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ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.
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Objective To analyze the core genes of lung ischemia-reperfusion injury and construct a competitive endogenous RNA (ceRNA) network. Methods Original data of GSE145989 were downloaded from the Gene Expression Omnibus (GEO) database as the training set, and the GSE172222 and GSE9634 datasets were used as the validation sets, and the differentially-expressed genes (DEG) were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Protein-protein interaction (PPI) network was constructed, and the core genes were screened, and the diagnostic values of these core genes and the immune infiltration levels of immune cells were evaluated. The ceRNA network was constructed and validated. The targeted drugs based on ceRNA network were assessed. Results A total of 179 DEG were identified, including 61 down-regulated and 118 up-regulated genes. GO analysis showed that DEGs were associated with multiple biological processes, such as cell migration, differentiation and regulation, etc. They were correlated with cell components, such as vesicle membrane, serosa and membrane raft, etc. They were also associated with multiple molecular functions, such as chemokine receptor, G protein-coupled receptor, immune receptor activity and antigen binding, etc. KEGG pathway enrichment analysis revealed that DEG were involved in tumor necrosis factor (TNF), Wnt, interleukin (IL)-17 and nuclear factor (NF)-κB signaling pathways, etc. PPI network suggested that CD8A, IL2RG, STAT1, CD3G and SYK were the core genes of lung ischemia-reperfusion injury. The ceRNA network prompted that miR-146a-3p, miR-28-5p and miR-593-3p were related to the expression level of CD3G. The miR-149-3p, miR-342-5p, miR-873-5p and miR-491-5p were correlated with the expression level of IL-2RG. The miR-194-3p, miR-512-3p, miR-377-3p and miR-590-3p were associated with the expression level of SYK. The miR-590-3p and miR-875-3p were related to the expression level of CD8A. The miR-143-5p, miR-1231, miR-590-3p and miR-875-3p were associated with the expression level of STAT1. There were 13 targeted drugs for CD3G, 4 targeted drugs for IL-2RG, 28 targeted drugs for SYK and 3 targeted drugs for lncRNA MUC2. No targeted drugs were identified for CD8A, STAT1 and other ceRNA network genes. Conclusions CD8A, IL2RG, STAT1, CD3G and SYK are the core genes of lung ischemia-reperfusion injury. The research and analysis of these core genes probably contribute to the diagnosis of lung ischemia-reperfusion injury and providing novel research ideas and therapeutic targets.
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【Objective】 To investigate the impact of long non-coding RNA (lncRNA) FGD5-AS1 on the malignant biolo-goical behavior of bladder cancer (BC) cells by regulating micro RNA (miR)-129-5p/cyclin dependent kinase 6 (CDK6) axis. 【Methods】 Human BC cell line T24 was cultured from tumor tissue and paracancerous tissue of 105 patients with confirmed BC. The expressions of FGD5-AS1, miR-129-5p and CDK6 mRNA in tissue samples and T24 cells were detected with RT-qPCR. T24 cells were randomly divided into control group, si-NC group, si-FGD5-AS1 group, si-FGD5-AS1+inhibitor NC group and si-FGD5-AS1+miR-129-5p inhibitor group. The cell viability, migration, invasion andapoptosis were detected with CCK-8, Wound healing test, Transwell assay and flow cytometry, respectively. The expressions of Bax, Bcl-2, Caspase3 and CDK6 were detected with Western blot. The relationship between FGD5-AS1 and miR-129-5p, between miR-129-5p and CDK6 were verified with double luciferase reporter gene experiment. 【Results】 FGD5-AS1 and CDK6 mRNA were highly expressed in BC tissue, while miR-129-5p was lowly expressed (P<0.05). After FGD5-AS1 silencing, the expression of FGD5-AS1,A450 value, cell scratch healing rate, cell invasion number, and expressions of Bcl-2 and CDK6 were significantly lower, while the apoptosis rate and expressions of miR-129-5p, Bax and Caspase3 were significantly higher (P<0.05). Inhibition of miR-129-5p expression reversed the effects of FGD5-AS1 silencing on various indexes of BC cells (P<0.05). FGD5-AS1 negatively regulated the expression of miR-129-5p, and miR-129-5p negatively regulated the expression of CDK6. 【Conclusion】 Silencing FGD5-AS1 may inhibit the expression of CDK6 protein by up-regulating miR-129-5p, thus inhibiting the proliferation, migration and invasion of BC cells and promoting cell apoptosis.
