miR-122-5p attenuates the inflammatory response by modulating microglia polarization after traumatic brain injury / 中华急诊医学杂志

Objective:To determine the effect of miR-122-5p on microglia polarization, apoptosis and inflammation after traumatic brain injury (TBI).Methods:A mouse model and an in vitro TBI model were established. Astrocytes were stimulated to synthesize and release exosomes by brain extracts. microRNA microarray analysis was used to analyze the significantly altered microRNAs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to detect the expression of miR-122-5p in the in vivo and in vitro TBI model. TUNEL, immunofluorescence, and Western blot were performed to detect the effects of miR-122-5p inhibitors on microglia apoptosis, microglia M1/M2 phenotype transformation and the activation of NLRP3 inflammasome pathway and the phosphorylation of NF-κB after TBI.Results:The results of microRNA microarray analysis showed that 83 miRNAs were downregulated significantly (altered more than 2 folds, P < 0.05), among which miR-122-5p was significantly down-regulated ( P < 0.01). Expression of miR-122-5p was significantly decreased in the in vivo and in vitro TBI model [(1.00±0.00) vs. (0.41±0.15), P < 0.001; (1.00±0.00) vs. (0.34±0.07), P < 0.001]. TUNEL and immunofluorescence showed that miR-122-5p inhibitor significantly alleviated microglia apoptosis[(8.03±1.30) vs. (3.17±0.34), P < 0.001] and promoted microglia M1→M2 phenotype transformation ,M1 phenotype polarization was reduced [(56.96±13.70) vs. (34.70±3.47), P =0.002] and M2 phenotype polarization was increased [(30.46±3.67) vs. (40.74±2.49), P =0.005]. Western blot showed that NLRP3 inflammasome activation was inhibited and NF-κB phosphorylation was decreased when miR-122-5p was downregulated[(0.77±0.10) vs. (0.51±0.11), P =0.02; (0.73±0.08) vs. (0.50±0.07), P =0.003]. Conclusions:miR-122-5p is downregulated in microglia and exosomes secreted by astrocytes after TBI. miR-122-5p inhibitor can attenuate the microglia inflammatory response after TBI by inhibiting the activation of NLRP3 inflammasome pathway and the phosphorylation of NF-κB, promoting the microglia M1→M2 phenotypic transformation and reducing microglia apoptosis, thereby reducing the microglia inflammatory injury after TBI.

Regorafenib regulates the proliferation and apoptosis of human hepatocellular carcinoma SMMC-7721 cells through miR-122 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究瑞戈非尼（regorafenib，Rego）对人肝癌SMMC-7721细胞增殖、凋亡的影响及其可能的机制。方法：将SMMC-7721细胞分为对照组及Rego组，分别用0、10 μmol/L Rego处理48 h后，流式细胞术检测各组细胞凋亡率，qPCR检测细胞中miR-122的表达。采用脂质体转染的方法将合成的hsa-miR-122-5p模拟物转染SMMC-7721细胞构建miR-122过表达的overExp-miR-122细胞，并将细胞分为对照组、Rego组、overExp-NC组、overExp-NC+Rego组、overExp-miR-122组及overExp-miR-122+Rego组，采用MTT法检测细胞活性，流式细胞术检测细胞凋亡率、WB法检测细胞中Bcl2、cleaved caspase-3、RAS、RAF1、p-ERK1蛋白表达水平。结果：与对照组相比，Rego处理后细胞凋亡率显著升高（P<0.05），且miR-122表达量显著上升（P<0.01）；与overExp-NC组比较，overExp-miR-122组细胞增殖抑制率、凋亡率和cleaved caspase-3表达均显著升高（均P<0.01），RAS蛋白表达显著下降（P<0.05），Bcl2、RAF、p-ERK1蛋白表达均显著下降（均P<0.01）；与overExp-miR-122组相比，overExp-miR-122+Rego组细胞中各检测指标变化进一步显著增加（P<0.01）。结论：Rego可抑制SMMC-7721细胞增殖、促进凋亡，其作用可能与调控miR-122、凋亡相关因子的表达和抑制RAS/RAF/ERK信号通路有关。

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy / 中西医结合学报

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Expression of exosomal miR-122 in patients with hepatitis C and its clinical function / 安徽医科大学学报

