Sequential expression of miR-221-3p and miR-338-3p in Schwann cells as a therapeutic strategy to promote nerve regeneration and functional recovery

The functional properties of endogenous Schwann cells(SCs)during nerve repair are dynamic.Optimizing the functional properties of SCs at different stages of nerve repair may have therapeutic benefit in improving the repair of damaged nerves.Previous studies showed that miR-221-3p promotes the proliferation and migration of SCs,and miR-338-3p promotes the myelination of SCs.In this study,we established rat models of sciatic nerve injury by bridging the transected sciatic nerve with a silicone tube.We injected a miR-221 lentiviral vector system together with a doxycycline-inducible Tet-On miR-338 lentiviral vector system into the cavity of nerve conduits of nerve stumps to sequentially regulate the biological function of endogenous SCs at different stages of nerve regeneration.We found that the biological function of SCs was sequentially regulated,the diameter and density of myelinated axons were increased,the expression levels of NF200 and myelin basic protein were increased,and the function of injured peripheral nerve was improved using this system.miRNA Target Prediction Database prediction,Nanopore whole transcriptome sequencing,quantitative PCR,and dual luciferase reporter gene assay results predicted and verified Cdkn1b and Nrp1 as target genes of miR-221-3p and miR-338-3p,respectively,and their regulatory effects on SCs were confirmed in vitro.In conclusion,here we established a new method to enhance nerve regeneration through sequential regulation of biological functions of endogenous SCs,which establishes a new concept and model for the treatment of peripheral nerve injury.The findings from this study will provide direct guiding significance for clinical treatment of sciatic nerve injury.

Antitumor activity of miR-188-3p in gastric cancer is achieved by targeting CBL expression and inactivating the AKT/mTOR signaling

BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.

Overexpression of miR-196b and HOXA10 characterize a poor-prognosis gastric cancer subtype

AIM:To identify molecular biologic differences between two gastric adenocarcinoma subgroups presenting different prognoses through the analysis of microRNA and protein expression.METHODS:Array technologies were used to generate1146 microRNAs and 124 proteins expression profiles of samples from 60 patients with gastric cancer.For the integrative analysis,we used established mRNA expression data published in our previous study.Whole mRNA expression levels were acquired from microarray data for 60 identical gastric cancer patients.Two gastric adenocarcinoma subgroups with distinct mRNA expression profiles presented distinctly different prognoses.MicroRNA and protein expression patterns were compared between gastric cancer tissue and normal gastric tissue and between two different prognostic groups.Aberrantly expressed microRNA,associated mRNA,and protein in patients with poor-prognosis gastric cancer were validated by quantitative reverse transcription polymerase chain reaction and immunochemistry in independent patients.RESULTS:We obtained the expression data of 1146microRNAs and 124 cancer-related proteins.Four microRNAs were aberrantly expressed in the two prognostic groups and in cancer vs non-cancer tissues(P<0.05).In the poor-prognosis group,miR-196b,miR-135b,and miR-93 were up-regulated and miR-29c*was down-regulated.miR-196b expression positively correlated with Homeobox A10(HOXA10)expression(r=0.726,P<0.001),which was significantly increased in poor-prognosis patients(P<0.001).Comparing gastric cancer with non-cancer tissues,46/124 proteins showed differential expression(P<0.05);COX2(P<0.001)and cyclin B1(P=0.017)were clearly overexpressed in the poor-prognosis group.CONCLUSION:Co-activation of miR-196b and HOXA10characterized a poor-prognosis subgroup of patients with gastric cancer.Elucidation of the biologic function of miR-196b and HOXA10 is warranted.