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Objective To investigate the role and mechanism of circular RNA SNRK (circSNRK) in ischemia-reperfusion injury (IRI). Methods A hypoxia-reoxygenation (IRI) cell model was established. The expression level of circSNRK after IRI treatment and the effect of overexpression of circSNRK on cell proliferation and apoptosis were detected. The targets of circSNRK were identified. HK2 cells were divided into the blank group (Mock group), IRI group, control plasmid+IRI group (IRI+NC group), human circSNRK overexpression+IRI group (IRI+circSNRK group), human circSNRK overexpression+IRI+protein kinase B (Akt) inhibitor group (IRI+circSNRK+MK2206 group) and control plasmid group (NC group). Cell proliferation and apoptosis were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the target of circSNRK were carried out. The expression levels of CDKN1A, Akt, B-cell lymphoma (Bcl)-2, cysteinyl aspartate specific proteinase (Caspase)-9 messenger RNA (mRNA), and those of p21, Bcl-2, Caspase-9, Akt and p-Akt proteins were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups, respectively. Cell proliferation and apoptosis were determined in the NC, IRI+NC, IRI+circSNRK and IRI+circSNRK+MK2206 groups. Results Compared with the Mock group, the expression level of circSNRK was lower, and cell proliferation capability of HK2 cells was decreased and cell apoptosis was increased in the IRI group. In the IRI+circSNRK group, cell proliferation capability was higher, whereas cell apoptosis was lower than those in the IRI+NC group. circSNRK could act on 648 targets through 51 microRNAs (miRNAs). GO enrichment analysis revealed that the targets of circSNRK were mainly enriched in biological processes (such as cell process and biological regulation), cell components (such as cell parts, cells and extracellular parts), and molecular functions (such as binding, binding proteins and enzymes). KEGG enrichment analysis showed that the targets of circSNRK were mainly enriched in cancer signaling pathway, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, miRNA in cancer and other related signaling pathways. Compared with the Mock group, the relative expression levels of CDKN1A and Caspase-9 mRNA were higher, the expression level of miR-99a-5p RNA was higher and the relative expression levels of Akt and Bcl-2 mRNA were lower in the IRI group. Compared with the IRI+NC group, the relative expression levels of CDKN1A and Caspase-9 mRNA were lower, those of Akt and Bcl-2 mRNA were higher, and the expression level of miR-99a-5p RNA was lower in the IRI+circSNRK group, and the differences were statistically significant (all P < 0.05). Compared with the Mock group, the expression levels of p21 and Caspase-9 proteins were higher, while those of p-Akt, Akt and Bcl-2 proteins were lower in the IRI group. Compared with the IRI+NC group, the expression levels of p21 and Caspase-9 proteins were lower, whereas those of p-Akt, Akt and Bcl-2 proteins were higher in the IRI+circSNRK group. The miR-99a-5p binding sites were observed in circSNRK and Akt. Compared with the NC group, cell proliferation capability was declined in the IRI+NC group. Compared with the IRI+NC group, cell proliferation capability was elevated in the IRI+circSNRK group. Compared with the IRI+circSNRK group, cell proliferation capability was declined in the IRI+circSNRK+MK2206 group (all P < 0.05). The cell apoptosis level in the IRI+NC group was higher than that in the NC group. The cell apoptosis level in the IRI+circSNRK group was lower compared with that in the IRI+NC group. The cell apoptosis level in the IRI+circSNRK+MK2206 group was higher than that in the IRI+circSNRK group. Conclusions Under IRI conditions, circSNRK may affect the proliferation and apoptosis of HK2 cells probably via the Akt signaling pathway.
