microRNA-199a-3p,DNMT3A,and aberrant DNA methylation in testicular cancer

It was previously demonstrated that miR-199a was downregulated in testicular germ cell tumor (TGCT), probably due to hypermethylation of its promoter. Further study found that re-expression of miR-199a in testicular cancer cells (NT2) led to suppression of cell growth, cancer migration, invasion and metastasis. More detailed analyses showed that these properties of miR-199a could be assigned to miR-199a-5p, one of its two derivatives. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this report, we identified DNA (cytosine-5)-methyltransferase 3A (DNMT3A), the de novo methyltransferase, as a direct target of miR-199a-3p using a 3′-UTR reporter assay. Transient expression of miR-199a-3p in NT2 cells led to decrease, while knocking down of miR-199a-3p in a normal human testicular cell line (HT) led to elevation, of DNMT3A2 (DNMT3A gene isoform 2) mRNA and protein levels. In clinical samples, DNMT3A2 was significantly overexpressed in malignant testicular tumor, and the expression of DNMT3A2 was inversely correlated with the expression of miR-199a-3p. However, DNMT3A did not affect miR-199a expression in NT2 cells. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation, partly through targeting DNMT3A. Overexpression of miR-199a-3p restored the expression of APC and MGMT tumor-suppressor genes in NT2 cells by affecting DNA methylation of their promoter regions. Our studies demonstrated the deregulation of miR-199a-3p expression in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potentially therapeutic use of synthetic miR-199a-3p oligonucleotides as effective hypomethylating compounds in the treatment of TGCT.     

LncRNA TMPO-AS1 serves as a ceRNA to promote osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis

Osteosarcoma (OS) is a common kind of aggressive tumor in bone which was mostly identified in children and adolescents with extremely high risk of death. Accumulating research works have displayed that long noncoding RNAs (lncRNAs) exert an essential role in the development of multiple cancers. It has been reported that TMPO-AS1 is an oncogene in cancers; nonetheless, its molecular mechanism in OS is totally unclear. Our present study elucidated that a remarkable overexpression of TMPO-AS1 was found in OS tissues and cells. Moreover, TMPO-AS1 depletion restrained Wnt/β-catenin pathway and cell proliferation as well as facilitated cell apoptosis. Further molecular mechanism investigations showed that TMPO-AS1 can sponge to miR-199a-5p. Moreover, miR-199a-5p was at a low level at OS cells. Importantly, miR-199a-5p's overexpression was associated with the OS cells' decreased proliferation and increased apoptosis. In addition, WNT7B was confirmed as a downstream gene of miR-199a-5p. Also the WNT7B expression was reversely modulated by miR-199a-5p and positively modulated by TMPO-AS1. Rescue experiments suggested that downregulated WNT7B rescued miR-199a-5p inhibitor-mediated repression on OS progression, but the treatment of LiCl counteracted the effect of WNT7B downregulation. In a word, TMPO-AS1 serves as a competing endogenous RNA to boost osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis, which provided an underlying therapeutic target for patients with OS.     

MicroRNA-506-3p initiates mesenchymal-to-epithelial transition and suppresses autophagy in osteosarcoma cells by directly targeting SPHK1

Osteosarcoma (OS) is the most common malignant bone tumor. In cancer cells, autophagy is related to epithelial-to-mesenchymal transition (EMT). Although microRNA (miR)-506-3p has been demonstrated to act as a tumor suppressor in OS, its role in regulating the EMT process and autophagy remains unknown. The results showed that miR-506-3p directly inhibited the expression of sphingosine kinase 1 (SPHK1) in 143B and SaOS-2 cells. The invasive capability of OS cells was reduced following miR-506-3p mimics transfection, and restored when SPHK1 was overexpressed simultaneously. Further, miR-506-3p mimics initiated mesenchymal-to-epithelial transition (MET) – E-cadherin expression was upregulated, whilst vimentin and fibronectin were downregulated. The basal autophagy flux (LC3II/I) was suppressed by miR-506-3p mimics. The alterations induced by miR-506-3p mimics were partly reversed by SPHK1 overexpression or treatment of rapamycin. Meanwhile, treatment of SPHK1-transfected cells with 3-methyladenine inhibited EMT. The data suggest that miR-506-3p initiates MET and suppresses autophagy in OS cells by targeting SPHK1.     