Objective To investigate the expression and the clinical function of exosomal miR-122 in patients with hepatitis C (HCV). Methods Forty HCV patients (28 HCV-1b and 12 non-HCV-1b) were enrolled in this study, serum and exosomal miR-122 and other conventional biomarkers reflecting liver function were measured.20healthy subjects were enrolled as control.ResultsThe expressions of exosomal miR-122 in HCV-1b and nonHCV-1b groups were higher than that in healthy group, but the former was slightly higher than the latter.Exosomal miR-122 in HCV group was weakly negatively related to AST and ALT levels.The expression of exosomal miR-122was consistent with that of serum miR-122.Besides, the serum miR-122 in sustained virological response (SVR) group was higher than that in non-sustained virological response (NR) group (P＜0.05), but statistical difference of exosomal miR-122 between SVR and NR groups was not found. Conclusion In HCV patients, exosomal miR-122 may be useful in evaluating hepatic impairment, but it cannot be used to evaluate the therapeutic effect.

miR-122-5p inhibits the proliferation of melanoma cells by targeting NOP14 / 南方医科大学学报

OBJECTIVE@#To investigate the expression profile of miR-122-5p in melanoma tissues and the effect of miR-122-5p on the proliferation, cell cycle and apoptosis of human melanoma cell lines SK-MEL-110 and A375.@*METHODS@#The expression profiles of miR-122-5p in melanoma and pigmented nevus tissues were detected using real-time fluorescence quantitative PCR (qRT-PCR). SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor or negative control inhibitor (NC) I were examined for miR-122- 5p expression using qRT-PCR and changes in cell proliferation, cell cycle and apoptosis using MTT assay or flow cytometry. NOP14 mRNA and protein expressions in the cells were detected using qRT- PCR and Western blotting, respectively. Luciferase reporter assay was used to confirm the identity of NOP14 as the direct target of miR-122-5p.@*RESULTS@#The relative expression of miR-122-5p in human pigmented nevus tissues and melanoma tissues was 1.23±0.270 and 7.65 ± 1.37, respectively. The relative expression of miR-122-5p in SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor was 0.21 ± 0.08 and 0.17 ± 0.05, respectively. miR-122-5p inhibitor obviously inhibited the cell proliferation and increased the percentage of cells in G1 stage in both SK-MEL-110 and A-375 cells, but did not cause obvious changes in the apoptosis of the two cells. miR-122-5p inhibitor did not significantly affect the expression level of NOP14 mRNA, but obviously increased the expression level of NOP14 protein. Luciferase reporter assay revealed a significantly lower luciferase activity in cells co-transfected with miR-122-5p mimics and wild-type psi-CHECK2-3'UTR plasmid than in the cells cotransfected with NC and wild-type psi-CHECK2-3'UTR plasmid (0.21 ± 0.14 0.56 ± 0.1, < 0.01).@*CONCLUSIONS@#miR-122-5p expression is upregulated in melanoma tissues, indicating its involvement in the development of melanoma. miR-122-5p inhibits the proliferation of SK-MEL-110 and A-375 cells possibly by affecting the cycle through NOP14.

Comparison on sensitivity and specificity of serum miR-122 as biomarkers of drug-induced liver injury / 中国药理学与毒理学杂志