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Gastric cancer （GC） is one of the most common malignant tumors in the digestive system， with high morbidity and mortality. Early clinical symptoms of GC are not obvious， and most of them have entered the advanced stage after discovery， which greatly reduces the clinical cure rate and affects the quality of life of patients， and the prognosis is very poor. In recent years， with the continuous exploration in the field of bioinformatics， it has been found that micro-RNA （miRNA） and long non-coding RNA （lncRNA） exist as non-coding RNA （ncRNA） without translation ability， and regulate the expression levels of related signal proteins by acting on a certain target， thereby activating or inhibiting a certain signaling pathway， which plays an important role in assisting diagnosis， guiding clinical medication， and judging prognosis in the progress of GC. Chinese medicine is easily accepted by patients because of its good curative effect and less side effects. In the present basic studies， with the interaction mechanism between miRNA， lncRNA and signaling pathways as the breakthrough point， various studies on the regulation of related signaling molecules and signaling pathways by Chinese medicine have been carried out. A large number of experimental data have proved that the development of GC is closely related to the interaction of miRNA， lncRNA， and related signaling pathways， and Chinese medicine， with multi-target， multi-mechanism， and multi-pathway characteristics， affects various signaling molecules and signaling pathways and intervenes in the progress of GC cells. This paper reviewed the basic research on lncRNA， miRNA molecules， and main signaling pathways involved in the occurrence and development of GC， and summarized specific molecular mechanisms of Chinese medicine in the regulation of each signaling pathway， hoping to provide references for modern research of Chinese medicine in the intervention of GC progress at the molecular level.
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Objective:To explore the molecular mechanism of pathological changes in osteoarthritis (OA) through applying bioinformatics to analyze the miRNA-mRNA regulatory network.Methods:MiRNA expression data from human serum samples were downloaded from the Gene Expression Omnibus database. Differentially-expressed miRNA was identified using the linear modelling package of the Bioconductor software suite. The target differentially-expressed mRNA was predicted using version 2.0 of the miRWalk database. Version 3.7.1 of the Cytoscape software was used to construct the miRNA-mRNA regulatory network for OA. The target genes were analyzed by using gene ontology and the Kyoto Encyclopedia of Genes and Genomes. The protein-protein interaction network was constructed and the core genes in osteoarthritis pathology were screened out.Results:A total of 7 differentially-expressed miRNAs (all down-regulated) and 900 mRNAs were identified, mostly involved in the negative regulation of protein binding, DNA binding, or transcription in the cell cycle. Ten core genes were screened out: MAPK1, TP53, MAPK14, CCND1, EP300, POLR2E, POLR2F, ABL1, RAC1 and SKIV2L2.Conclusions:Multiple miRNAs, target genes and signaling pathways are involved in the development of OA. The miRNA-mRNA regulatory network identified provides new ideas for exploring the molecular mechanism of OA′s pathology and its clinical diagnosis and treatment.
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Objective:To study the expression of micro RNA-155(miR-155) and IFN-γ in lung tissue in a neonatal rat model of acute respiratory distress syndrome(ARDS)lung injury by intraperitoneal injection of lipopolysaccharide(LPS).Methods:Eighty neonatal SD rats on the 7th day after birth were assigned to the experimental group(LPS group)and control group(isotonic NaCl group), with 40 rats in each group.LPS solution(4 mg/kg)was injected into the abdominal cavity of neonatal SD rats in the experimental group to establish an animal model of neonatal acute respiratory distress syndrome(NARDS). The control group was established by isotonic NaCl solution(4 ml/kg)in the same way.The lung tissue samples were taken at 3 h, 6 h, 12 h and 24 h after drug administration to observe the surface changes.Then the lung sections were stained with HE to observe the pathological changes and score the lung tissue injury.Finally, the expression levels of miR-155 and IFN-γ in the lung tissue were tested by RT-PCR and ELISA techniques, respectively.Results:(1)At the beginning of the experiment, the neonatal rats in the experimental group gradually showed the clinical manifestations of ARDS, and the macroscopic observation, pathological changes and lung tissue injury scores of the lung tissues suggested the appearance of NARDS lung injury, indicating that the model was successful.(2)The expression levels of miR-155(1.33±0.12 vs 0.95±0.02、1.77±0.17 vs 0.96±0.01、2.18±0.09 vs 0.96±0.02 and 2.43±0.06 vs 0.96±0.02)and IFN-γ(370.79±13.89 vs 273.03±11.44、424.24±10.11vs270.70±13.05、466.63±6.57 vs 268.11±7.88 and 519.13±7.09 vs 272.97±12.54)ng/L in the lung tissue of rats between the experimental group and the control group were significantly different( P<0.01), and the difference was statistically significant among the groups in the experimental group( F values were 165.983 and 408.574, P<0.01). The expression levels of miR-155 and IFN-γ in the lung tissue of the experimental group increased gradually over time and showed an increasing trend. Conclusion:After the successful establishment of NARDS animal model, the expression levels of miR-155 and IFN-γ in the lung tissue of NARDS rats have significantly increased and showed a sequential pattern.MiR-155 is expected to become an early biomarker for the diagnosis of NARDS.