Bone marrow-derived mesenchymal stem cell-derived exosomal microRNA-208a promotes osteosarcoma cell proliferation,migration, and invasion

A recent study has discovered that mesenchymal stem cells (MSCs) are recruited into tumors and MSC-derived exosomes in a novel mechanism of cell-to-cell communication in human cancers. Here, in this study, we explore the impact of the microRNA-208a (miR-208a)-enriched exosomes derived from bone marrow-derived mesenchymal stem cells (BMSCs) on osteosarcoma cells. Human osteosarcoma cells MG-63 and Saos-2 were exposed to BMSCs-derived exosomes treated with either miR-208a mimic or inhibitor. The MTT assay, transwell migration assay, and soft agar colony formation assay were used to evaluate the viability, migration, and clonogenicity of osteosarcoma cells. Bioinformatics analysis and dual-luciferase reporter gene assays validated the targeted relationship between miR-208a and PDCD4. Western blot assay was used to detect the expression of PDCD4 and related proteins in the ERK1/2 pathway in osteosarcoma cells. BMSCs communicated with osteosarcoma cells via exosomes. Ectopic expression of miR-208a was shown to increase the viability, migration, and clonogenicity of osteosarcoma cells. Analysis of the exosomal content identified miR-208a as a mediator of the exosomal effects on osteosarcoma cells in part via downregulation of PDCD4 and activating the ERK1/2 pathway. In summary, our study illuminates that BMSC-derived exosomal miR-208a enhances the progression of osteosarcoma.     

miR-199a-3p/5p regulate tumorgenesis via targeting Rheb in non-small cell lung cancer

Lung cancer is one of the deadliest cancers, in which non-small cell lung cancer (NSCLC) accounting for 85% and has a low survival rate of 5 years. Dysregulation of microRNAs (miRNAs) can participate in tumor regulation and many major diseases. In this study, we found that miR-199a-3p/5p were down-expressed in NSCLC tissue samples, cell lines, and the patient sample database. MiR-199a-3p/5p overexpression could significantly suppress cell proliferation, migration ability and promote apoptosis. Through software prediction, ras homolog enriched in brain (Rheb) was identified as a common target of miR-199a-3p and miR-199a-5p, which participated in regulating mTOR signaling pathway. The same effect of inhibiting NSCLC appeared after down-regulating the expression of Rheb. Furthermore, our findings revealed that miR-199a can significantly inhibit tumor growth and metastasis in vivo, which fully demonstrates that miR-199a plays a tumor suppressive role in NSCLC. In addition, miR-199a-3p/5p has been shown to enhance the sensitivity of gefitinib to EGFR-T790M in NSCLC. Collectively, these results prove that miR-199a-3p/5p can act as cancer suppressor genes to inhibit the mTOR signaling pathway by targeting Rheb, which in turn inhibits the regulatory process of NSCLC. Thus, to investigate the anti-cancer effect of pre-miR-199a/Rheb/mTOR axis in NSCLC, miR-199a-3p and miR-199a-5p have the potential to become an early diagnostic marker or therapeutic target for NSCLC.     

microRNA-34a promotes DNA damage and mitotic catastrophe

Efficient and error-free DNA repair is critical for safeguarding genome integrity, yet it is also linked to radio- and chemoresistance of malignant tumors. miR-34a, a potent tumor suppressor, influences a large set of p53-regulated genes and contributes to p53-mediated apoptosis. However, the effects of miR-34a on the processes of DNA damage and repair are not entirely understood. We explored tet-inducible miR-34a-expressing human p53 wild-type and R273H p53 mutant GBM cell lines, and found that miR-34a influences the broad spectrum of 53BP1-mediated DNA damage response. It escalates both post-irradiation and endogenous DNA damage, abrogates radiation-induced G₂/M arrest and drastically increases the number of irradiated cells undergoing mitotic catastrophe. Furthermore, miR-34a downregulates 53BP1 and inhibits its recruitment to the sites of DNA double-strand breaks. We conclude that whereas miR-34a counteracts DNA repair, it also contributes to the p53-independent elimination of distressed cells, thus preventing the rise of genomic instability in tumor cell populations. These properties of miR-34a can potentially be exploited for DNA damage-effecting therapies of malignancies.     

MicroRNA-29a-3p regulates abdominal aortic aneurysm development and progression via direct interaction with PTEN

Various research studies have been conducted in deducing the role of microRNAs (miRNAs) in the pathogenesis and physiological processes of various systematic diseases. This study aims at demonstration of the important role played by miR-29a-3p, through association with phosphatase and tensin homolog (PTEN), in the regulation of abdominal aortic aneurysm development and progression. Quantitative real-time polymerase chain reaction (RT-qPCR) examined miRNA-19a-3p and PMEPA1 expression in multiplied vascular smooth muscle cells (VSMCs). Cell transfection upregulated or downregulated the genes and cell counting kit-8 assay determined cellular viability. RT-qPCR detected cellular proliferation and cell death using the cell proliferation and apoptosis biomarkers Ki87 and proliferating cell nuclear antigen, caspase-8 and caspase-3, respectively. Furthermore, luciferase assay analyzed the luciferase activity and western blot analysis determined miRNA-19a-3p and PMEPA1 protein expression in proliferation and apoptosis biomarkers. TargetScan 4.2 online software ( www.targetscan.org ) was used to perform the bioinformatics analysis so as to forecast the putative targets of miR-29a-3p and PTEN. The results inferred that there was an increased expression of miRNA-29a-3p found in AAA-mimic cells with increased cellular viability and significant pathological apoptosis. Further, when the expression of miRNA-29a-3p was downregulated, it reduced the cell viability of AAA cells. On the basis of the gene interplays, it can be understood that the PTEN was directly targeted by miRNA-29a-3p so as to regulate the AAA progression. Thus, PTEN was found to strengthen the proliferation effect of miRNA-29a-3p in AAA cells. The current study thus shed more insights about the molecular mechanistic roles of miRNA-29a-3p and PTEN, opening doors for novel therapeutic approach to AAA.     

