Discovery of miRNA and target signal molecules involved in inhibition of chlorogenic acid on N-acetyl-p-aminophenol-induced hepatotoxicity based on microRNA array / 中国中药杂志

This study aims to observe the effect of chlorogenic acid(CGA) on microRNA(miRNA) in the process of protecting against N-acetyl-p-aminophenol(APAP)-induced liver injury. Eighteen C57BL/6 mice were randomly assigned into a normal group, a model group(APAP, 300 mg·kg~(-1)), and a CGA(40 mg·kg~(-1)) group. Hepatotoxicity of mice was induced by intragastric administration of APAP(300 mg·kg~(-1)). The mice in the CGA group were administrated with CGA(40 mg·kg~(-1)) by gavage 1 h after APAP administration. The mice were sacrificed 6 h after APAP administration, and plasma and liver tissue samples were collected for the determination of serum alanine/aspartate aminotransferase(ALT/AST) level and observation of liver histopathology, respectively. MiRNA array combined with real-time PCR was employed to discover important miRNAs. The target genes of miRNAs were predicted via miRWalk and TargetScan 7.2, verified by real-time PCR, and then subjected to functional annotation and signaling pathway enrichment. The results showed that CGA administration lowered the serum ALT/AST level elevated by APAP and alleviate the liver injury. Nine potential miRNAs were screened out from the microarray. The expression of miR-2137 and miR-451a in the liver tissue was verified by real-time PCR. The expression of miR-2137 and miR-451a was significantly up-regulated after APAP administration, and such up-regulated expression was significantly down-regulated after CGA administration, consistent with the array results. The target genes of miR-2137 and miR-451a were predicted and verified. Eleven target genes were involved in the process of CGA protecting against APAP-induced liver injury. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment with DAVID and R language showed that the 11 target genes were enriched in Rho protein-related signal transduction, vascular patterning-related biological processes, binding to transcription factors, and Rho guanyl-nucleotide exchange factor activity. The results indicated that miR-2137 and miR-451a played an important role in the inhibition of CGA on APAP-induced hepatotoxicity.

The Role and Mechanism of MiR-451 in Multidrug Resistance of Leukemia Cell Line K562/A02 / 中国实验血液学杂志

OBJECTIVE@#To detect the differential expressions of miR-451, ABCB1 and ABCC2 in drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, and explore the regulatory relationship between miR-451 and the expressions of ABCB1 and ABCC2 , and the mechanism of miR-451 involved in drug resistance in leukemia.@*METHODS@#CCK-8 assay was used to detect the drug resistance of K562/A02 and K562 cells. Quantitative Real-time PCR (qRT-PCR) was used to verify the differential expressions of miR-451 in K562 and K562/A02 cells. MiR-451 mimic and negative control (miR-NC), miR-451 inhibitor and negative control (miR-inNC) were transfected into K562 and K562/A02 cells respectively, then qRT-PCR and Western blot were used to detect the expression levels of mRNA and protein of ABCB1 and ABCC2 in K562 and K562/A02 cells and the transfected groups.@*RESULTS@#The drug resistance of K562/A02 cells to adriamycin was 177 times higher than that of its parent cell line K562. Compared with K562 cells, the expression of miR-451 in K562/A02 cells was significantly higher (P <0.001), and the mRNA and protein expression levels of ABCB1 and ABCC2 in K562/A02 cells were significantly higher than those in K562 cells (P <0.001). After transfected with miR-451 inhibitor, the expression of miR-451 was significantly down-regulated in K562/A02 cells (P <0.001), the sensitivity to chemotherapy drugs was significantly enhanced (P <0.05), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly decreased (P <0.01). After transfected with miR-451 mimic, the expression of miR-451 was significantly upregulated in K562 cells (P <0.001), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly increased (P <0.01).@*CONCLUSION@#There are significant differences in the expressions of miR-451, ABCB1 and ABCC2 between the drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, which suggests that miR-451 may affect the drug resistance of leukemia cells by regulating the expression of ABCB1 and ABCC2.

Shexiang Baoxin Pill Regulates Intimal Hyperplasia, Migration, and Apoptosis after Platelet-Derived Growth Factor-BB-Stimulation of Vascular Smooth Muscle Cells via miR-451 / 中国结合医学杂志