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Posttransplant lymphoproliferative disease (PTLD) is a series of heterogeneous lymphoproliferative diseases and a severe complication after solid organ transplantation in children. Over 70% of PTLD is associated with Epstein-Barr virus (EBV). EBV-related B-cell lymphoma is also the main malignant tumor after pediatric organ transplantation. EBV-related PTLD is still a challenge in pediatric solid organ transplantation, which is mainly caused by immune function damage induced by immune suppression after transplantation. However, the specific mechanism remains elusive. In recent years, biomarkers have been developed to guide the diagnosis and individualized treatment of EBV-related PTLD, which possesses excellent application prospect. In this article, research progresses on the incidence of EBV-related PTLD in solid organ transplantation and its biomarkers were reviewed, aiming to explore novel ideas for clinical diagnosis and treatment.
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Objective:To investigate the effects of micro RNA(miR)-296-3p on cell proliferation, migration and invasion of tongue squamous cell carcinoma and its underlying mechanism.Methods:Normal oral epithelial mucosal keratinocytes HOK was used as the control. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of miR-296-3p and nucleoprotein 1(NUPR1) mRNA in tongue squamous cell carcinoma cell lines CAL27, SCC15 and SCC9, and Western blot was used to detect the expression of NUPR1 protein. CAL27 cells were divided into NC group, miR-con group, miR-296-3p group, si-con group, si-NUPR1 group, miR-296-3p+pcDNA group and miR-296-3p+pcDNA-NUPR1 group, then CCK8 method was used to detect the cell activity. Transwell was used to detect the cell migration and invasion. Western blot was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), matrixmetallo proteinase-2(MMP-2) and matrixmetallo proteinase-9(MMP-9). Dual-luciferase reporter assay system was implemented to verify the relationship between miR-296-3pand NUPR1.Results:Compared with the HOK cells, the content of miR-296-3p in the tongue squamous cell line CAL27, SCC15 and SCC9 groups was significantly reduced (0.54 ± 0.08, 0.38 ± 0.05, 0.59 ± 0.07 vs. 1.04 ± 0.12, t = 10.401, 15.231 , 9.718, P<0.05), while the expression levels of NUPR1mRNA (5.94 ± 0.40, 4.48 ± 0.45, 5.19 ± 0.48 vs.0.94 ± 0.12, t = 35.918, 22.803, 25.769) and protein (0.79 ± 0.09, 0.54 ± 0.05, 0.62 ± 0.08 vs. 0.28 ± 0.04, t = 15.535, 12.182, 11.404) were significantly increased (all P<0.05). Compared with those in the NC group or miR-con group, CAL27 cell activity, migration number, invasion number and the protein expression of PCNA, MMP-2 and MMP-9 in the miR-296-3p group were decreased (all P<0.05). Compared with those of the NC group or the si-con group, the CAL27 cell activity, migration number, number of invasions and the protein expression of PCNA, MMP-2 and MMP-9 in the si-NUPR1 group were decreased (all P<0.05). miR-296-3p negatively regulatedthe expression of NUPR1 in CAL27 cells. Compared with the miR-296-3p+pcDNA group, CAL27 cell viability (138.34 ± 5.73 vs. 98.54 ± 5.89, t = 14.530), migration number (95.28 ± 7.56 vs. 67.92 ± 5.23, t = 8.929), invasion number (184.53 ± 17.57 to 101.26 ± 10.64, t = 12.162) and the protein expression of PCNA (0.68 ± 0.07 to 0.35 ± 0.06, t = 10.738), MMP-2 (0.43 ± 0.05 to 0.29 ± 0.04, t = 6.559) and MMP-9 (0.58 ± 0.06 vs. 0.33 ± 0.08, t = 7.500) in the miR-296-3p+pcDNA-NUPR1 group were increased (all P<0.05). Conclusions:miR-296-3p may inhibitthe proliferation, migration and invasion of tongue squamous cell carcinoma cells by negatively regulating NUPR1.