Expression of miR-155 in nasopharyngeal carcinoma and its effect on proliferation of human nasopharyngeal carcinoma cells

Objective:To investigate the expression of microRNA-155(miR-155)in nasopharyngeal carcinoma and its effect on the proliferation of nasopharyngeal carcinoma cells.Methods:Patients with nasopharyngeal carcinoma who underwent surgical resection in our hospital from September 2016 to September 2018 were selected.miR-155 was detected by real-time fluorescence quantitative PCR in nasopharyngeal carcinoma tissues,normal tissues and nasopharyngeal carcinoma cell lines(CEN1),which were resected in our hospital from September 2016 to September 2018.The expression of NP69 in normal nasopharyngeal epithelial cells was detected by silencing miR-155,and its effect on the proliferation of human nasopharyngeal carcinoma cells was detected.Results:The expression of miR-155 in nasopharyngeal carcinoma tissue and cell line CEN1 was significantly higher than that in normal tissues.The expression level of NP69 in nasopharyngeal epithelial cells was significantly higher than that in nasopharyngeal epithelial cells(t=8.560,P=0.000;t=42.386,P=0.000).The expression of miR-155 was correlated with TNM stage,pathological grade,extent of invasion and lymph node metastasis in nasopharyngeal carcinoma(P<0.05),but not with age and gender(P>0.05).The expression of miR-155 after transfection of miR-155-inhibitor was significantly lower than that of the control group and the blank control group(F=35.63,P=0.003).The growth rate of nasopharyngeal carcinoma cells silenced by miR-155 was significantly slower than that of control group and blank control group(P<0.05).Conclusion:The high expression of miR-155 in human nasopharyngeal carcinoma tissues and cells can inhibit the proliferation of nasopharyngeal carcinoma cells,and it is of great significance for the treatment of nasopharyngeal carcinoma.

MiR-16 regulates CCND1 and CCND2 expression contributing to cardiac hypertrophy

MicroRNAs(miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression post-transcriptionally. Recent studies have demonstrated that miRNAs are involved in the pathogenesis of hypertrophy.We investigated miR-16 expression and their potential roles in a rat model of hypertrophy induced by abdominal artery constriction (AAC).miR-16 expression was significantly decreased, and CCND1 and CCND2 protein were markedly increased without obvious change of its mRNA level after hypertrophy induction.CCND1 and CCND2 levels were increased without changing their transcript levels in neonatal rat ventricular cardiomyocytes(NRVC) induced by PE,and miR-16 was down-regulated in this process with significantly up-regulatedβ-MHC,ANF and MLC-2 expression.Conversely,introduction of functional miR-16,CCND1 siRNA or CCND2 siRNA into NRVCs could repress cardiomyocyte hypertrophy.These results implicate that miR-16 is involved in contributing to cardiac hypertrophy,one of the mechanisms may be resulted from post-transcriptional regulation of CCND1 and CCND2.

食管癌组织中miR-3651表达与其IPCL分型及病理分级的关系

目的探讨食管癌组织中miR-3651表达与其IPCL分型及病理分级的关系。方法分析2019年1月至2020年12月东莞市人民医院确诊并收治的82例食管癌患者,选取患者的食管癌组织以及癌旁组织,对食管癌组织进行IPCL分型和病理分级,并采用实时定量聚合酶链反应(Real-time PCR)法检测癌旁组织以及各IPCL分型以及病理分级的食管癌组织的miR-3651表达;采用spearman分析法分析各组患者IPCL分型及病理分级结果与miR-3651水平的相关性;采用单因素分析以及Cox比例风险回归分析研究食管癌预后不良的影响因素。结果食管癌组织中miR-3651相对表达量小于癌旁组织的miR-3651相对表达量(t=18.839,P<0.05);各IPCL分型及病理分级之间食管癌组织的miR-3651相对表达量比较,差异有统计学意义(F=8.411,t=7.490,P<0.05);食道癌患者癌组织中miR-3651表达水平与患者食管癌组织的IPCL分型及病理分级呈正相关(P<0.05);miR-3651高表达者术后1年总生存率低于miR-3651低表达者,差异有统计学意义(χ^(2)=4.623,P<0.05);Cox回归分析结果显示,浸润性食管癌、IPCL分型Ⅴ_(3A)型、病理分级中-高分化以及miR-3651表达≥0.528为食管癌患者预后不良的独立危险因素(P<0.05)。结论食管浅表性病变患者的食管癌组织IPCL分型以及病理分级与miR-3651表达水平呈正相关;miR-3651表达水平检验可以为医生对食管癌的早期诊断提供理论依据。

