MicroRNA-200c Promotes Suppressive Potential of Myeloid-Derived Suppressor Cells by Modulating PTEN and FOG2 Expression

Myeloid-derived suppressor cells (MDSCs) constitute one of the major populations that potently suppress anti-tumor immune responses and favor tumor growth in tumor microenvironment. However, the mechanism(s) regulating the differentiation and suppressive function of tumor-associated MDSCs remain(s) unclear. Here, we identified a microRNA-200c (miR-200c), whose expression was dramatically induced by tumor-derived factors. Meanwhile, we also demonstrated that GM-CSF was a main inducer of miR-200c in tumor environment, and miR-200c in turn promoted the expansion and immune suppressive activity of MDSCs via targeting phosphatase and tensin homolog (PTEN) and friend of Gata 2 (FOG2), which can lead to STAT3 and PI3K/Akt activation. Finally, we examined in vivo suppressive function of miR-200c transfected MDSCs and found that miR-200c could remarkably promote tumor growth via modifying MDSCs. Thus, GM-CSF induced miR-200c in tumor environment plays a critical role in governing the expansion and functions of tumor-associated MDSCs and serves as a potential target in immunotherapy against tumor.     

MicroRNA-494 is required for the accumulation and functions of tumor-expanded myeloid-derived suppressor cells via targeting of PTEN

Myeloid-derived suppressor cells (MDSCs) potently suppress the anti-tumor immune responses and also orchestrate the tumor microenvironment that favors tumor angiogenesis and metastasis. The molecular networks regulating the accumulation and functions of tumor-expanded MDSCs are largely unknown. In this study, we identified microRNA-494 (miR-494), whose expression was dramatically induced by tumor-derived factors, as an essential player in regulating the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway. TGF-β1 was found to be the main tumor-derived factor responsible for the upregulation of miR-494 in MDSCs. Expression of miR-494 not only enhanced CXCR4-mediated MDSC chemotaxis but also altered the intrinsic apoptotic/survival signal by targeting of PTEN, thus contributing to the accumulation of MDSCs in tumor tissues. Consequently, downregulation of PTEN resulted in increased activity of the Akt pathway and the subsequent upregulation of MMPs for facilitation of tumor cell invasion and metastasis. Knockdown of miR-494 significantly reversed the activity of MDSCs and inhibited the tumor growth and metastasis of 4T1 murine breast cancer in vivo. Collectively, our findings reveal that TGF-β1-induced miR-494 expression in MDSCs plays a critical role in the molecular events governing the accumulation and functions of tumor-expanded MDSCs and might be identified as a potential target in cancer therapy.     

Both miR-17-5p and miR-20a alleviate suppressive potential of myeloid-derived suppressor cells by modulating STAT3 expression

Myeloid-derived suppressor cells (MDSCs) were one of the major components of the immune suppressive network. STAT3 has an important role in regulating the suppressive potential of MDSCs. In this study, we found that the expression of STAT3 could be modulated by both miR-17-5p and miR-20a. The transfection of miR-17-5p or miR-20a remarkably reduces the expression of reactive oxygen species and the production of H(2)O(2), which are regulated by STAT3. MDSCs transfected with miR-17-5p or miR-20a are less able to suppress Ag-specific CD4 and CD8 T cells. Importantly, both miR-17-5p and miR-20a alleviate the suppressive function of MDSCs in vivo. The expression of miR-17-5p and miR-20a in tumor-associated MDSCs was found to be lower than in Gr1(+)CD11b(+) cells isolated from the spleens of disease-free mice. Tumor-associated factor downregulates the expression of both miR-17-5p and miR-20a. The modulation of miR-17-5p and miR-20a expression may be important for the process by which patients with a tumor can overcome the immune tolerance mediated by MDSCs. Our results suggest that miR-17-5p and miR-20a could potentially be used for immunotherapy against diseases, especially cancer, by blocking STAT3 expression.     

