hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTSmiRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSIONOur data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

miR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis

AIM: To profile expression of microRNAs (miRNAs) in gastric cancer cells and investigate the effect of miR-374b-5p on gastric cancer cell invasion and metastasis.

Endometrial MicroRNA Signature during the Window of Implantation Changed in Patients with Repeated Implantation Failure

Background： At present, a diagnostic tool with high specificity for impaired endometrial receptivity, which may lead to implantation failure, remains to be developed. We aimed to assess the different endometrial microRNA （miRNA） signatures for impaired endometrial receptivity by microarray analysis. Methods： A total of 12 repeated implantation failure （RIF） patients and I0 infertile patients, who conceived and delivered after one embryo transfer attempt, were recruited as RIF and control groups, respectively. Endometrial specimens from the window of implantation （WOI） were collected from these two groups. MiRNA microarray was conducted on seven and five samples from the RIF and control groups, respectively. Comparative, functional, and network analyses were performed for the microarray results. Quantitative real-time polymerase chain reaction （PCR） was performed on other samples to validate the expression of specific miRNAs. Results： Compared with those in the control group, the expression levels of 105 miRNAs in the RIF group were found to be significantly up- or down-regulated （at least 2-fold） by microarray analysis. The most relevant miRNA functional sets of these dysregulated miRNAs were miR-30 family, human embryonic stern cell regulation, epithelial-mesenchymal transition, and miRNA tumor suppressors by tool for annotations ofmicroRNA analysis. Network regulatory analysis found 176 miRNA-mRNA interactions, and the top 3 core miRNAs were has-miR-4668-5p, has-miR-429, and has-miR-5088. Expression levels of the 18 selected miRNAs in new samples by real-time PCR were found to be regulated with the same trend, as the result ofmicroarray analysis. Conclusions： There is a significant different expression of certain miRNAs in the WOI endometrium for RIF patients. These miRNAs may contribute to impaired endometrial receptivity.

Differentially Expressed microRNAs as Potential Markers for Vital Reaction of Burned Skin

The identification of antemortem burns and postmortem burns is essential in forensic practice.In this study,microRNA(miRNA)microarray analysis was conducted to identify differentially expressed miRNAs in the skin of an experimental burn model.Microarray analysis revealed 24 differentially expressed miRNAs in antemortem burned mice skin,with 19 miRNAs significantly upregulated and 5 downregulated.Based on the intersection predicted using three databases(Targetscan,microRNA.org,and PITA),293 potential miRNA targets were identified.These dysregulated miRNAs and their predicted targets were further analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases.Several functional categories and signaling pathways were enriched,including the“fc epsilon ri signaling pathway,”“endometrial cancer,”and“mTOR signaling pathway.”Expression patterns of 10 differentially expressed miRNAs were verified by reverse transcription‑quantitative polymerase chain reaction in mice skins.The results agreed with the results of microarray analysis.These findings suggest that differentially expressed miRNAs revealed by microarray are potential markers for forensic molecular diagnosis of antemortem burns.

MicroRNA Profiling of Transgenic Mice with Myocardial Overexpression of Nucleolin

Background：Nucleolin （NCL） is the most abundant RNA-binding protein in the cell nucleolus and plays an important role in chromatin stability,ribosome assembly,ribosomal RNA maturation,ribosomal DNA transcription,nucleocytoplasmic transport,and regulation of RNA stability and translation efficiency.In addition to its anti-apoptotic properties,the underlying mechanisms associated with NCL-related roles in different cellular processes remain unclear.In this study,the effect of NCL on microRNA （miRNA） expression was evaluated by generating transgenic mice with myocardial overexpression of NCL and by analyzing microarrays of mature and precursor miRNAs from mice.Methods：Using microinjection ofalpha-MyHc clone 26-NCL plasmids,we generated transgenic mice with myocardial overexpression of NCL firstly,and then mature and precursor miRNAs expression profiles were analyzed in NCL transgenic mice （n =3） and wild-type （WT） mice （n =3） by miRNA microarrays.Statistical Package for the Social Sciences version 16.0 software （SPSS,Inc.,Chicago,IL,USA） was used to perform Student＇s t-test,and statistical significance was determined at P 〈 0.05.Results：Several miRNAs were found to be differentially expressed,of which 11 were upregulated and 4 were downregulated in transgenic mice with myocardial overexpression of NCL compared to those in WT mice.Several differentially expressed miRNAs were subsequently confirmed and quantified by real-time quantitative reverse transcription-polymerase chain reaction.Bioinformatics analysis was used for the prediction of miRNA targets.Furthermore,in vitro experiments showed that NCL regulated miR-21 expression following hydrogen peroxide preconditioning.Conclusions：Myocardial-protection mechanisms exerted by NCL might be mediated by the miRNAs identified in this study.

The miRNA expression profile of the uveal melanoma

The miRNA expression profile was initially established to investigate its corresponding function in human uveal melanoma. The miRNA expression profile in human uveal melanoma was analyzed by a micro chip technique.The hsa-miRNA expression between four uveal melanomas and four normal uveal tissues was compared.Based on the bioinformatic approach,chip data was analyzed to select out differentially expressed candidate hsa-miRNAs.Real-time quantitative PCR(RT-PCR) was used to confirm the candidate hsa-miRNAs expression in all samples.The results of miRNA microarray chips that matched with RT-PCR were considered as the miRNA expression which was significantly different between normal tissue and uveal melanomas.In four uveal melanomas,expressions of miRNA-20a,miRNA-106a,miRNA-17,miRNA-21,and miRNA-34a were significantly up-regulated,while miRNA-145 and miRNA-204 expression were significantly down-regulated.We used miRNA microarray analysis as a fast,efficient technology to study biological information.The differentially expressed miRNAs may be involved in uveal melanoma pathogenesis,and may help promote the diagnosis and treatment for uveal melanoma.

miRNA expression profile during fluid shear stress-induced osteogenic differentiation in MC3T3-E1 cells

Background Mechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress （FSS） is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells. Methods MC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm2 using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs （miRNAs） were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase （ALP） activity assay was performed at 24th, 48th, and 72th hours after FSS treatment, and Alizarin Red Staining was checked at day 12. Results One hour of FSS at 12 dyn/cm2 induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated. Conclusion The short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be invovled in FSS-induced pre-osteoblast differentiation.