MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells via targeting of mTOR

Objectives

To investigate the biological functions of microRNA-144-3p with respect to proliferation and apoptosis of human salivary adenoid carcinoma cell lines via mTOR.

Results

After transfection of microRNA-144-3p agomir, cell viability assays confirmed that the salivary adenoid carcinoma cell (SACC) proliferation was inhibited and apoptosis was induced. Dual luciferase reporter assay validated that the mammalian target of rapamycin (mTOR) was a direct target of miR-144-3p. Western blot, immunofluorescent analysis and a xenograft mouse model of adenoid cystic carcinoma indicated that miR-144-3p was a tumor suppressor and repressed mTOR expression and signaling in SACCs.

Conclusions

MicroRNA-144-3p inhibits proliferation and induces apoptosis of human salivary adenoid carcinoma cells by downregulating mTOR expression in vitro and in vivo.

     

涎腺腺样囊性癌细胞蛋白激酶C的活性阻抑对其侵袭能力的影响

用蛋白激酶Ｃ（ＰＫＣ） 抑制剂Ｈ７ 作用于人涎腺腺样囊性癌ＳＡＣＣ８３ 系, 利用扫描电镜观察对人羊膜基底膜侵袭能力的影响。癌细胞接种于羊膜基底膜表面４８－ ７２ 小时后, 在扫描电镜下可以观察到对照组癌细胞侵袭现象, 侵袭的细胞伸出丝状伪足且侵入基底膜内, 伪足周围的基底膜断裂和被溶解, 而实验组癌细胞侵袭现象很难发现。细胞附于基底膜表面, 细胞表面较光滑且无明显伪足伸出, 基底膜无明显受损改变。实验结果表明, 降低涎腺腺样囊性癌细胞的ＰＫＣ活性可明显降低其侵袭能力, ＰＫＣ与涎腺腺样囊性癌的侵袭能力有密切的关系     

Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.     

miRNA-7-5p inhibits melanoma cell migration and invasion

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of melanoma. However, the precise mechanistic role of many of these miRNAs remains unclear. We have investigated the functional role of miR-7-5p in melanoma, and demonstrate that miR-7-5p expression is reduced in metastatic melanoma-derived cell lines compared with primary melanoma cells, and that when ectopically expressed miR-7-5p significantly inhibits melanoma cell migration and invasion. Additionally, we report that insulin receptor substrate-2 (IRS-2) is a target of miR-7-5p in melanoma cells, and using RNA interference (RNAi) we provide evidence that IRS-2 activates protein kinase B (Akt), and promotes melanoma cell migration. Thus, miR-7-5p may represent a novel tumor suppressor miRNA in melanoma, acting at least in part via its inhibition of IRS-2 expression and oncogenic Akt signaling.     

MicroRNA-143 regulates adipocyte differentiation

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.     

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.     

MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting MAPK–Snai2 pathway

Background

To date microRNAs and their contribution to the onset and propagation of salivary adenoid cystic carcinoma (SACC) are limited. The objective of this study was to identify miR-181a and its mechanism in the metastasis of SACC.

Methods

At first microarray and quantitative RT-PCR were used to investigate microRNA profiles and miR-181a in paired SACC cell lines with different metastatic potential. Then the effect of miR-181a on metastatic potential of SACC was investigated. MiR-181a target genes and Snai2 promoter activity were investigated using luciferase reporter gene assays. Western blot was used to detect MAPK–Snai2 pathway-related protein level.

Results

A panel of deregulated microRNAs (including miR-181a) was identified in paired of SACC cell lines. Functional analysis indicated that miR-181a inhibited SACC cell migration, invasion and proliferation in vitro, and it suppressed tumor growth and lung metastasis in vivo. Direct targeting of miR-181a to MAP2K1, MAPK1 and Snai2 was confirmed by luciferase reporter gene assays. MiR-181a mimic inhibited the expression of MAP2K1, MAPK1 and Snai2 in SACC cells. MAP2K1 or MAPK1 siRNA suppressed Snai2 gene promoter activity and reduced Snai2 expression and the metastatic potential of SACC cells.