Effect of microRNA-34a in cell cycle, differentiation, and apoptosis: a review

The tumor suppressor gene p53 was shown to directly regulate the expression of microRNA-34a (miR-34a). miR-34a regulates a plethora of target proteins, which are involved in cell cycle, apoptosis, differentiation, and cellular development.miR-34a resides in the region of chromosome 1p36.23, which is commonly deleted in many tumor types, while it results in the loss expression of miR-34a. The promoters of the miR-34a gene subject to inactivation by CpG methylation also induce the loss expression of miR-34a. Ectopic miR-34a expression induces apoptosis, cell cycle arrest, and differentiation or reduces migration. This review summarizes the progress regarding the role of miR-34a in cell cycle, differentiation, and apoptosis.     

MicroRNA-34a induces apoptosis in the human glioma cell line,A172, through enhanced ROS production and NOX2 expression

Background

MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma.

Methods

The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot.

Results

MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells.

Conclusion

MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.     

miR-34a/c induce caprine endometrial epithelial cell apoptosis by regulating circ-8073/CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways

microRNAs (miRNAs) and circular RNAs (circRNAs) are important for endometrial receptivity establishment and embryo implantation in mammals. miR-34a and miR-34c are highly expressed in caprine receptive endometrium (RE). Herein, the functions and mechanisms of miR-34a/c in caprine endometrial epithelial cell (CEEC) apoptosis and RE establishment were investigated. miR-34a/c downregulated the expression level of centrosomal protein 55 (CEP55) and were sponged by circRNA8073 (circ-8073), thereby exhibiting a negative interaction in CEEC. miR-34a/c induced CEEC apoptosis by targeting circ-8073/CEP55 through the regulation of the RAS/RAF/MEK/ERK and phosphoitide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways. Positive and negative feedback loops and cross-talk were documented between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. miR-34a/c regulated the levels of RE marker genes, including forkhead box M1, vascular endothelial growth factor, and osteopontin (OPN). These results suggest that miR-34a/c not only induce CEEC apoptosis by binding to circ-8073 and CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways, but may also regulate RE establishment in dairy goats.     

miR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.     

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors. However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.     

Fluid shear stress regulates osteoblast proliferation and apoptosis via the lncRNA TUG1/miR-34a/FGFR1 axis

LncRNAs and microRNAs play critical roles in osteoblast differentiation and bone formation. However, their exact roles in osteoblasts under fluid shear stress (FSS) and the possible mechanisms remain unclear. The aim of this study was to explore whether and how miR-34a regulates osteoblast proliferation and apoptosis under FSS. In this study, FSS down-regulated miR-34a levels of MC3T3-E1 cells. MiR-34a up-regulation attenuated FSS-induced promotion of proliferation and suppression of apoptosis. Luciferase reporter assay revealed that miR-34a directly targeted FGFR1. Moreover, miR-34a regulated osteoblast proliferation and apoptosis via FGFR1. Further, we validated that lncRNA TUG1 acted as a competing endogenous RNA (ceRNA) to interact with miR-34a and up-regulate FGFR1 protein expression. Furthermore, lncRNA TUG1 could promote proliferation and inhibit apoptosis. Taken together, our study revealed the key role of the lncRNA TUG1/miR-34a/FGFR1 axis in FSS-regulated osteoblast proliferation and apoptosis and may provide potential therapeutic targets for osteoporosis.     

C-type lectin receptor Dectin3 deficiency balances the accumulation and function of FoxO1-mediated LOX-1+ M-MDSCs in relieving lupus-like symptoms