OBJECTIVE@#To investigate the regulatory roles of Shexiang Baoxin Pill (SXBXW) in neointimal formation and vascular smooth muscle cells (VSMCs) invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury (platelet-derived growth factor (PDGF)-BB-stimulated) in vitro.@*METHODS@#VSMCs were randomly assigned to 5 groups: blank, PDGF-BB (20 ng/mL+ 0.1% DMSO), SXBXW-L (PDGF-BB 20 ng/mL + SXBXW low dose 0.625 g/L), SXBXW-M (PDGF-BB 20 ng/mL + SXBXW medium dose 1.25 g/L) and SXBXW-H (PDGF-BB 20 ng/mL+ SXBXW high dose 2.5 g/L) group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and bromodeoxyuridine (BrdU) incorporation assay, the migration effects were detected by Transwell assay, cell apoptosis rate was measured by the Annexin V/propidium iodide (PI) apoptosis kit. The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining. To validate the effects of miR-451 in regulating proliferation, migration and apoptosis treated with SXBXW, miR-451 overexpression experiments were performed, the VSMCs were exposed to PDGF-BB 20 ng/mL + 0.1% DMSO and later divided into 4 groups: mimic-NC (multiplicity of infection, MOI=50), SXBXW (1.25 g/L) + mimic-NC, mimic-miR451 (MOI=50), and SXBXW (1.25 g/L) + mimic-miR451, and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.@*RESULTS@#PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration. SXBXW inhibited phenotypic switching, proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs. In addition, miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation. SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs (P<0.05). Compared with SXBXW + mimic-NC and mimic-miR451 groups, the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and p53 was further reduced in SXBXW + mimic-miR451 group, while activating transcription factor 2 (ATF2) was increased in VSMCs (P<0.05).@*CONCLUSION@#SXBXW regulated proliferation, migration and apoptosis via activation of miR-451 through ATF2, p53 and Ywhaz in PDGF-BB-stimulated VSMCs.

28-hydroxy-3-oxoolan-12-en-2-oic acid and miR-451 synergistically inhibit the proliferation and migration of gastric cancer AGS cells and its possible mechanism / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探索南蛇藤提取物齐墩果烷型五环三萜（28-hydroxy-3-oxoolean-12-en-2-oic acid）协同miR-451对人胃癌AGS细胞增殖、迁移的影响及其可能的分子机制。方法：用miR-451过表达慢病毒感染AGS细胞，并用盐酸多西环素（DOX）10或100 ng/ml诱导24 h，构建过表达miR-451的细胞AGS/miR-451+。采用10、20、40、80、160 μmol/L的齐墩果烷型五环三萜处理AGS/miR-451+细胞，MTT法、划痕实验分别检测细胞增殖和迁移能力的变化，WB法检测细胞中mTOR通路及凋亡相关蛋白表达水平的变化。结果：成功构建过表达miR-451的AGS/miR-451+细胞。与未加药对照组相比，齐墩果烷型五环三萜处理后AGS/miR-451+细胞的增殖抑制率均呈时间和浓度依赖性升高（P<0.05或P<0.01），细胞迁移率均显著降低（P<0.05或P<0.01）。齐墩果烷型五环三萜处理组细胞中，mTOR 信号通路相关蛋白的表达量均有所降低（P<0.05或P<0.01）；凋亡相关蛋白中，Bcl2的表达量下降，BAX、caspase-3、caspase-1及细胞色素c的表达量升高（P<0.05或P<0.01）。结论：齐墩果烷型五环三萜能够协同miR-451抑制人胃癌AGS细胞的增殖与迁移，其机制可能与影响凋亡和mTOR信号通路相关蛋白的表达有关。

New research progress of miR-144 in cardiovascular diseases / 解放军医学杂志

MicroRNA is a type of widely distributed endogenous small single-stranded non-coding RNA that regulates important biological processes. It complements the target mRNA sequence, degrades the target mRNA and inhibits protein translation, and plays a regulatory role at the transcription level. Studies have confirmed that microRNAs in circulation are present in various body fluids in a stable form and can be used as biomarkers of disease. miR-144 is highly conservative in structure, exists in the form of miR-144/miR-451 gene cluster in the genome, and participates in the occurrence and development of erythroid differentiation, tumors, and cardiovascular diseases. With the continuous progress of life sciences, miR-144 pathogenic mechanism and role in cardiovascular disease have been gradually revealed. This article gathers the relationship between miR-144 and the pathogenesis of cardiovascular disease and the diagnosis and treatment plan, starting from the relationship between miR-144 and coronary atherosclerotic heart disease, arrhythmia and structural heart disease, combing related research progress, with a view to providing new ideas and methods for the diagnosis and treatment of cardiovascular diseases.

