MiR-183 regulates the differentiation of osteoblasts in the development of osteoporosis by targeting Smad4

ObjectiveTo discuss the effect of miR-183 on osteoblast differentiation in the osteoporosis progression via targeting Smad4.MethodsOsteoporosis models were constructed on ovariectomized (OVX) mice to determine the expression of miR-183 and Smad4. Then, MC3T3-E1 cells and primary osteoblasts were divided into Mock, miR-control, miR-183 mimic, miR-183 inhibitor, siSmad4 and miR-183 inhibitor + siSmad4 groups. Alkaline phosphatase (ALP) staining were performed to determine ALP activity, alizarin red staining to evaluate the calcium deposit, while qRT-PCR and Western blotting were used to determine the expression of related molecules. Besides, MC3T3-E1 cells transfected with miR-control or miR-183 mimic were cultured with or without TGF-β1 to verify whether miR-183 regulates the TGF-β signaling pathway.ResultsMiR-183 was up-regulated with decreased Smad4 in the femur of OVX mice, and dual luciferase reporter gene assay showed that Smad4 was a target of miR-183. As compared to Mock group, MC3T3-E1 cells and primary osteoblasts in the miR-183 mimic group and siSmad4 group had significant reductions of OCN, OPN, Runx2 and Osx, as well as decreased ALP activity and calcium deposit. Contrarily, miR-183 and Smad4 were up-regulated and down-regulated respectively. However, cells in the miR-183 inhibitor group manifested the opposite changes. Besides, osteoblast differentiation in the miR-183 inhibitor + siSmad4 group was weakened evidently when compared to miR-183 inhibitor group. Pathway analysis indicated that miR-183 regulated osteogenic differentiation via TGF-β signaling pathway.ConclusionMiR-183 was up-regulated in osteoporosis, and miR-183 overexpression can inhibit osteoblast differentiation by targetedly down-regulating TGF-β pathway member Smad4 to trigger osteoporosis.     

MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN

MiR-195, which exhibits a proliferation-inhibiting role in different tumors, has been reported to be down-regulated in the ectopic endometrium. The aim of this study was to determine the impact of miR-195 on the biological characteristic of the endometrial stromal cells (ESCs). MiR-195 has been presumed to target the 3’-untranslated regions (3’-UTR) of Fractalkine (FKN), which also plays important roles in endometriosis. Fluorescence reporter assays showed that miR-195 effectively binds to the 3’-UTR of FKN. The normal ESCs showed a significant higher miR-195 expression than that of eutopic and ectopic ESCs associated with endometriosis, while the FKN expression showed opposite results. MiR-195 mimics inhibited proliferation and growth and induced apoptosis of eutopic ESCs, and these effects were abolished by FKN-siRNA. miR-195 could decrease the expression of survivin, matrix metalloproteinase-9 (MMP9) and up-regulate the expression of CD82, tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 of eutopic ESCs by targeting FKN. Our study has demonstrated for the first time that miR-195 plays important roles in regulating the functions of ESCs through targeting FKN. The information may be useful for developing a new therapeutic strategy for endometriosis.     

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

RETRACTED: Long noncoding RNA TUG1 facilitates cell ovarian cancer progression through targeting MiR-29b-3p/MDM2 axis

Ovarian cancer (OC) is one of the most aggressive female cancers in the world. OC trends to be diagnosed at an advanced stage with abdominal metastasis. Our study explored the biological function and underlying mechanism of lncRNA on OC cell proliferation and migration. The expression of turine up-regulated gene 1 (TUG1) in human OC tissues and cell lines was measured by qRT-PCR. OC cell proliferation, viability, migration, and invasion were measured by MTT assays, colony formation assays, and transwell assays in vitro. Furthermore, the nude mice xenograft model was established to determine the effects of TUG1 in vivo. The relationship between TUG1 and miR-29b-3p, as well as miR-29b-3p and MDM2 were identified using the luciferase reporter assays. We showed that the expression of TUG1 and MDM2 were significantly increased, but the expression of miR-29b-3p was remarkably decreased in OC tissues and cell lines. Knockdown of TUG1 strongly inhibited the ability of cell proliferation, colony formation, migration, and invasion in vitro. The relationship between TUG1 and miR-29b-3p, or miR-29b-3p and MDM2 were predicted by StarBase and miRanda online software. Besides, miR-29b-3p reversed the positive effect of TUG1 on the OC cell proliferation, migration, and invasion through inhibiting MDM2 expression and increasing p53 phosphorylation level. Moreover, knockdown of TUG1 suppressed tumor growth in vivo. Taken all together, this study shows that TUG1 plays a crucial oncogenic role and facilitates cell proliferation, migration, and invasion in OC through regulating miR-29b-3p/MDM2 axis.     