The tumor suppressor role and ceRNA network of miR-1294 in cancer

miRNAs are endogenous small RNAs that are important regulators of gene expression.miR-1294 was found to be significantly down-regulated in 15 cancers and regulated by 21 upstream regulators.miR-1294 affects the proliferation,migration,invasion,and apoptosis of cancer cells.The target genes of miR-1294 are involved in the PI3K/AKT/mTOR,RAS,and JAK/STAT signaling pathways.Six target genes of miR-1294 are the targets of a variety of drugs.Low expression of miR-1294 is associated with resistance to cisplatin and TMZ and a poorer prognosis in patients with ESCC,GC,EOC,PDAC,or NSCLC.Therefore,this work outlines the molecular mechanisms and provides a basis for the clinical significance of the tumor suppressor miR-1294 in cancer.

Expression and Clinical Significance of miRNA-495 in the Peripheral Blood of Acute Myeloid Leukemia Patients

Objective:To analyze the expression and clinical significance of miRNA-495 in the peripheral blood of patients with acute myeloid leukemia.Methods:Fifty-six patients with acute myeloid leukemia and 56 healthy controls were selected.Fasting venous blood was drawn and centrifuged,and the plasma was collected.The target miRNA was directly amplified and reverse transcribed into cDNA.The expression of plasma miR-495 was detected by qRT-PCR.Results:The expression level of miRNA-495 in newly diagnosed acute myeloid leukemia(AML[ND])and relapsed/refractory acute myeloid leukemia(AML[RR])was significantly lower than that in complete remission(AML[CR])and normal control group(Control)(p<0.0001).There was no significant difference between AML(ND)group and AML(RR)group(p>0.05).The area under the ROC curve(AUC)of miRNA-495 was 0.9503,the 95%confidence interval was 0.9113–0.9892(p<0.0001),the standard error was 0.020,the sensitivity and specificity were 91.1%and 92.9%,respectively,and the Jordan index was 0.857.There was no significant difference between the expression level of miRNA-495 and gender,age,leukocyte count,hemoglobin,and platelet count(p>0.05).However,it was found related to the proportion of primitive bone marrow cells in patients(p=0.017).Conclusion:The decreased expression of plasma miR-495 in AML patients can be used as a new indicator for the diagnosis and prognosis of AML.

Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway

AIM: To determine if mir-30 d inhibits the autophagy response to Helicobacter pylori(H. pylori) invasion and increases H. pylori intracellular survival.METHODS: The expression of mir-30 d was detected by quantitative polymerase chain reaction(PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30 d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the m RNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay.RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30 d expression(P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30 d was found to repress the autophagy process, whereas mir-30 d inhibitor increased autophagy responseto H. pylori invasion. mir-30 d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3′ untranslated region(UTR) of all five tested genes(ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30 d mimic transfection(P < 0.05, vs control cells without mir-30 d mimic treatment). Mir-30 d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells.CONCLUSION: Mir-30 d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.

miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

MicroRNAs(miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically(P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased(P<0.001). In addition, the mRNA expression of miR-10b was negatively(P<0.01) correlated with DAZAP1 mRNA expression(r=–0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated(P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted(P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8(CCK-8) assay and the 5-Ethynyl-2′-deoxyuridine(EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect(P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted(P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.

Increased levels of miR-3099 induced by peripheral nerve injury promote Schwann cell proliferation and migration

