurane treatment markedly reduced tumorigenic ability, which was improved by knockdown of miR-34a. Sevoflurane weakens proliferative and migratory abilities in GC by upregulating miR-34a and downregulating TGIF2.

MicroRNA-26a inhibits wound healing through decreased keratinocytes migration by regulating ITGA5 through PI3K/AKT signaling pathway.

Background: Keratinocyte migration is essential for skin wound healing and recent studies demonstrated that microRNAs (miRNAs) are involved in the differentiation, migration and apoptosis in keratinocytes. However, the function of miR-26a in wound healing remains to be largely explored.Methods: Northern blot and quantitative reverse transcriptase PCR (qRT-PCR) were used to detect the miR-26a expression and Western blot was used to detect integrin α-5 (ITGA5), phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and p-AKT protein expression in immortalized human keratinocyte cell line HaCaT and normal human epidermal keratinocytes (NHEK) after 2 ng/ml transforming growth factor-β1 (TGF-β1) treatment for 0, 6, 12 and 24 h. Transwell assay and Wound healing assay were introduced to measure the cell migration of HaCaT cells. TargetScan online database, luciferase reporter assay and RNA immunoprecipitation (RIP) were employed to confirm the relationship between miR-26a and ITGA5.Results: The RNA expression of miR-26a was down-regulated and ITGA5 protein expression was up-regulated by TGF-β1 treatment in HaCaT and NHEK cells in a time-dependent manner. MiR-26a overexpression inhibited the migration of HaCaT cells induced by TGF-β1 while miR-26a inhibitor enhanced the migration. ITGA5 was a downstream target mRNA and regulated by miR-26a. ITGA5 overexpression reversed the inhibitory effect of miR-26a on migration in HaCaT, while ITGA5 knockdown attenuated the stimulative effect of miR-26a inhibitor in HaCaT via PI3K/AKT signaling pathway.Conclusion: MiR-26a overexpression inhibited TGF-β1 induced HaCaT cells migration via down-regulating ITGA5 through activating the PI3K/AKT signaling pathway.

Abstract 4818: miR-34-AGO-TNRC6A complex transcriptionally up-regulates BLU tumor suppressor via binding of a novel promoter-associated lncRNA in lung cancer

Abstract We focused on genes whose expression is induced by miR-34a, a potent tumor suppressor microRNA (miRNA) in lung cancer and many other types of cancer, and sought to elucidate the mechanisms underlying the tumor suppressor function of miR-34a. We found that the miR-34a inhibited the proliferation of lung cancer cells by inducing expression of the BLU(also known as ZMYND10) tumor suppressor. The next-generation sequencing analysis of RNA extracted from lung cancer tissues identified a novel divergent lncRNA expressed from promoter of BLUthat include two predicted miR-34 binding sites. Interestingly, miR-34a failed to induce the BLUtranscription in the mutants lacking these miR-34 binding sites in the lncRNA. In addition, the miR-34-AGO-TNRC6A complex induced transcription of BLUby binding to the lncRNA. This non-canonical miRNA pathway, which induces gene expression via cooperation between an miRNA and promoter-associated lncRNA, is a previously unknown tumor suppression mechanism of miR-34a. Citation Format: Shinichiro Ohno, Keiki Oikawa, Yuichirou Harada, Masahiko Kuroda. miR-34-AGO-TNRC6A complex transcriptionally up-regulates BLU tumor suppressor via binding of a novel promoter-associated lncRNA in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4818.

MicroRNA-34a Inhibition Alleviates Lung Injury in Cecal Ligation and Puncture Induced Septic Mice.