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@#AIM: To investigate the clinical value of microRNA(miRNA, miR)-27 expression in patients with diabetic retinopathy(DR).<p>METHODS: A total of DR 80 patients(DR group)treated between January 2019 and January 2020 were retrospectively reviewed. Meanwhile, 40 patients with simple type 2 diabetes mellitus(T2DM)(T2DM group)and 40 normal healthy persons(control group)were enrolled, and plasma RNA was extracted. Real time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)was adopted to determine plasma miR-27 expression, and enzyme-linked immunosorbent assay was performed to determine the vascular endothelial growth factor(VEGF)level. Plasma miR-27 and serum VEGF expression in different groups and in patients with different severities of DR was comparatively analyzed. Multivariate Logistic regression analysis was performed to screen factors influencing the expression of miR-27 in patients with DR, and Pearson correlation analysis of miR-27, serum VEGF and blood glucose indexes was conducted. Meanwhile, significance of miR-27 in pathogenesis of DR was summarized.<p>RESULTS: DR group had the highest plasma miR-27 and serum VEGF levels, followed by T2DM group, and then the control group(<i>P</i><0.05). Proliferative diabetic retinopathy(PDR)patients had higher levels of plasma miR-27, serum VEGF, fasting blood glucose and glycated hemoglobin than those with non-proliferative diabetic retinopathy(NPDR)(<i>P</i><0.05). It was found that course of disease(<i>OR</i>=3.206), fasting blood glucose(<i>OR</i>=2.570), glycated hemoglobin(<i>OR</i>=2.787), VEGF(<i>OR</i>=3.442)and severity of DR(<i>OR</i>=5.842)were influencing factors of plasma miR-27 expression in DR patients(<i>P</i><0.05). In DR patients, relative expression of plasma miR-27 was positively correlated with serum VEGF, fasting blood glucose and glycated hemoglobin(<i>r</i>=0.548, 0.398, 0.522, all <i>P</i><0.05).<p>CONCLUSION: DR patients have higher plasma miR-27 expression level than those with simple T2DM and normal healthy people. The duration of diabetes, fasting blood glucose, glycated hemoglobin and severity of DR all affect the expression of miR-27. Besides, miR-27 is positively correlated with serum VEGF, glycated hemoglobin and fasting blood glucose. It is speculated that miR-27 may mediate the pathogenesis and progression of DR by regulating glucose metabolism and promoting angiogenesis.
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@#MicroRNAs(miRNAs)are one of the most important regulatory factors of gene expression, which involved in the growth, development, differentiation and apoptosis of various cells, tissues and organs. TRPM3 is located in human chromosome 9 and belongs to M sub-family of the transient receptor potential(TRP)channels. MiR-204 is located on TRPM3 intron 6 and participates in the regulation of post-transcriptional gene expression through cleavage or translation inhibition of target mRNAs. Studies have shown that TRPM3/miR-204 complex locus plays an important role in the occurrence and development of eye diseases such as cataract, glaucoma, corneal neovascularization, corneal wound healing, retinal diseases, optic nerve diseases and so on. In this paper, the biological function of TRPM3/miR-204, its expression and regulation in the eyes and its correlation with a variety of ophthalmic diseases are reviewed.
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Objective: To investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot. Results: Compared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased ( P0.05). Conclusion: miR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.
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Objective: To investigate the effect of miR-125b on the ventricular remodeling after myocardial infarction in the rats via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, and to elucidate its mechanism. Methods: A total of 75 rats were divided into sham operation group, myocardial infarction group and miR-125b inhibitor group, with 25 rats in each group. The acute myocardial infarction models were established in the rats in myocardial infarction group and miR-125b inhibitor group. The rats in miR-125b inhibitor group was injected with miR-125b inhibitor via the tail vein. The rats were sacrificed 4 weeks later, the body weig hts were recorded; the hearts were obtained, and the big vessel and left and right auricular appendix were cut out; the heart weights were weighed. The heart weight/body weight (HW/BW), (left ventricle + ventricular septum) weight/body weight (LV+S)/BW, (left ventricular + ventricular septum) weight/heart weight (LV+S)/HW were calculated. HE staining was used to observe the morphological changes of myocardium tissue. Masson staining was used to observe the myocardial fibrosis, and the collagen volume integral of myocardium was calculated. Immunofluorescence staining was used to observe the expression levels of type I collagen and type III collagen proteins in the myocardium tissue of the rats. Western blotting method was used to detect the expression levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), PI3K, Akt and phosphorylated Akt (p-Akt) in the peripheral region of myocardial infarction of the rats. Results: The HE staining results showed that the myocardial cells in sham operation group were arranged neatly; the myocardial cells in myocardial infaction group had hypertrophy and disordered arrangement; the myocardial cells in miR-125b inhibitor group were arranged neatly. Compared with sham operation group, the HW/BW, (LV + S)/BW and (LV + S)/HW of the rats in myocardial infarction group were elevated (P<0.05), the collagen volume integral was increased (P<0.05), the protein expression levels of type I collagen and type III collagen were increased (P<0.05), the expression levels ANP and BNP proteins were increased (P<0.05), and the expression levels of PI3K and p-Akt proteins were decreased (P<0.05). Compared with myocardial infarction group, the HW/BW, (LV+S)/BW and (LV+S)/HW of the rats in miR-125 b inhibitor group were decreased (P<0.05), the collagen volume integral was decreased (P<0.05), the expression levels of type I collagen and type III collagen proteins were decreased (P<0.05), the expression levels of ANP and BNP proteins were decreased (P<0.05), and the expression levels of PI3K and p-Akt proteins were increased (P<0.05). Conclusion: Inhibition of miR-125b can inhibit ventricular remodeling after myocardial infarction in the rats by inhibiting the PI3K/Akt signaling pathway, and plays an important role in reversing ventricular remodeling after myocardial infarction.
