microRNA-501-5p promotes cell proliferation and migration in gastric cancer by downregulating LPAR1

In spite of the achievement in treatment, the gastric cancer (GC) mortality still remains high. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play a crucial part in tumor progression. In this study, we explored the expression and function of microRNA-501-5p (miR-501-5p) in GC cell lines. Quantitative real-time polymerase chain reaction assay results suggested that miR-501-5p was significantly upregulated in GC tissues and cell lines. And, the Cell Counting Kit-8 colony formation and cell migration assay results showed that the downregulation of miR-501-5p decreased GC cell proliferation and migration. Besides that, we found that GC cell cycle was arrested in G2 phase and cell apoptosis rate was increased by silencing the expression of miR-501-5p in GC cell lines using the flow cytometry. We also found that miR-501-5p could directly target lysophosphatidic acid receptor 1 (LPAR1) and negatively regulate LPAR1 expression in GC cell lines by performing dual-luciferase reporter gene assay and Western blot analysis. And, LPAR1 was significantly downregulated in GC tissues and inversely correlated with miR-501-5p expression. Furthermore, LPAR1 downregulation promoted cell proliferation and migration, which were attenuated by cotransfection of miR-501-5p inhibitor in GC cells. In conclusion, miR-501-5p can promote GC cell proliferation and migration by targeting and downregulating LPAR1. miR-501-5p/LPAR1 may become a potential therapeutic target for GC treatment.     

CircRNA circPDSS1 promotes the gastric cancer progression by sponging miR-186-5p and modulating NEK2

The aim of this study is to investigate the regulatory mechanism of circPDSS1/miR-186-5p/NEK2 axis on the viability and proliferation in gastric cancer (GC) cell line. Differentially expressed circRNAs, miRNAs, and mRNAs in GC tissues and paracarcinoma tissues were analyzed using gene chips GSE83521, GSE89143, and GSE93415. Then, the expression of circPDSS1, miR-186-5p, and NEK2 was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was adopted to explore the association between the circPDSS1 expression and the prognosis of GC. The effect of circPDSS1 on GC cell cycle and apoptosis was verified with the flow cytometry. Targeting relationships among circPDSS1, miR-186-5p, and NEK2 were predicted via bioinformatics analysis and demonstrated by the dual-luciferase reporter assay. Our results showed that circPDSS1 and NEK2 were high-expressed whereas miR-186-5p was low-expressed in GC tissues and cells. CircPDSS1 promoted GC cell cycle and inhibited apoptosis by sponging miR-186-5p, while miR-186-5p inhibited cell cycle and promoted apoptosis by targeting NEK2. Thus, circPDSS1 acts as a tumor promoter by regulating miR-186-5p and NEK2, which could be a potential biomarker and therapeutic target for the management of GC.     

The MicroRNA-217 Functions as a Potential Tumor Suppressor in Gastric Cancer by Targeting GPC5

Gastric cancer (GC) is one of the most common malignancies worldwide. Emerging evidence has shown that aberrant expression of microRNAs (miRNAs) plays important roles in cancer progression. However, little is known about the potential role of miR-217 in GC. In this study, we investigated the role of miR-217 on GC cell proliferation and invasion. The expression of miR-217 was down-regulated in GC cells and human GC tissues. Enforced expression of miR-217 inhibited GC cells proliferation and invasion. Moreover, Glypican-5 (GPC5), a new ocncogene, was identified as the potential target of miR-217. In addition, overexpression of miR-217 impaired GPC5-induced promotion of proliferation and invasion in GC cells. In conclusion, these findings revealed that miR-217 functioned as a tumor suppressor and inhibited the proliferation and invasion of GC cells by targeting GPC5, which might consequently serve as a therapeutic target for GC patients.     