Sepsis is the leading cause of death in intensive care units. MicroRNA-34a (miR-34a) is involved in sepsis progression, while its underlying mechanisms on sepsis-induced lung injury remain obscure. Oxidative stress, pyroptosis, and inhibition of autophagy can result in organ injury. MiR-34a has been reported to regulate oxidative stress and autophagy via inhibiting silent information regulator T1 (SIRT1) and autophagy gene 4B (ATG4B) signaling. This study aimed at identifying the function of miR-34a in oxidative stress, inflammation, pyroptosis, and autophagy in sepsis-induced lung injury. Male 8-week-old C57BL/6 mice were subjected to cecal ligation and puncture and treated with miR-34a antagomir/agomir. Survival (n = 10), histopathological changes (n = 6), and lung wet-to-dry ratio (n = 6) were recorded and assayed. Other detection (n = 6) was performed to investigate the level of oxidative stress, inflammation, pyroptosis, and autophagy in lung tissues. Results showed that miR-34a down-regulation ameliorated lung injury in septic mice as reflected by decreased lung injury scores (decrease from 3.00 ± 0.32 to 2.00 ± 0.32) and wet-to-dry ratio (0.36-fold decrease). MiR-34a down-regulation also decreased reactive oxygen species accumulation (0.36-fold decrease), and promoted superoxide dismutase activity and the expression of SIRT1 (1.24-fold increase), heme oxygenase-1 and nuclear factor erythroid 2 like 2 to inhibit oxidative stress in septic mice. Moreover, miR-34a down-regulation suppressed inflammatory response and pyroptosis in septic mice, as evidenced by decreased level of pro-inflammatory factors including tumor necrosis factor α, interleukin-6 (IL-6), IL-1β, and IL-18, activity of caspase-1 (0.51-fold decrease) and expression of nucleotide-binding domain and leucine-rich repeat protein-3 (0.48-fold decrease), apoptosis-associated speck-like protein containing a CARD, cleaved-caspase-1, and cleaved-gasdermin D (0.36-fold decrease), and increased level of anti-inflammatory factors IL-10. MiR-34a down-regulation also enhanced autophagy in septic mice as evidenced by more autolysosomes and elevated expressions of ATG4B (0.90-fold increase), beclin1, ATG9, and LC3 II/I. Among these experiments, miR-34a up-regulation showed opposite effects on oxidative stress, inflammatory response, pyroptosis, and autophagy in septic mice. Additionally, miR-34a could bind to the 3′-untranslated region of SIRT1 and ATG4B. In conclusion, our findings demonstrated that miR-34a was implicated in oxidative stress, inflammation, pyroptosis, and autophagy in the development of sepsis. MiR-34a inhibition had a potential to alleviate sepsis-induced lung injury.

Targeting of CD38 by the Tumor Suppressor miR-26a Serves as a Novel Potential Therapeutic Agent in Multiple Myeloma.

Multiple myeloma is an incurable refractory hematologic malignancy arising from plasma cells in the bone marrow. Here we investigated miR-26a function in multiple myeloma and tested single-wall carbon nanotube delivery of miR-26a in vitro and in vivo. miR-26a was downregulated in patients with multiple myeloma cells compared with plasma cells from healthy donors. miR-26a overexpression inhibited proliferation and migration and induced apoptosis in multiple myeloma cell lines. To identify the targets of miR-26a, RPMI8226-V-miR-26-GFP and RPMI8226-V-GFP cells were cultured using stable isotope labeling by amino acids in cell culture (SILAC) medium, followed by mass spectrometry analysis. In multiple myeloma cells overexpressing miR-26a, CD38 protein was downregulated and subsequently confirmed to be a direct target of miR-26a. Depletion of CD38 in multiple myeloma cells duplicated the multiple myeloma inhibition observed with exogenous expression of miR-26a, whereas restoration of CD38 overcame the inhibition of miR-26a in multiple myeloma cells. In a human multiple myeloma xenograft mouse model, overexpression of miR-26a inhibited CD38 expression, provoked cell apoptosis, and inhibited cell proliferation. Daratumumab is the first CD38 antibody drug for monotherapy and combination therapy for patients with multiple myeloma, but eventually resistance develops. In multiple myeloma cells, CD38 remained at low level during daratumumab treatment, but a high-quality response is sustained. In daratumumab-resistant multiple myeloma cells, CD38 expression was completely restored but failed to correlate with daratumumab-induced cell death. Therefore, a therapeutic strategy to confer selection pressure to maintain low CD38 expression in multiple myeloma cells may have clinical benefit. SIGNIFICANCE: These results highlight the tumor suppressor function of miR-26a via its targeting of CD38 and suggest the therapeutic potential of miR-26a in patients with multiple myeloma.

MiR-26a as a novel potential therapeutic via inhibit CD38 translation and may overcome datatumumab resistance in multiple myeloma(MM)

Abstract Daratumumab is the first CD38 antibody drug for MM patients therapy. CD38 remains at a low level during daratumumab treatment and shows a sustained high quality response. In daratumumab resistant MM cells, CD38 expression was found either totally restored or at even higher levels. Nevertheless, a strategy to pressure the MM cells to maintain low CD-38 expression may have clinical benefit. miR-26a is usually downregulated in cancer and has been show to have potential clinical use. Here, we investigated miR-26a function in MM and tested single-wall carbon nanotube delivery of miR-26a in vitro and in vivo. GSE16558 dataset analysis showed miR-26a was downregulated in patient MM cells compared with plasma cells from healthy donors. miR-26a overexpression inhibited proliferation, repressed migration, and induced apoptosis in MM cell lines. To identify the targets of miR-26a, RPMI8226-V-miR-26-GFP and RPMI8226-V-GFP cells were cultured using Stable isotope labeling by amino acids in cell culture (SILAC) medium followed by mass spectrometry analysis. CD38 protein was found downregulated in MM cells overexpressing miR-26a and subsequently confirmed to be a direct target of miR-26a by reporter assay and immunoblotting. In addition, when overexpressed, miR-26a had a synergistic effect with bortezomib (BTZ) or melphalan (MEL) treatment. To determine whether the inhibition of MM was due to inhibited CD38, we knocked down CD38 in MM cells and duplicated the MM inhibition observed with exogenous expression of miR-26a, whereas restoration of CD38 overcame the inhibition of miR-26a in MM cells. Furthermore, miR-26a inhibited CD38 expression provoked cell apoptosis and inhibited cell proliferation in human MM xenograft mouse models.