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Stanniocalcin-1 (STC1) is secreted by the variety of tissues having a major role in the regulation of calcium ions in the involuting mammary gland. The present work aims to sequence and structural characterization as well as expression profiling of STC1 gene in buffalo. Polymorphism identified in the 3-untranslated region (UTR) was analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping in riverine and swamp buffaloes. Expression profiling of STC1 was performed in different lactation stages of mammary gland and peripheral blood mononuclear cells to study the impact of 3'-UTR polymorphism on its expression. Different polymorphic sites were detected in the entire coding and noncoding regions of riverine and swamp buffaloes, including two INDELs. An identified polymorphic nucleotide locus A324G, having target sites for two miRNAs, namely bta-miR-2382 and bta-miR-1343, reported in cattle, was genotyped by PCR-RFLP to reveal variable allelic distribution among swamp and riverine buffaloes. Gene expression profiling across buffalo mammary tissues representing different lactation stages showed maximum expression of the STC1 gene in the involuting mammary gland. Ruminants’ specific genetic variation has been observed in STC1 and its implication in buffalo mammary gland involution as well as coregulation of gene expression throughmiRNA binding in the 3'-UTR is suggested.
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Objective@#To analyze the biological effects of miRNA-155 in the cardiac myocyte apoptosis.@*Methods@#The mouse-derived macrophage cell line RAW264.7 was treated by different concentration or different stage of oxidized low density lipoprotein (ox-LDL), and transfected by miR-155 mimic (M group), miR-155 mimics-NC (M-NC group), miR-155 inhibitor (I group) or miR-155 inhibitor-NC (I-NC group), respectively. The cell viability was measured by CCK-8 assay, cell apopotosis was measured by TUNEL and flow cytometry.@*Results@#The ox-LDL induced cell viability of Raw264.7 cells decreased and the expression of miR-155 increased in dose and time dependent manner, after treatment with different concentration of ox-LDL (10, 20, 40, 80, 160 mg/L) or 80 mg/L of ox-LDL with different stage (6, 12, 24, 48, 72 h). The expression of miR-155 increased significantly. Raw264.7 cell viability decreased significantly, compared to that of the blank control. The difference between two groups had statistical significance (P<0.05). The cell viability in M group was significantly higher than that in M-NC group, in I group was significantly higher than that in I-NC group, and the difference between two groups was statistical significance (P<0.05). Compared to that in the M-NC group, the cell apoptosis rate in M group was significantly increased (P<0.05), compared to that in the I-NC group, the cell apoptosis rate in I group were significantly decreased (P<0.05).@*Conclusions@#miR-155 can enhance the Raw264.7 cell apoptosis induced by ox-LDL.
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Objective To analyze the biological effects of miRNA-155 in the cardiac myocyte apoptosis. Methods The mouse-derived macrophage cell line RAW264.7 was treated by different concentration or different stage of oxidized low density lipoprotein (ox-LDL), and transfected by miR-155 mimic (M group), miR-155 mimics-NC (M-NC group), miR-155 inhibitor (I group) or miR-155 inhibitor-NC (I-NC group), respectively. The cell viability was measured by CCK-8 assay, cell apopotosis was measured by TUNEL and flow cytometry. Results The ox-LDL induced cell viability of Raw264.7 cells decreased and the expression of miR-155 increased in dose and time dependent manner, after treatment with different concentration of ox-LDL (10, 20, 40, 80, 160 mg/L) or 80 mg/L of ox-LDL with different stage (6, 12, 24, 48, 72 h). The expression of miR-155 increased significantly. Raw264.7 cell viability decreased significantly, compared to that of the blank control. The difference between two groups had statistical significance (P＜0.05). The cell viability in M group was significantly higher than that in M-NC group, in I group was significantly higher than that in I-NC group, and the difference between two groups was statistical significance (P＜0.05). Compared to that in the M-NC group, the cell apoptosis rate in M group was significantly increased (P＜0.05), compared to that in the I-NC group, the cell apoptosis rate in I group were significantly decreased (P＜0.05). Conclusions miR-155 can enhance the Raw264.7 cell apoptosis induced by ox-LDL.