MicroRNAs(miRNAs) can regulate the modulation of the phenotype of Schwann cells. Numerous novel miRNAs have been discovered and identified in rat sciatic nerve segments, including miR-3099. In the current study, miR-3099 expression levels following peripheral nerve injury were measured in the proximal stumps of rat sciatic nerves after surgical crush. Real-time reverse transcription-polymerase chain reaction was used to determine miR-3099 expression in the crushed nerve segment at 0, 1, 4, 7, and 14 days post sciatic nerve injury, which was consistent with Solexa sequencing outcomes. Expression of miR-3099 was up-regulated following peripheral nerve injury. EdU and transwell chamber assays were used to observe the effect of miR-3099 on Schwann cell proliferation and migration. The results showed that increased miR-3099 expression promoted the proliferation and migration of Schwann cells. However, reduced miR-3099 expression suppressed the proliferation and migration of Schwann cells. The potential target genes of miR-3099 were also investigated by bioinformatic tools and high-throughput outcomes. miR-3099 targets genes Aqp4, St8 sia2, Tnfsf15, and Zbtb16 and affects the proliferation and migration of Schwann cells. This study examined the levels of miR-3099 at different time points following peripheral nerve injury. Our results confirmed that increased miR-3099 level induced by peripheral nerve injury can promote the proliferation and migration of Schwann cells.

Elevated miR-33a and miR-224 in steatotic chronic hepatitis Cliver biopsies

AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues.

miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3' untranslated region of CCND2 and CCNE2

BACKGROUND： The deficiency of liver regeneration needs to be addressed in the fields of liver surgery, split liver transplan- tation and living donor liver transplantation. Researches of microRNAs would broaden our understandings on the mecha- nisms of various diseases. Our previous research confirmed that miR-26a regulated liver regeneration in mice; however, the relationship between miR-26a and its target, directly or in- directly, remains unclear. Therefore, the present study further investigated the mechanism of miR-26a in regulating mouse hepatocyte proliferation. METHODS： An established mouse liver cell line, Nctc-1469, was transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a- EGFP or AdS-EGFP vector. Cell proliferation was assessed by MTS, cell apoptosis and cell cycle by flow cytometry, and gene expression by Western blotting and quantitative real-time PCR. Dual-luciferase reporter assays were used to test targets of miR-26a. RESULTS： Compared with the Ad5-EGFP group, Ad5-anti- miR-26a-EGFP down-regulated miR-26a and increased prolif- eration of hepatocytes, with more cells entering the G1 phase of cell cycle （82.70%＋1.45% vs 75.80%＋_3.92%）, and decreased apoptosis （5.50%＋0.35% vs 6.73%_＋0.42%）. CCND2 and CCNE2 were the direct targeted genes of miR-26a, miR-26a down- regulation up-regulated CCND2 and CCNE2 expressions and down-regulated p53 expression in Nctc-1469 cells. On the con- trary, miR-26a over-expression showed the opposite results. CONCLUSIONS： miR-26a regulated mouse hepatocyte pro- liferation by directly targeting the 3＇ untranslated regions of cyclin D2/cyclin E2; miR-26a also regulated p53-mediated apoptosis. Our data suggested that miR-26a may be a promis- ing regulator in liver regeneration.

Identification of miR-802-5p and its involvement in type 2 diabetes mellitus

MicroRNAs(miRNA)are recently discovered endogenous,small noncoding RNAs(of 22 nucleotides)that play pivotal roles in gene regulation.They are involved in post-transcriptional control of gene expression.miRNAs are emerging as important regulators of cell proliferation,development,cancer formation,stress responses,cell death and physiological conditions.Increasing evidence has demonstrated the human miRNAs bind to their target mRNA sequences with perfect or near-perfect sequence complementarily.This provides a powerful strategy for discovering potential type 2 diabetes mellitus(T2DM)targets and gives the probability to exploit them for diagnostic and therapeutic causes.About 6%of the world population is affected by T2DM,and it is recognized as a global epidemic by the World Health Organization.At present there is no valid biomarker to control or manage T2DM.Therefore,the present study applied a mature sequence of miRNAs from publicly accessible databases to identify the miRNA from T2DM expressed sequence tags,and the results are detailed and discussed below.