Recent studies indicate that Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) can function as the signal of pattern recognition receptors, which play a pivotal role in the pathogenesis of the autoimmune disease. Systemic lupus erythematosus (SLE) is a classic autoimmune disease. Previous reports mainly focused on the potential role of TLRs in regulating the development of SLE, but little is known about the role of CLRs in the progression of SLE. Our previous studies showed that the inflammation-mediated accumulation of myeloid-derived suppressor cells (MDSCs) including granulocytic (G-MDSCs) and monocytic (M-MDSCs) participated in the pathogenesis of lupus. Mice deficient in Card9 (the downstream molecule of CLRs) were more susceptible to colitis-associated cancer via promoting the expansion of MDSCs. Whether the abnormal activation of CLRs regulates the expansion of MDSCs to participate in the pathogenesis of lupus remains unknown. In the present study, the expressions of CLRs were examined in both SLE patients and mouse models, revealing the expression of Dectin3 was positively correlated with SLEDAI. Dectin3 deficiency retarded the lupus-like disease by regulating the expansion and function of MDSCs. The mechanistic analysis revealed that Dectin3 deficiency promoted FoxO1-mediated apoptosis of MDSCs. Syk-Akt1-mediated nuclear transfer of FoxO1 increased in Dectin3-deficient MDSCs. Notedly, the accumulation of M-MDSCs mainly decreased in Dectin3^−/− lupus mice, and the nuclear transfer of FoxO1 negatively correlated with the expression of LOX-1 on M-MDSCs. The silencing of FoxO1 expression in Dectin3^−/− mice promoted the expansion of LOX-1⁺ M-MDSCs in vivo, and LOX-1⁺ M-MDSCs increased the differentiation of Th17 cells. Both LOX-1 expression on M-MDSCs and Dectin3 expression on MDSCs increased in patients with SLE. These data indicated that increased LOX-1⁺ M-MDSCs were related to the exacerbation of SLE development and might be potential target cells for the treatment of SLE.Subject terms: Cell signalling, Autoimmunity, Cell death and immune response     

MicroRNA-34a regulation of endothelial senescence

Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelial cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.     

Retracted: MicroRNA-34a inhibit hepatocellular carcinoma progression by repressing hexokinase-1

Hepatocellular carcinoma (HCC) is known as a frequent type of primary cancer in the liver, and it is the third-most common cause of cancer-related death all over the world. However, the molecular mechanism in the progression of HCC is still unclear. The current study was designed to investigate the expression and function of microRNA-34a (miR-34a) in HCC. In HCC tissues and cells, the expression levels of miR-34a were analyzed by quantitative real-time polymerase chain reaction. The association between the level of miR-34a and hexokinase (HK)-1 was also investigated via luciferase reporter assay. Cell viability and proliferation were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. To assess whether miR-34a can limit tumor growth in vivo, animal models and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used for examining the role of miR-34a on the development of HCC and cell apoptosis. The expression level of miR-34a was reduced in HCC samples and cells. The expression of miR-34a was associated with the viability and proliferation capacity of HCC cells, and miR-34a could inhibit HCC cells proliferation by inhibiting HK1. In the mouse model of HCC, volumes and weight of the tumors were significantly decreased by transfection with miR-34a mimic compared with the control group. Furthermore, miR-34a mimics could induce apoptosis in a greater proportion of cells compared with the control group. Taken together, the data may provide some novel insights into the molecular mechanism of miR-34a and HK1 in the progression of HCC. Thus, miR-34a/HK1 axis might be a novel promising therapeutic target for treating HCC.     

Omega-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid enhance dexamethasone sensitivity in multiple myeloma cells by the p53/miR-34a/Bcl-2 axis

Dexamethasone is widely used in multiple myeloma (MM) for its cytotoxic effects on lymphoid cells. However, many MM patients are resistant to dexamethasone, although some can benefit from dexamethasone treatment. In this study, we noted that ω-3 polyunsaturated fatty acids (PUFAs) enhanced the dexamethasone sensitivity of MM cells by inducing cell apoptosis. q-PCR analysis revealed that miR-34a could be significantly induced by PUFAs in U266 and primary MM cells. Transfection with miR-34a antagonist or miR-34a agomir could restore or suppress the dexamethasone sensitivity in U266 cells. Both luciferase reporter assay and Western blot showed that Bcl-2 is the direct target of miR-34a in MM cells. In addition, we observed that PUFAs induced p53 protein expression in MM cells under dexamethasone administration. Furthermore, suppressing p53 by its inhibitor, Pifithrin-α, regulated the miR-34a expression and modulated the sensitivity to dexamethasone in U266 cells. In summary, these results suggest that PUFAs enhance dexamethasone sensitivity to MM cells through the p53/miR-34a axis with a likely contribution of Bcl-2 suppression.