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Objective To investigate the role of microRNA-10b (miR-10b) in the proliferation and invasion potential of osteosarcoma cell lines MG-63 and the exact underlying mechanism.Methods The expression level of miR-10b in human osteosarcoma tissue samples and adjacent normal bone tissues were detected by relative quantitative real-time PCR (qRT-PCR).miR-10b mimic and siRNA against Twist (Twist siRNA) were transfected into human osteosarcoma cell lines MG-63 respectively using lipofactamine 2000, and RT-PCR was used to detect the mRNA expression levels of miR-10b and Twist, and Twist protein expression level was detected by Western blot.The effect of miR-10b mimic and Twist siRNA on proliferation of MG-63 were detected by MTT[3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2 H-tetrazolium].The in-vitro cell invasion ability was determined by Transwell invasion assays after up-regulating miR-10b or knocking down of Twist.Results The expression levels of miR-10b was higher in human osteosarcoma tissue samples compared with adjacent normal bone tissues, the differences were extremely statistical significance (P＜0.01).miR-10b directly up regulated the mRNA and protein expression levels of Twist, the differences were significant (P＜0.05).In addition, miR-10b had enhanced the cell invasion and the proliferation (P＜0.05), whereas the proliferation and invasion ability of MG-63 which transfected by both miR-10b mimic and Twist siRNA were significantly reduced than that transfected by miR-10b mimic (P＜0.05).Conclusion miR-10b in MG-63 promotes the proliferation and invasion potential of human osteosarcoma cell lines MG-63, at least partly through the upregulation of Twist gene.
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MicroRNAs (miRNAs) are endogenous,single-stranded,noncoding RNAs with 18 to 24 nucleotides in length.miRNAs play important regulatory roles by targeting mRNAs for cleavage or translational repression.Thus,they negatively regulate gene expression at the post-transcriptional level.miRNA can degrade target mRNA or inhibit the translational level of their target mRNAs,which in turn affects cell differentiation,proliferation and apoptosis,and thus plays an important role in the regulation in the occurrence of diseases and the development of the organism.Recent studies have shown that miRNA also plays an important regulatory role in the development of thyroid-associated ophthalmopathy (TAO).This article reviewed the regulatory effect of miRNA in the development of TAO,providing a new insight in the pathogenesis and treatment of TAO.
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Objective To study the effect of microRNA-34a(miR-34a) on the biological behavior of uveal melanoma cells and its mechanism.Methods Uveal melanoma M23 cells were used as research objects,miR-34a mimics and mimics negative control were transfected into the cells respectively as miR-34a transfection group and the negative control group,and the non-transfected cells served as the normal control group.The overexpression effect was validated by real-time PCR.MTT assay was used to detect cell proliferation.Cell invasion and migration were detected by Transwell test.Target gene prediction library predicted target genes of miR-34a,and the target gene was identified by luciferase activity report.Real-time PCR and Western blot were used to detect the mRNA and protein expression of target genes.MiR-34a mimics and microphthalmia-associtated transcription factor (MITF) overexpression vectors were cotransfected into M23 cells.Cell proliferation,invasion and migration abilities were detected by MTT assay and Transwell test,respectively.The mRNA and protein expressions of MITF were detected by real-time PCR and Western blot.Results The expression of miR-34a in M23 cells transfected with miR-34a mimics increased.The cell proliferation (A570),number of invasive cells and migrating cells were significantly different among the miR-34a transfection group,negative control group and normal control group (F =18.000,P =0.003;F =20.345,P =0.002;F=15.717,P=0.004).The proliferation,invasion and migration ability of M23 cells in the miR-34a transfection group were significantly decreased compared with the negative control group and normal control group (all at P＜0.05).Target gene prediction library and luciferase activity report showed that MITF was the target gene of miR-34a.The relative expression levels of MITF mRNA and protein were 0.45 ±0.06 and 0.36± 0.04 in the miR-34a transfection group,0.99± 0.11 and 0.62 ± 0.05 in the negative control group,1.00 ± 0.07 and 0.63 ± 0.08 in the normal control group,respectively,and compared with the negative control group and normal control group,the expression of MITF in miR-34a transfection group were significantly decreased (all at P＜0.05).Cell proliferation (A570),the number of invasived cells and the number of migrated cells were 0.35±0.02,29.48±3.20 and 41.87±5.82 in the miR-34a + MITF group,0.26 ± 0.03,18.53 ± 1.47 and 27.64 ± 2.45 in the miR-34a + Vector group,respectively,the proliferation,invasion and migration ability of the cell.s in the miR-34a+MITF group was significantly higher than that in the miR-34a+Vector group (all at P＜0.05).Conclusions miR-34a can inhibit the malignant phenotype of uveal melanoma cells by inhibiting the expression of the target gene MITF.