下调microRNA-34a增强对人脂肪干细胞成骨诱导分化作用的初步探索

目的:颅颌面临界骨缺损是整复外科常见疾患,常常给患者的身心带来障碍和压力。为解决骨形成不足这一难题,我们拟通过表观遗传修饰的手段,调节microRNA-34a在人脂肪干细胞中的表达,来探讨microRNA-34a对人脂肪干细胞成骨诱导分化的影响。方法:分离培养原代人脂肪干细胞,以慢病毒为转染载体,分三组对其进行上调、下调microRNA-34a的表达及阴性对照。然后对其进行成骨诱导培养,于诱导的第7天行碱性磷酸酶(ALP)染色,第14天行茜素红(Alizarin Red)染色,定性比较转染后人脂肪干细胞成骨诱导分化的效果。结果:成功分离和培养原代人脂肪干细胞,并以慢病毒高效率转染调控microRNA-34a的表达。对比碱性磷酸酶和茜素红染色可见下调microRNA-34a组成骨效率最高,其次是阴性对照组,上调组效率最低。结论:1.人脂肪干细胞通过慢病毒转染获得高效率的表观遗传修饰;2.下调microRNA-34a的表达对人脂肪干细胞成骨的诱导分化有一定的促进作用。     

Mechanism of histone deacetylase inhibitor Trichostatin A induced apoptosis in human osteosarcoma cells

Although histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in malignancy, how they exert their effect on osteosarcoama cells is as yet unclear. This study was undertaken to investigate the underlying mechanism of a HDAC inhibitor Trichostatin A (TSA)-induced apoptosis in a osteosarcoma cell line HOS. We observed that TSA treatment decreased the viability of the cells and prominently increased acetylation of histone H3. Evidence was obtained indicating that TSA induced apoptosis of HOS cells as follows: (1) Generation of DNA fragmentation; (2) activation of procaspase-3; (3) cleavage of PARP; and (4) increase of DNA hypoploidy. The reduction of MMP and the release of cytochrome c to cytosol were also shown, indicating that TSA induces apoptosis in HOS cells in a histone acetylation- and mitochondria-dependent fashions. We also examined whether TSA can sensitize HOS cells to the action of an antitumor agent genistein. The combination therapy of TSA and genistein showed synergistic anticancer effect indicating that TSA can be considered as a novel therapeutic strategy for osteosarcoma not only from its direct apoptosis-inducing activity but also from the possibility of sensitization to other antitumor agents.     

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.     

miR-34a expands myeloid-derived suppressor cells via apoptosis inhibition

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population and show significant expansion under pathological conditions. microRNA plays important roles in many biological processes, whether microRNAs have a function in the expansion of MDSCs is still not very clear. In this study, miR-34a overexpression can induce the expansion of MDSCs in bone marrow chimera and transgenic mice model. The experimental results suggest that miR-34a inhibited the apoptosis of MDSCs but did not affect the proliferation of MDSCs. The distinct mRNA microarray profiles of MDSCs of wild type and miR-34a over-expressing MDSCs combined with the target prediction of miR-34a suggest that miR-34a may target genes such as p2rx7, Tia1, and plekhf1 to inhibit the apoptosis of MDSCs. Taken together, miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis of MDSCs.     

microRNA-33-3p involved in selenium deficiency-induced apoptosis via targeting ADAM10 in the chicken kidney

Selenium (Se) deficiency induces typical clinical and pathological changes and causes various pathological responses at the molecular level in several different chicken organs; the kidney is one of the target organs of Se deficiency. To explore the mechanisms that underlie the effects of microRNA-33-3p (miR-33-3p) on Se deficiency-induced kidney apoptosis, 60 chickens were randomly divided into two groups (30 chickens per group). We found that Se deficiency increased the expression of miR-33-3p in the chicken kidney. A disintegrin and metalloprotease domain 10 (ADAM10) was verified to be a target of miR-33-3p in the chicken kidney. The overexpression of miR-33-3p decreased the expression levels of β-catenin, cyclinD1, T-cell factor (TCF), c-myc, survivin, and Bcl-2; it increased the expression levels of E-cadherin, Bak, Bax, and caspase-3; and it increased the number of chicken kidney cells in the G0/G1 phase. In addition, Se deficiency caused the ultrastructure of the kidney to develop apoptotic characteristics. The results of flow cytometry analysis and AO/EB staining showed that the number of apoptotic chicken kidney cells increased in the miR-33-3p mimic group. All these results suggest that Se deficiency-induced cell cycle arrest and apoptosis in vivo and in vitro in the chicken kidney via the regulation of miR-33-3p, which targets ADAM10.     

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.