LncRNA OGFRP1 promotes angiogenesis and epithelial-mesenchymal transition in colorectal cancer cells through miR-423-5p/CTCF axis

ObjectiveTo investigate the mechanism of lncRNA OGFRP1 affecting angiogenesis and epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC) and provide a new target for the treatment of CRC.MethodsThe expressions of OGFRP1, miR-423-5p, and CTCF were measured in CRC cell lines (HT29, LoVo, HCT116, SW620, and SW480) and normal colonic epithelial cells NCM460. Gain and loss of function experiments were performed on HCT116 and SW620 cells, after which the proliferation, apoptosis, EMT, invasion, and migration of the cells were measured using CCK-8 and colony formation assays, flow cytometry, Western blotting, Transwell, and scratch assay. The transfected cells were incubated with human umbilical vein endothelial cells (HUVECs) to assess angiogenesis using tube formation assay. ELISA was performed to detect VEGF in the conditioned medium of HCT116 and SW620 cells. The interactions among OGFRP1, CTCF and miR-423-5p were validated by dual-luciferase reporter assay.ResultsCRC cell lines had increased expression levels of OGFRP1 and CTCF and a suppressed expression level of miR-423-5p when compared with NCM460 cells. Suppression on OGFRP1 or CTCF and overexpression of miR-423-5p led to inhibited proliferation, EMT, invasion and migration and increased apoptosis of HCT116 and SW620 cells. HUVECs incubated with cells transfected with si-OGFRP1, si-CTCF or miR-423-5p mimic had suppressed angiogenesis ability. The effect of OGFRP1 suppression in CRC cells could be counteracted by miR-423-5p inhibition. Both CTCF and OGFRP1 could bind to miR-423-5p.ConclusionOGFRP1 promotes proliferation, EMT, and angiogenesis in CRC through miR-423-5p/CTCF axis.     

MiR-378 as a biomarker for response to anti-angiogenic treatment in ovarian cancer

Objective

To determine the role of miR-378 as a biomarker for anti-angiogenic therapy response in ovarian cancer.

Methods

Expression of miR-378 was analyzed in ovarian cancer cell lines and human tumors vs. normal ovarian epithelial cells by qRT-PCR. After miR-378 transfection in SKOV3 cells, dysregulated genes were identified using microarray. Data from The Cancer Genome Atlas (TCGA) was utilized to correlate miR-378 expression with progression-free survival (PFS) among patients treated with anti-angiogenic therapy by using Kaplan–Meier and Cox proportional hazards.

Results

MiR-378 was overexpressed in ovarian cancer cells and tumors vs. normal ovarian epithelial cells. Overexpressing miR-378 in ovarian cancer cells altered expression of genes associated with angiogenesis (ALCAM, EHD1, ELK3, TLN1), apoptosis (RPN2, HIPK3), and cell cycle regulation (SWAP-70, LSM14A, RDX). In the TCGA dataset, low vs. high miR-378 expression was associated with longer PFS in a subset of patients with recurrent ovarian cancer treated with bevacizumab (9.2 vs. 4.2 months; p = 0.04). On multivariate analysis, miR-378 expression was an independent predictor for PFS after anti-angiogenic treatment (HR = 2.04, 95% CI: 1.12–3.72; p = 0.02). Furthermore, expression levels of two miR-378 targets (ALCAM and EHD1) were associated with PFS in this subgroup of patients who received anti-angiogenic therapy (9.4 vs. 4.2 months, p = 0.04 for high vs. low ALCAM; 7.9 vs. 2.3 months, p < 0.01 for low vs. high EHD1).

Conclusions

Our data suggest that miR-378 is overexpressed in ovarian cancer cells and tumors vs. normal ovarian epithelial cells. MiR-378 and its downstream targets may serve as markers for response to anti-angiogenic therapy.     

MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord

Glia-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. Our recent study demonstrated that TNF receptor associated factor-6 (TRAF6) is expressed in spinal astrocytes and contributes to the maintenance of spinal nerve ligation (SNL)-induced neuropathic pain. MicroRNA (miR)-146a is a key regulator of the innate immune response and was shown to target TRAF6 and reduce inflammation. In this study, we found that in cultured astrocytes, TNF-α, IL-1β, or lipopolysaccharide (LPS) induced rapid TRAF6 upregulation and delayed miR-146a-5p upregulation. In addition, miR-146a-5p mimic blocked LPS-induced TRAF6 upregulation, as well as LPS-induced c-Jun N-terminal kinase (JNK) activation and chemokine CCL2 expression in astrocytes. Notably, LPS incubation with astrocytes enhanced the DNA binding activity of AP-1 to the promoters of mir-146a and ccl2. TRAF6 siRNA or JNK inhibitor SP600125 significantly reduced LPS-induced miR-146a-5p increase in astrocytes. In vivo, intrathecal injection of TNF-α or LPS increased spinal TRAF6 expression. Pretreatment with miR-146a-5p mimic alleviated TNF-α- or LPS-induced mechanical allodynia and reduced TRAF6 expression. Finally, SNL induced miR-146a-5p upregulation in the spinal cord at 10 and 21 days. Intrathecal injection of miR-146a-5p mimic attenuated SNL-induced mechanical allodynia and decreased spinal TRAF6 expression. Taken together, the results suggest that (1) miR-146a-5p attenuates neuropathic pain partly through inhibition of TRAF6 and its downstream JNK/CCL2 signaling, (2) miR-146a-5p is increased by the activation of TRAF6/JNK pathway. Hence, miR-146a-5p may be a novel treatment for chronic neuropathic pain.     

Serum microRNA-133b is associated with low ceruloplasmin levels in Parkinson's disease

IntroductionThe cause of low serum ceruloplasmin levels in Parkinson's disease (PD) remains to be clarified. In this study, we explored serum miR-133b expression to determine whether it correlates with serum ceruloplasmin level in PD patients.MethodsForty-six patients with PD and forty-six control subjects were evaluated for miR-133b expression using qRT-PCR. The serum ceruloplasmin levels in all of the subjects were also determined.ResultsSerum miR-133b expression levels were significantly decreased in PD patients compared with those in the control subjects. Furthermore, PD patients with low serum ceruloplasmin levels also exhibited significantly lower expression of miR-133b compared with that of patients with normal ceruloplasmin levels. MiR-133b expression was correlated with the ceruloplasmin level in patients with PD, whereas no correlation was found between miR-133b and disease severity or motor phenotype.ConclusionOur observations suggest that miR-133b might be involved in ceruloplasmin dysmetabolism in PD patients and a further investigation is warranted to confirm this hypothesis.     

长链非编码RNA ZFAS1在食管癌中的表达及作用机制研究

目的探讨长链非编码RNA锌指结构反义转录本1（zinc finger antisense 1,ZFAS1）在食管癌中的表达及作用机制。方法采用实时荧光定量PCR法分别检测癌旁组织和食管癌组织、正常食管上皮细胞和食管癌细胞的ZFAS1表达水平,通过细胞增殖活性检测、细胞划痕实验及流式细胞仪检测细胞凋亡技术,评价干扰食管癌细胞表达ZFAS1对细胞增殖、迁移及凋亡的影响,采用荧光素酶报告基因验证微小RNA（microRNA,miRNA）miR-302b-3p是ZFAS1的作用靶点,Western蛋白印迹法检测ZFAS1及miR-302b-3p作用下c-Jun氨基末端激酶2（c-Jun N-terminal kinase 2,JNK2）、胰岛素样生长因子1受体（insulin-like growth factor-1 receptor,IGF-1R）、白细胞介素-1受体相关激酶4（interleukin-1 receptor-associated kinase 4,IRAK-4）和上皮细胞激酶（epithelial cell kinase 2,EphA2）蛋白的表达变化。结果与癌旁组织相比,食管癌组织表达ZFAS1水平升高（t=19.40,P<0.001）,与正常食管上皮细胞相比,食管癌细胞表达ZFAS1水平增加（t=13.80,P<0.001）,通过抑制细胞表达ZFAS1发现,与NC干扰组相比,显著降低ZFAS1干扰组细胞增殖活性（t值分别=2.933,8.568,10.04,均P<0.05,）及迁移能力（t=13.96,P<0.001）,且细胞凋亡率升高（t=10.68,P<0.001）。ZFAS1可通过靶向miR-302b-3p调节JNK2、IGF-1R、IRAK4和EphA2蛋白的表达。结论ZFAS1在食管癌组织中表达显著增加,具有促进食管癌细胞增殖、迁移,抑制凋亡的能力,且ZFAS1可通过靶向miR-302b-3p激活JNK2、IGF-1R、IRAK4和EphA2,有望成为治疗食管癌的潜在靶点。