Conclusions

Our results indicate that miR-181a plays an important role in the metastasis of SACC, and may serve as a novel therapeutic target for SACC. MiR-181a regulates the MAPK–Snai2 pathway both through direct cis-regulatory mechanism and through indirect trans-regulatory mechanism.

General significance

To our knowledge, this is the first study revealing that miR-181a deregulation mediated the metastasis of SACC by regulating MAPK–Snai2 pathway.     

miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation

MicroRNAs are small non-coding RNAs that regulate protein expression by binding 3′UTRs of target mRNAs, thereby inhibiting translation. Similar to siRNAs, miRNAs are cleaved by Dicer. Mouse and ES cell Dicer mutants demonstrate that microRNAs are necessary for embryonic development and cellular differentiation. However, technical obstacles and the relative infancy of this field have resulted in few data on the functional significance of individual microRNAs. We present evidence that miR-17 family members, miR-17-5p, miR-20a, miR-93, and miR-106a, are differentially expressed in developing mouse embryos and function to control differentiation of stem cells. Specifically, miR-93 localizes to differentiating primitive endoderm and trophectoderm of the blastocyst. We also observe high miR-93 and miR-17-5p expression within the mesoderm of gastrulating embryos. Using an ES cell model system, we demonstrate that modulation of these miRNAs delays or enhances differentiation into the germ layers. Additionally, we demonstrate that these miRNAs regulate STAT3 mRNA in vitro. We suggest that STAT3, a known ES cell regulator, is one target mRNA responsible for the effects of these miRNAs on cellular differentiation.     

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.     

Identification of potential miRNA biomarkers for traumatic osteonecrosis of femoral head

Traumatic osteonecrosis of femoral head (TONFH) is a common orthopedic disease caused by physical injury in hip. However, the unclear pathogenesis mechanism of TONFH and lacking of simple noninvasive early diagnosis method cause the necessity of hip replacement for most patients with TONFH. In this study, we aimed to identify circulating microRNAs (miRNAs) by integrated bioinformatics analyses as potential biomarker of TONFH. mRNA expression profiles were downloaded from the Gene Expression Omnibus database. Then we combined two miRNA screen methods: Weighted gene co-expression network analysis and fold change based differentially expressed miRNAs analysis. As a result, we identified 14 key miRNAs as potential biomarkers for TONFH. Besides, 302 target genes of these miRNAs were obtained and the miRNA–mRNA interaction network was constructed. Furthermore, the results of Kyoto Encyclopedia of Gene and Genome pathway analysis, Gene Ontology function analysis, protein–protein interaction (PPI) network analysis and PPI network module analysis showed close correlation between these 14 key miRNAs and TONFH. Then we established receiver operating characteristic curves and identified 6-miRNA signature with highly diagnosis value including miR-93-5p (area under the curve [AUC] = 0.93), miR-1324 (AUC = 0.92), miR-4666a-3p (AUC = 0.92), miR-5011-3p (AUC = 0.92), and miR-320a (AUC = 0.89), miR-185-5p (AUC = 0.89). Finally, the results of quantitative real-time polymerase chain reaction confirmed the significantly higher expression of miR-93-5p and miR-320a in the serum of patients with ONFH. These circulating miRNAs could serve as candidate early diagnosis markers and potential treatment targets of TONFH.     

MicroRNAs: new players in heart failure

MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the most exciting areas of modern medical science. miRNAs modulate a large and complex regulatory network of gene expression of the majority of the protein-coding genes. Currently, evidences suggest that miRNAs play a crucial role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and miR-208a are highly expressed in the heart and strongly associated with the development of cardiac hypertrophy. Recent data indicate that these miRNAs as well as miR-206 change their expression quickly in response to physical activity. The differential regulation of miRNAs in response to exercise suggests a potential value of circulating miRNAs (c-miRNAs) as biomarkers of physiological mediators of the cardiovascular adaptation induced by exercise. Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of miRNAs levels using techniques such as ‘miR mimics’ and ‘antagomiRs’ is becoming evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.     