OBJECTIVE To make a comparison of sensitivity and specificity between serum miR-122 and traditional biomarkers of drug-induced liver injury. METHODS Acetaminophen (APAP, 1250 mg · kg-1, ig), α-naphthylisothiocyanate (ANIT, 150 mg · kg-1, ig), methionine-choline deficient diet (MCDD, free feeding) and carbon tetrachloride (CCl4, 50%, ip) were used to establish hepatocellular injury, cholestasis, steatosis and fibrosis models, respectively, which were used to evaluate the sensitivity of serum miR-122 as a biomarker of drug-induced liver injury, when compared with the traditional biomarkers. Isoprenaline hydrochloride (IH) and gentamicin (GM) were used to establish myocardial and renal injury models, respectively, which were used to evaluate the specificity of serum miR-122, when compared with the tradi-tional biomarkers. Serum and liver tissues were collected at different time points during the study. Tradi-tional biomarkers such as glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT) and total bilirubin (TBIL) were measured with an automatic biochemistry analyzer. MiR-122 was detected with real- time quantitative PCR. Histopathological examination with HE staining was performed for liver tissues. RESULTS Serum miR-122 increased significantly earlier [miR-122 was signifi-cantly increased (>2 fold) at 1.5 h, 3 h, 2 weeks and 4 weeks after treatment in the four models respec-tively, while no significant increase (<2 fold) was observed for GPT, GOT and TBIL at 6 h, 12 h, 3 weeks and 6 weeks after treatment in the four models respectively] and more signficantly (the maximum fold change for miR-122 was 235.8, 202.7, 73.8 and 600.3 for the four models, respectively. For the GPT, the maximum fold change was 9.5, 3.9, 2.5 and 6.6, respectively; 6.0, 2.4, 1.4 and 3.5 respectively for GOT; 2.6, 2.3, 1.2 and 1.8 respectively for TBIL) than that of traditional biomarkers in hepatocellular injury, cholestasis, steatosis and fibrosis models, when compared with the control group. In the myocar-dial injury model, a significant increase of GOT was observed after IH treatment (2.1 fold), while no change was observed for serum miR-122. In the renal injury model, no false positive results were observed for serum miR-122. CONCLUSION Serum miR-122 can be used as a biomarker of drug-induced liver injury and serum miR-122 is more sensitive and specific than traditional biomarkers (such as GPT, GOT and TBIL).

Inhibitory effect of oxymatrine on hepatitis B virus and its effect on expression of mircroRNA-122 / 实用医学杂志

Objective To investigate whether miR-122 is a target of oxymatrine against HBV. Methods HepG2.2.15 cells were incubated with culture medium containing different concentrations of oxymatrine. The cyto-toxicity of oxymatrine was determined by cck-8 assay. The surface antigen of HBV (HBsAg),antigenof HBV (HBeAg)and HBV DNA in supernatant and intracellular miR-122 were determined in HepG2.2.15 cells after incu-bation with culture medium containing oxymatrine for 72 h. Results The survival percentage of HepG2.2.15 cells under different concentrations of oxymatrine was higher than 95% when the concentration of oxymatrine was lower than 4 mg/mL. After treatment with oxymatrine for 72 h,the secretion of HBsAg and HBeAg,the level of HBV DNA in the supernatant were reduced. The intracellular miR-122 in oxymatrine experience group was 3.5 times higher than that in the negative control group. Conclusions Oxymatrine might be used to inhibit viral replication and antigen expression by enhancing the expression of miR-122 in HepG2.2.15 cells.

Effects of miR-122a on blood-spinal cordbarrier after spinal cord ischemia-reperfusion injury in rats / 中国药理学通报

Aim To investigate the effects of miR-122a on blood-spinal cord barrier after spinal cord ischemia-reperfusion injury in rats.Methods Thirty-six SD rats were randomly divided into three groups:group of sham(S group),group of control(C group)and group of miR-122a antagomir(M group).Rats in S group were subjected to exposure of aorta arch but without occlusion.Spinal ischemia-reperfusion injury was induced by clamping the aorta arch for 14 min in C group and M group.Rats in M group and C group were intrathecally injected with miR-122a antagomir or antagomir control daily for three times after injury.The miR-122a expression in injured spinal cord tissue was detected by real-time PCR.The occludin expression in injured spinal cord tissue was detected by Western blot.The permeability of blood-spinal cord barrier was examined using evans blue as a vascular tracer.The neurological motor function was evaluated by Basso Beattie Bresnahan score.Results Compared with S group,the expression of miR-122a was increased,the expression of occludin was decreased,the permeability of blood-spinal cord barrier was increased,and neurological motor function score was decreased significantly in C group(P<0.05).Compared with C group,the expression of miR-122a was decreased,the expression of occludin was increased,the permeability of blood-spinal cord barrier was decreased,and neurological motor function score was increased significantly in M group(P<0.05).Conclusion miR-122a can regulate the expression of occludin and change the permeability of blood-spinal cord barrier.