Efficacy of green tea extract on PC3 prostate cancer cells through upregulation of miR-195 expression and suppression of epithelial to mesenchymal transition

OBJECTIVE: To evaluate anticancer efficacy of green tea extract(GTE) on PC3 prostate cancer cells. METHODS: By using quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot methods, the expression of miR-195 and the epithelial to mesenchymal transition(EMT) markers such as E-cadherin and vimentin was analyzed. RESULTS: Based on the results of 2D and 3D cell culture models, the inhibition of EMT and up regulation of miR-195 expression were detected. CONCLUSIONS: Our findings will be helpful to design anti-tumor regimens with natural product original, and more studies will be required to identify the related mechanisms involving anticancer activities of green tea via miRNAs.

Construction and Identification of a Human Liver Specific MicroRNA Eukaryotic Expression Vector

MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction （PCR） from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular ＆ Molecular Immunology.

mi R-122 negatively correlates with liver fibrosis as detected by histology and FibroScan

AIM: To investigate whether expression of selected mi RNAs obtained from fibrotic liver biopsies correlate with fibrosis stage.METHODS: Altogether, 52 patients were enrolled in the study representing various etiologic backgrounds of fibrosis: 24 cases with chronic hepatitis infections(types B, C), 19 with autoimmune liver diseases(autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, overlapping syndrome cases), and 9 of mixed etiology(alcoholic and nonalcoholic steatosis, cryptogenic cases). Severity of fibrosis was determined by both histologic staging using the METAVIR scoring system and noninvasive transient elastography. Following RNAisolation, expression levels of mi R-21, mi R-122, mi R-214, mi R-221, mi R-222, and mi R-224 were determined using Taq Man Micro RNA Assays applying mi R-140 as the reference. Selection of mi RNAs was based on their characteristic up- or downregulation observed in hepatocellular carcinoma. Relative expression of mi RNAs was correlated with fibrosis stage and liver stiffness(LS) value measured by transient elastography, as well as with serum alanine aminotransferase(ALT) level.RESULTS: The expression of individual mi RNAs showed deregulated patterns in stages F1-F4 as compared with stage F0, but only the reduced level of mi R-122 in stage F4 was statistically significant(P < 0.04). When analyzing mi RNA expression in relation to fibrosis, levels of mi R-122 and mi R-221 showed negative correlations with fibrosis stage, and mi R-122 was found to correlate negatively and mi R-224 positively with LS values(all P < 0.05). ALT levels displayed a positive correlation with mi R-21(P < 0.04). Negative correlations were observed in the fibrosis samples of mixed etiology between mi R-122 and fibrosis stage and LS values(P < 0.05), and in the samples of chronic viral hepatitis, between mi R-221 and fibrosis stage(P < 0.01), whereas mi R-21 showed positive correlation with ALT values in the samples of autoimmune liver diseases(P < 0.03). The results also revealed a strong correlation between fibrosis stage and LS values(P < 0.01) when etiology of fibrosis was not taken into account.CONCLUSION: Reduced expression of mi R-122 in advanced fibrosis and its correlation with fibrosis stage and LS values seem to be characteristic of hepatic fibrosis of various etiologies.

Construction and detection of expression vectors of microRNA-9a in BmN cells

MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

The microRNA,miR-29c,participates in muscle development through targeting the YY1 gene and is associated with postmortem muscle pH in pigs

Previous studies indicated that miR-29c is important for muscle development in mice and human,but its role in pigs is unknown.In this study,we detected the expression of miR-29c in Meishan longissimus lumborum(LL)muscle.The results showed that miR-29c was gradually upregulated during development of skeletal muscle in pig.Moreover,the expression of YY1 and Akt3 genes,which were confirmed to be targeted by miR-29c in mice,was decreased along with muscle development.Furthermore,the expression level of miR-29c was significantly higher in adult Meishan pigs than Large White pigs,while the expression of YY1 and Akt3 genes was significantly lower in Meishan pigs.These results indicated that the expression pattern of miR-29c was opposite to that of YY1 and Akt3 genes in pigs.Also,the luciferase assay indicated that miR-29s can target the YY1 gene in pigs.In addition,we identified a T to C mutation in the primary transcript of miR-29c,which was associated with the postmortem muscle pH in pigs.Based on these results,we concluded that miR-29c is also important in skeletal muscle development of pigs.