MicroRNA-590-5p regulates proliferation and invasion in human hepatocellular carcinoma cells by targeting TGF-β RII

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that can regulate gene expression by binding to gene elements, such as the gene promotor 5'UTR, mainly in the 3'UTR of mRNA. One miRNA targets many mRNAs, which can be regulated by many miRNAs, leading to a complex metabolic network. In our study, we found that the expression level of miR-590-5p is higher in the human hepatocellular carcinoma cell line HepG2 than in the normal hepatocellular cell line L02. Downregulation of miR-590-5p inhibited proliferation and invasion of hepatocellular carcinoma cells (HCCs). We also showed that expression of TGF-beta RII, which has been regarded as a regulator of tumor proliferation, invasion, and migration in hepatocellular carcinoma, is regulated by miRNA-590-5p. In addition, miR-590-5p downregulated the expression of TGF-beta RII by targeting the 3'UTR of mRNA. We also found that downregulation of miR-590-5p was associated with an elevation of TGF-beta RII and inhibition of proliferation and invasion in HepG2 cells. Furthermore, overexpression of miR-590-5p was associated with upregulation of TGF-beta RII and could promote proliferation and invasion in L02 cells. In conclusion, we determined that TGF-beta RII is a novel target of miRNA-590-5p. Thus, the role of TGF-beta RII in regulating proliferation and invasion of human HCCs is controlled by miR-590-5p. In other words, miR-590-5p promotes proliferation and invasion in human HCCs by directly targeting TGF-beta RII.     

MicroRNA调控动物脂肪细胞的分化

MicroRNA (miRNA)属于非编码小调节RNA,在动物细胞的增殖、分化、凋亡和代谢等许多生物学过程中具重要作用.研究显示大量miRNA也参与动物脂肪细胞的分化调节,在前体脂肪细胞向成熟脂肪细胞的分化过程中具有多种功能.目前的研究结果表明,这些miRNA在脂肪细胞分化的早期或后期通过其靶基因发挥功能,如miR-17-92和miR-143分别通过其靶基因Rb 2/p 130和ERK 5/BMK 1调节脂肪细胞分化,过表达可促进体外培养的脂肪细胞分化.因此,了解更多miRNA在脂肪细胞分化中的功能,可以加深对动物脂肪形成分子机制的理解,并有可能将其作为脂类代谢性疾病治疗的潜在靶点.     

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.     

靶向调控血管生成素miRNA的筛选和验证

血管生成素是一个重要的促血管生成因子,在细胞增殖、迁移和凋亡等过程中均发挥重要作用,但其具体的分子机制尚待阐明.miRNA是一类长约22 nt的小RNA,在转录后水平调控基因的表达,广泛参与各种生物学过程.本文探索了可直接调控血管生成素表达的miRNA,希望为阐明血管生成素的作用机制提供线索.首先,我们利用数据库预测得到8个可能靶向结合血管生成素mRNA 3′端非编码区的miRNA;然后,用实验方法验证它们与血管生成素的靶向关系,发现miR-1208、miR-196b、miR-296、miR-409-3p、miR-570和miR-641这6个miRNA可以不同程度地抑制血管生成素的mRNA和蛋白质表达水平,但只有miR-196b、miR-296、miR-409-3p和miR-641可以直接结合血管生成素mRNA的3′端非编码区;进而,在血管内皮细胞中分别过表达这4个miRNA,发现miR-196b、miR-409-3p和miR-641可以抑制血管内皮细胞的细胞增殖,而miR-196b、miR-296和miR-409-3p可以抑制血管内皮细胞的管腔形成.以上结果表明,细胞内有多个miRNA调控血管生成素的表达,它们可能协调调节血管生成,抑或在血管生成的不同阶段发挥作用.我们的工作还为“一种mRNA可被多种microRNA调节,而一种microRNA可调节多种mRNA”假说提供了部分证据.