The effects of MOTILIPERM on cisplatin induced testicular toxicity in Sprague–Dawley rats

Genome-wide miRNA expression profile has identified microRNA (miR)-96 as one of upregulated miRNAs in clinical bladder cancer (BC) tissues compared to normal bladder tissues. The aim of this study was to confirm the expression pattern of miR-96 in BC tissues and to investigate its involvement in carcinogenesis. Quantitative real-time PCR was performed to detect the expression levels of miR-96 in 60 BC and 40 normal control tissues. Bioinformatics prediction combined with luciferase reporter assay were used to verify whether the cyclin-dependent kinase inhibitor CDKN1A was a potential target gene of miR-96. Cell counting kit-8 and apoptosis assays were further performed to evaluate the effects of miR-96-CDKN1A axis on cell proliferation and apoptosis of BC cell lines. We validated that miR-96 was significantly increased in both human BC tissues and cell lines. According to the data of miRTarBase, CDKN1A might be a candidate target gene of miR-96. In addition, luciferase reporter and Western blot assays respectively demonstrated that miR-96 could bind to the putative seed region in CDKN1A mRNA 3′UTR, and significantly reduce the expression level of CDKN1A protein. Moreover, we found that the inhibition of miR-96 expression remarkably decreased cell proliferation and promoted cell apoptosis of BC cell lines, which was consistent with the findings observed following the introduction of CDKN1A cDNA without 3′UTR restored miR-96. Our data reveal that miR-96 may function as an onco-miRNA in BC. Upregulation of miR-96 may contribute to aggressive malignancy partly through suppressing CDKN1A protein expression in BC cells.     

MicroRNA-135b/CAMK2D Axis Contribute to Malignant Progression of Gastric Cancer through EMT Process Remodeling

There is a continued need for investigating the roles of microRNAs (miRNAs) and their targets on the progression of gastric cancer (GC), especially metastasis. Here, we performed an integrated study to identify dysregulated miRNAs critical for GC development and progression. miR-135b was determined as a promising biomarker for GC. The expression level of miR-135b was increased among GC cell lines, patient tumor tissues, serum samples, and correlation with aggravation of the GC patients. The in vitro functional assays demonstrated overexpression of miR-135b promoted cell proliferation, migration and invasion in GC, while miR-135b inhibition led to the opposite results. CAMK2D was found to be the direct target of miR-135b, serving as a tumor suppressor in GC cells. Based on our and public datasets, we confirmed the attenuation of CAMK2D expression in GC tissues. And, the expression levels of miR-135b and CAMK2D were closely associated with prognosis of GC patients. Ectopic expression of miR-135b resulted in the down-regulation of CAMK2D. Additionally, CAMK2D was a prerequisite for miR-135b to promote GC cells proliferation and migration by regulating the EMT process, which was confirmed by the in vivo experiments. Importantly, in vivo injection of miR-135b antagomir significantly repressed the tumor growth and metastasis of xenograft models, which suggested that the miR-135b antagomir were promising for clinical applications. Taken together, these results indicate that miR-135b/CAMK2D axis drives GC progression by EMT process remodeling, suggesting that miR-135b may be utilized as a new therapeutic target and prognostic marker for GC patients.     

miR-25 promotes gastric cancer cells growth and motility by targeting RECK

Gastric cancer (GC) is the second leading cause of cancer-related death worldwide. Recently, accumulating evidence suggests that microRNAs (miRNAs) play prominent roles in tumorigenesis and metastasis. Here, we confirmed that miR-25 was significantly increased in human GC tissues and cell lines. Forced expression of miR-25 remarkably enhanced cell proliferation, migration, and invasion in GC cells, whereas inhibition of miR-25 by inhibitor caused significant suppression of proliferation and significant increase of apoptosis. Moreover, inhibition of miR-25 significantly decreased migration and invasion of GC cells. Finally, reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was found to be a target of miR-25. Overexpression of RECK could significantly reverse the oncogenic effect of miR-25. Taken together, miR-25 might promote GC cells growth and motility partially by targeting RECK.     

MicroRNA-638 inhibits human airway smooth muscle cell proliferation and migration through targeting cyclin D1 and NOR1