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Objective@#To explore the correlation between expression level of miRNAs and pulmonary fibrosis on the basis of comparison the differential expression of miRNAs in rat pulmonary fibrosis induced by nano SiO2 and micron SiO2.@*Methods@#Thirty-six healthy male SD rats weighting 180-220 g were randomly divided into 3 groups. They were instilled intratracheally with 1 ml suspension of saline, 25 mg/ml nanosized SiO2 and microsized SiO2 particles and sacrificed at 60 d and 90 d postexposure from each group with six rats. The change of pathological morphology and ultrastructure of lung were observed by optical and transmission electron microscopy. The differentially expressed microRNAs in lung tissue of the rats after instilled intrachcally nanosized SiO2 and microsized SiO2 particles at 60 d and 90 d were determined by Illumina HiSeq 2 000 sequencing technique. Target prediction for miRNAs was conducted by databases of Target-scan. Function-significant enrichment analysis and signal pathway analysis for predicted target genes were respectively conducted by the GO and the KEGG, then target genes related to pulmonary fibrosis were screened out.@*Results@#Light microscope examination showed that wide bronchi, vessels, interlobular septa and slight fibrous connective tissue proliferation at 60 d and 90 d postexposure in 25 mg/ml nanosized SiO2 group. A few fused nodules at 30 d postexposure, a lot of fused nodules at 60 d postexposure, fibrous cell nodules and compensatory emphysema around alveolar at 90 d postexposure in 25 mg/mL microsized SiO2 group were observed. Electron microscopy demonstrated swelling and vacuolar degeneration of osmiophilic lamellar bodies in type Ⅱ alveolar epithelial cells, collagen fiber and elastic fiber hyperplasia in pulmonary interstitial at 60 d, 90 d postexposure in 25 mg/ml nanosized SiO2 group. Increased and vacuoloid changed osmiophilic lamellar bodies in type Ⅱ alveolar epithelial cells, collagen fiber and elastic fiber hyperplasia in the interstitial at 60 d, 90 d postexposure in 25 mg/ml microsized SiO2 group were observed. Comparing to saline control group, the number of miRNA up-regulated expression was 50, 70, and down-regulated expression was 22 and 24 at 60 d, 90 d postexposure in 25 mg/ml nanosized SiO2 group respectively. There were 91,70 miRNAs up-regulated expression and 34,78 miRNAs down-regulated expression at 60 d, 90 d postexposure in 25 mg/ml microscale SiO2 group. The common miRNA of differential up-regulated expression are miRNA-18a and miRNA-702-3p, down-regulated expression are miRNA-541, miRNA-127 and miRNA-379 both in nanosized SiO2 and microscale SiO2 group. The target genes related to pulmonary fibrosis were CTGF, IGF, BMP7, FGF7, TGF-β RIII, IGF1R and TGF-β1 respectively. Their biologic functions are to regulate signal pathway of TGF-β, MAPK and Wnt, and activation of fibroblast.@*Conclusion@#These findings suggested that same dose of nanosized SiO2 particles could cause mainly characterized by pulmonary interstitial fibrosis differing from silicotic nodule caused by microsized SiO2. miRNA-18a, miRNA-702-3p, miRNA-541, miRNA-127 and miRNA-379 may play a role in the process of pulmonary fibrosis in nanosized SiO2 and microscale SiO2 by regulating its target genes.