Inhibitory effect of miR-451 on proliferation of hepatic carcinoma HepG2 cells and its prospect in hepatic carcinoma diagnosis and prognosis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the mRNA molecular targets for diagnosis of hepatic carcionoma and to investigate their functional roles in proliferation and cell cycle of hepatic cancer cells. Methods: Based on the statistical analysis of miRNA expression data from 377 hepatic carcionoma samples and 37 adjacent non-cancerous samples in TCGAdatabase, a group of 33 differentially expressed miRNAs were identified.A further screen of these differentially expressed miRNAs was performed using the receiver operating characteristic curve (ROC curve) and Kaplan-Meier survival analysis; and with referring to the current publications, miR-451 was screened as the study subject. HepG2 cells were transfected with pLVX-shRNA2-miR-451 to over-express miR-451. The effect of miR-451 over-expression on the proliferation of HepG2 cell was determined by CCK-8 assay; while the effect on cell cycles was detected by flow cytometry. Results: The expression of miR-451 in the adjacent non-cancerous tissues was significantly lower than that in cancer tissues ([473.40±390.24] vs [1 990.47±2 118.04], P<0.05). MiR-451 could be used as an early diagnostic biomarker of hepatic carcionoma, with a high ROC value of 0.91 (sensitivity 0.89, specificity 0.87). The results of in vitro experiments showed that the proliferation of HepG2 cells was significantly decreased after miR-451 over-expression (48 h: [0.69±0.04] vs [1.08±0.05]; 72 h: [0.76±0.07] vs [1.52± 0.02]; all P<0.01), and a large number of cells were blocked in S phase(P<0.05). Conclusion: miR-451 has the potential to be used as a biomarker for hepatic carcionoma diagnosis and prognosis; moreover, it also exhibits the inhibitory effect on proliferation of hepatic cancer cells.

Expressions and significances of prognosis by MiR-451a in nasopharyngeal carcinoma in Guangxi / 实用医学杂志

Objective To investigate the expression level and the significances of prognosis by miR-451a in nasopharyngeal carcinoma (NPC) in Guangxi. Methods The expressions of miR-451a in 89 cases of nasopharyngeal carcinoma were detected by real time RT-PCR. The relation among the expression level , the clinicopathologic features of NPC and its prognosis were analyzed. Results The expression of miR-451a were found in all of nasopharyngeal carcinoma. The expression level of miR-451a in nasopharyngeal carcinoma was negative correlated to overall survival and disease free survival (P = 0.01,P = 0.04). Conclusions miR-451a may play a key role in detection of nasopharyngeal carcinoma with poor prognosis.

The expression of miR-451 and its clinical significance in gastric cancer / 天津医药

Objective To investigate the expression of miR-451 and its clinical significance in gastric cancer. Meth?ods The miR-451 expression was detected in 92 samples of gastric cancer and 47 sapmles of normal stomach mucosa. The correlations of miR-451 expression with clinicopathologic characteristics, overall survival and disease-free survival were an?alyzed in patients with gastric cancer. Results The overexpression rate of miR-451 was 38.04%in gastric cancer, which was significantly lower than 76.60%in adjacent normal tissues (χ2=18.503,P0.05). The overall survival rate was significantly higher in patients with high expression of miR-451 than that in patients with low expression of miR-451(69.2%vs 39.1%, P=0.003). Conclusion miR-451 is associated with the malignant transformation, progression, and prognosis of gastric cancer, and which can be used as a potential target of therapy and a biomarker of diagnosis and prognosis of gastric cancer in the future.

Changes of miR-451 expression during erythroid differentiation of K562 cells / 第二军医大学学报

Objective: To observe the expression of miR-451 during erythroid differentiation of K562 cells and investigate the role of miR-451 in erythroid differentiation. Methods: MiR-451 expression was analyzed in different tissues (the liver, bone marrow, erythrocytes, and white blood cells) of mice, human erythrocytes, and chicken red blood cells by Northern blotting. Erythroid differentiation of K562 cells was assessed by DAB staining and RT-PCR of heamoglobin mRNA before and 36 h after hemin induction (50 μmol/L). The expression of miR-451 in K562 cells was further explored by Northern blotting and stem-loop RT-PCR before and 24,48,72,and 96 h after hemin induction (50 μmol/L). Results: Expression of miR-451 was only identified in the erythrocytes, not in white blood cells, hepatic cells or bone marrow of mice. MiR-451 expression was also detected in human erythrocytes and chicken erythrocytes with nuclei. Two bands were detected in the human and mouse erythrocytes by Northern blotting, indicating that, in addition to the one reported by Sanger's miRBase, there was another miR-451 sequence which had additional uracil residue in 3′ terminal. Hemin induced differentiation of K562 cells and DAB staining showed more positive cells after induction (P<0.05); the expression of γ, δ and -̇globin mRNA was also increased after treatment with hemin (P<0.05). Although Northern blotting revealed no changes in miR-451 expression in K562 cells after hemin induction, more sensitive stem-loop RT-PCR showed that miR-451 expression, which maintained at lower level in un-induced K562 cells, was increased during erythroid differentiation 24 h after hemin induction. With the upregulation of δ-globin protein, the expression of miR-451 reached its peak 72 h later. Conclusion: miR-451 is specifically and highly expressed in erythroid terminal differentiation. Two different sequences of miR-415 (one with and additional uracil residue) are present in the human and mouse erythrocytes, and their expression is elevated during the erythroid differentiation of K562 cells.