Abnormal airway smooth muscle cell (ASMC) proliferation and migration contribute significantly to increased ASM mass associated with asthma. MicroRNA (miR)-638 is a primate-specific miRNA that plays important roles in development, DNA damage repair, hematopoiesis, and tumorigenesis. Although it is highly expressed in ASMCs, its function in ASM remodeling remains unknown. In the current study, we found that in response to various mitogenic stimuli, including platelet-derived growth factor-two B chains (PDGF-BB), transforming growth factor β1, and fetal bovine serum, the expression of miR-638, as determined by quantitative real-time polymerase chain reaction (qRT-PCR), was significantly downregulated in the proliferative human ASMCs. Both gain- and loss-of-function studies were performed to study the role of miR-638 in ASMC proliferation and migration. We found that adenovirus-mediated miR-638 overexpression markedly inhibits ASMC proliferation and migration, while ablation of miR-638 by anti-miR-638 markedly increases cell proliferation and migration, as determined by WST-8 proliferation and scratch wound assays. Dual-luciferase reporter assay, qRT-PCR, and immunoblot analysis were used to investigate the effects of miR-638 on the expression of the downstream target genes in ASMCs. Our results demonstrated that miR-638 overexpression significantly reduced the expression of downstream target cyclin D1 and NOR1, both of which have been shown to be essential for cell proliferation and migration. Together, our study provides the first in vitro evidence highlighting the antiproliferative and antimigratory roles of miR-638 in human ASMC remodeling and suggests that targeted overexpression of miR-638 in ASMCs may provide a novel therapeutic strategy for preventing ASM hyperplasia associated with asthma.     

microRNA-203 suppresses bladder cancer development by repressing bcl-w expression

It is increasingly clear that microRNAs (miRNAs) play an important role in many diseases, including tumorigenesis. However, the mechanisms by which miRNAs regulate bladder cancer development remain poorly understood. Here, we evaluated the expression of microRNA-203 (miR-203) in bladder cancer tissues using real-time PCR, and defined the target genes and biologically functional effect using luciferase reporter assay, flow cytometry and western blot analysis. We first verified that the expression of miR-203 was decreased in bladder cancer tissues. Moreover, ectopic expression of miR-203 promoted the apoptosis of human bladder cancer cell lines and inhibited cell proliferation, whereas its depletion increased cell growth. We further verified that miR-203 directly targeted 3'-untranslated region of the bcl-w gene, and decreased its expression in vitro and in vivo. Western blot analysis also showed that the expression level of miR-203 was negatively correlated with bcl-w level in tumor tissues. These data suggest an important role for miR-203 in the molecular etiology of bladder cancer and implicate the potential application of miR-203 in bladder cancer therapy.     

miR-362靶向ZNF644基因调控猪未成熟支持细胞的增殖和凋亡

睾丸组织中未成熟支持细胞的增殖能力决定成熟支持细胞的数量,进而制约成年雄性动物的精子生成能力。研究表明microRNA (miRNA)参与调控猪未成熟支持细胞的增殖和凋亡,但大部分鉴定出的miRNA功能仍不明确。本文基于前期RNA-seq数据筛选结果,研究了miR-362对猪未成熟支持细胞增殖和凋亡的调控作用。首先利用生物信息学方法预测miR-362的靶基因,通过qRT-PCR技术检测miR-362和ZNF644基因在不同发育阶段的猪睾丸组织中的表达水平以及在猪未成熟支持细胞中过表达或抑制表达miR-362后ZNF644基因的表达水平,采用双荧光素酶报告基因系统验证miR-362与ZNF644基因之间的靶向关系。结果显示,miR-362与ZNF644基因3′UTR具有一个潜在的结合位点,miR-362和ZNF644基因在猪睾丸组织中的mRNA表达水平显著负相关(r=-0.723, P<0.01),miR-362和psiCHECK2-ZNF644-WT 3′UTR共转染组的双荧光活性显著降低,且miR-362显著调节ZNF644基因的表达水平,表明miR-362靶向ZNF644基因并抑制其表达水平。为进一步检测过表达miR-362或抑制表达ZNF644基因对猪未成熟支持细胞增殖和凋亡的影响,通过流式细胞术检测细胞周期,CCK8和EdU试剂盒检测细胞增殖情况,Annexin V-FITC/PI方法和qRT-PCR技术检测细胞凋亡情况及凋亡相关基因的表达水平。结果表明,过表达miR-362后,猪未成熟支持细胞周期被阻滞在G₁期,抑制表达ZNF644基因后,猪未成熟支持细胞被阻滞在G₂期,细胞增殖能力显著减弱,细胞凋亡率显著提高,细胞凋亡相关基因呈促进凋亡的差异表达。本研究结果证实miR-362靶向ZNF644基因抑制猪未成熟支持细胞的增殖而促进其凋亡,为深入研究miR-362在猪精子生成过程中的生物学功能提供了理论基础。     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