RNA G-quadruplex regulates microRNA-26a biogenesis and function

RNA G-quadruplexes (RG4s) appear to be important in post-transcriptional gene regulation, but their pathophysiological functions remain unknown. MicroRNA-26a (miR-26a) is emerging as a therapeutic target for various human diseases, however the mechanisms underlying endogenous miR-26a regulation are poorly understood. Herein, we study the role of RG4 in miR-26a expression and function invitro and invivo. Putative RG4s within liver-enriched miRNAs were predicted by bioinformatic analysis, and the presence of an RG4 structure in the miR-26a-1 precursor (pre-miR-26a-1) was further analyzed by biophysical and biochemical methods. RG4 stabilizers, pre-miR-26a-1 overexpression plasmids, and luciferase reporter assays were used to assess the effect of RG4 on pre-miR-26a-1 maturation. Both miR-26a knock-in and knockout mouse models were employed to investigate the influence of this RG4 on miR-26a expression and function. Moreover, the interaction between RG4 in pre-miR-26a-1 and DEAH-box helicase 36 (DHX36) was determined by biophysical and molecular methods. Finally, miR-26a processing and DHX36 expression were quantified in the livers of obese mice. We identify a guanine-rich sequence in pre-miR-26a-1 that can fold into an RG4 structure. This RG4 impairs pre-miR-26a-1 maturation, resulting in a decrease in miR-26a expression and subsequently an increase in miR-26a cognate targets. In line with known miR-26a functions, this RG4 can regulate hepatic insulin sensitivity and lipid metabolism invitro and invivo. Furthermore, we reveal that DHX36 can bind and unwind this RG4 structure, thereby enhancing miR-26a maturation. Intriguingly, there is a concordant decrease of miR-26a maturation and DHX36 expression in obese mouse livers. Our findings define a dynamic DHX36/RG4/miR-26a regulatory axis during obesity, highlighting an important role of RG4 in physiology and pathology. Specific RNA sequences called G-quadruplexes (orRG4) appear to be important in post-transcriptional gene regulation. Obesity leads to the formation of these RG4 structures in pre-miR-26a-1 molecules, impairing the maturation and function of miR-26a, which has emerged as a therapeutic target in several diseases. This contributes to hepatic insulin resistance and the dysregulation of liver metabolism.

Downregulation of miR-34a Promotes Proliferation and Inhibits Apoptosis of Rat Osteoarthritic Cartilage Cells by Activating PI3K/Akt Pathway.

ObjectiveTo elucidate the expression and function of miR-34a in rat osteoarthritic cartilage cells, and further to explore its mechanism.Material and MethodsRat model of osteoarthritis was constructed and knee joint cartilage cells were isolated in vitro. Immunocytochemical staining was used for identification. qRT-PCR was used to detect the expression of miR-34a in cartilaginous tissues and cartilage cells. Cartilage cells were divided into blank control (BC), negative control (NC), miR-34a inhibitor (34aI), osteoarthritis model (OA), osteoarthritis model + negative control (OA + NC) and osteoarthritis model + miR-34a inhibitor (OA + 34aI) groups. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was studied by flow cytometry and Western blot. PI3K/AKT-pathway-related proteins were also analyzed by Western blot. To further validate the effect of miR-34a on the PI3K/Akt pathway, the cartilage cells were divided into blank control (BC), osteoarthritis model (OA), osteoarthritis model + miR-34a inhibitor (OA + 34aI), osteoarthritis model + PI3K activator (OA + IGF-1) and osteoarthritis model + miR-34a inhibitor + PI3K inhibitor (OA + 34aI + LY) groups, the experiments above were repeated.ResultsThe expression of miR-34a in cartilaginous tissues and cells of osteoarthritis model was significantly higher than that in normal (p < 0.05). After silencing miR-34a gene, the cell proliferation and proteins expression of PI3K/Akt pathway were increased, while the apoptosis rate and expression of apoptosis-related proteins were decreased. Addition of PI3K activator also evidently promoted proliferation and inhibited apoptosis. The protein expression of Bax, Cleaved caspase-3 and Cleaved caspase-9 were dramatically decreased, while the ratios of p-PI3K/PI3K and p-Akt/Akt were increased in OA + IGF-1 group.ConclusionDownregulation of miR-34a regulated proliferation and apoptosis of cartilage cells by activating PI3K/Akt pathway, providing a potential therapeutic approach for the treatment of osteoarthritis.