MiR-483-5p suppresses the proliferation of glioma cells via directly targeting ERK1

MicroRNAs (miRNAs) exhibit tumor-specific expression signatures and play crucial roles in tumorigenesis by targeting oncogenes. Here, through analyzing the miRNA-array profiles of human glioblastoma tissues and the adjacent normal brain tissues, we found miR-483-5p was significantly down-regulated in gliomas, which was confirmed in both human glioma specimens and cell lines. The overexpression of miR-483-5p suppressed glioma cell proliferation and induced a G0/G1 arrest. In contrast, miR-483-5p inhibition promoted cell proliferation. Furthermore, by a dual-luciferase reporter assay and expression analysis, we identified extracellular signal-regulated kinase 1 (ERK1) as a direct target of miR-483-5p. ERK1 knockdown can block cell proliferation induced by miR-483-5p inhibition. Thus, our findings provide the first evidence that miR-483-5p can serve as a tumor suppressor in gliomas.     

MicroRNA-135a Inhibits Cell Proliferation by Targeting Bmi1 in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies. MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy. In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines. The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line. We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a. The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis. We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC. Its expression was inversely correlated with miR-135a expression in PDAC. Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3''-untranslated region (3''-UTR) of Bmi1. Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor. miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.     

miR-4510 acts as a tumor suppressor in gastrointestinal stromal tumor by targeting APOC2

Dysregulation of microRNAs (miRNAs) expression has been demonstrated in gastrointestinal stromal tumor (GIST). In this study, we aimed to determine the differential miRNAs expression in GISTs and explore the functional mechanism of differential miRNAs in GIST cells. We measured differential miRNAs in six pairs of GIST tissues and matched adjacent tissues through a high-throughput sequencing, which was confirmed in 64 pairs of GIST tissues and adjacent tissues by real-time polymerase chain reaction. We found that miR-4510 expression was significantly downregulated in GIST tissues compared to matched control tissues. Luciferase reporter assay identified apolipoprotein C-II (APOC2) as a direct target of miR-4510. Overexpression of miR-4510 inhibited the mRNA and protein expression of APOC2. In addition, overexpression of miR-4510 suppressed GIST cell proliferation, migration, and invasion. Overexpression of miR-4510 also inhibited the phosphorylation of AKT and ERK1/2, reduced the expression of matrix metallopeptidase 2 (MMP2) and MMP9. APOC2 knockdown mimicked the effect of miR-4510 overexpression. Further investigation confirmed that APOC2 was notably upregulated in GIST tissues compared to adjacent control tissues. These results suggested that miR-4510 downregulation could promote GIST progression, including growth, invasion, and metastasis, through increasing APOC2 expression.     

MicroRNA-30a functions as tumor suppressor and inhibits the proliferation and invasion of prostate cancer cells by down-regulation of SIX1

Increasing reports have demonstrated that aberrant expression of microRNAs (miRNAs) is found in multiple human cancers. Many studies have shown that down-regulated level of miR-30a is in a variety of cancers including prostate cancer (PCa). However, the precise mechanisms of miR-30a in PCa have not been well explored. In this study, we investigated the biological functions and molecular mechanism of miR-30a in PCa cell lines, discussing whether it could be a therapeutic biomarker of PCa in the future. We found that miR-30a is down-regulated in PCa tissues and cell lines. Moreover, the low level of miR-30a was associated with increased expression of SIX1 in PCa tissues and cell lines. Up-regulation of miR-30a significantly inhibited proliferation of PCa cells. In addition, invasion of PCa cells was suppressed by overexpression of miR-30a. However, down-regulation of miR-30a promoted cell growth and invasion of PCa cells. Bioinformatics analysis predicted that the SIX1 was a potential target gene of miR-30a. Next, luciferase reporter assay confirmed that miR-30a could directly target SIX1. Consistent with the effect of miR-30a, down-regulation of SIX1 by siRNA inhibited proliferation and invasion of PCa cells. Overexpression of SIX1 in PCa cells partially reversed the effect of miR-30a mimic. In conclusion, introduction of miR-30a dramatically inhibited proliferation and invasion of PCa cells by down-regulating SIX1 expression, and that down-regulation of SIX1 was essential for inhibition of cell growth and invasion of PCa cells by overexpression of miR-30a.     

miR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G₁ cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.