Diagnostic Value of Serum miR-122,ALT and HBV-DNA in Patients with Chronic Hepatitis B / 现代检验医学杂志

Objective To explore the clinical significance of serum miR-122,ALT and HBV-DNA in patients with chronic hepatitis B(CHB).Methods 900 patients of CHB were selected in Zaozhuang Mining Group Hospital from January 2014 to January 2016,and the serum levels of miR-122,ALT and HBV-DNA were detected,and analysed the relevance among the three and chronic hepatitis B.Results Serum markers of hepatitis B test showed that the HBV-DNA load in patients with chronic hepatitis B or early hepatitis infection was significantly higher than that of other patients (F=12.355,P=0.000),and the ALT content in patients with CHB was significantly higher than that of other patients (F=24.654,P =0.000).There was no significant correlation between ALT and HBV-DNA and HBV-M pattern.With the increase of HBV load,the proportion of hepatitis B patients with ALT ＞ 80 U/L increased gradually within limits (P＜0.05).Correlation analysis showed that ALT content was positively correlated with HBV loading (r=0.919,P =0.034).With the increase of HBV load,the level of serum miR-122 in patients with CHB was gradually increased (F=1463.435,P=0.000).Correlation analysis showed that serum miR-122 was positively correlated with HBV (r=0.975,P=0.012).Conclusion There was a correlation between serum miR-122,ALT and HBV-DNA in patients with CHB.The three indicators have good sensitivity and specificity in the diagnosis of CHB,so can use the three joint detection in the diagnosis in patients CHB,in order to improve the diagnostic accuracy and help patients better treatment.

Impact of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep2 / 中国免疫学杂志

Objective:To study the effect of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep-2.Meth-ods:The oligomucleotide of miR-122a was transfected into laryngeal carcinoma cell line Hep 2 cells,which were devided into three groups of A(miR-122a transfection),B(miR-122a inhibitor),C(miR-122a-NC inhibitor) and group of D(blank control).The expres-sion of miR-122a was defected by RT-PCR,and relevant protein expression was evaluated by Western blot .The cell proliferation and cell cycle were determined by MTT assy and flow cytometry ,respectively.Results:Compared to group D,miR-122a expression in Hep2 cells was obviously elevated atter miR-122a-transfected.The proliferation of Hep2 cells in group A was significantly inhibited and the cell cycle arrested at G1/G0 phase.The protein expression of CDC42 was downregulated with decreased expressions of CDK 4 and cyclin D1 in group A.Conclusion:miR-122a inhibits the proliferation activity of Hep 2 cells,suggesting that miR-122a can be taken as a po-tential candidate for gene therapy of laryngeal carcinoma .

PBMCs microRNA-122 level correlates with virological responses to pegylated interferon alpha therapy in patients with hepatitis C genotype 1b / 中华实验和临床病毒学杂志

Objective@#To clarify the predictive power of PBMCs miR-122, as well as other clinical factors, for response to IFNα therapy in chronic HCV infected patients.@*Methods@#A total of 40 patients chronically infected with HCV genotype 1b were enrolled. All the patients received pegylated interferon alpha (PEG-IFN α) in combination with ribavirin for 48 weeks. To perform the analyses, the patients were compared in terms of achieving sustained virological response (SVR) or not (NSVR) at 24th week after antiviral treatment.@*Results@#SVR rate was 72.5% (29/40) and NSVR rate was 27.5% (11/40). SVR group experienced significantly lower HCV viral load, total bilirubin (TBIL), alpha fetal protein (AFP), fibroscan and laminin (LN) compared with NSVR group before treatment (P<0.05). PBMCs miR-122 expression level was also lower in SVR group than that in SNVR group, although the difference was not statistically significant (P>0.05). and there was no significant change of miR-122 level from baseline to the last available measurement between SVR group and NSVR group. However, no significant association was found between baseline PBMCs miR-122 and HCV viral load, body mass index (BMI), alanine transaminase (ALT), aspartate transaminase (AST), degree of liver fibrosis, respectively.@*Conclusions@#Our result suggest that PBMCs miR-122 level is not an efficient biomarker to predict response to IFN alpha therapy in chronic HCV patients. However, baseline HCV viral load, TBIL, AFP and fibroscan may serve as predictive factors.

MicroRNA-122 regulates caspase-8 and promotes the apoptosis of mouse cardiomyocytes

Cardiomyocyte apoptosis plays key roles in the pathogenesis of heart diseases such as myocardial infarction. MicroRNAs are important regulators of gene expression, which are also involved in the regulation of cardiomyocyte apoptosis. However, cardiomyocyte apoptosis regulated by microRNA (miR)-122 is largely unexplored. The aim of this study focused on the role of miR-122 in cardiomyocyte apoptosis. Cardiomyocytes were isolated from neonatal mice and primarily cultured. MiR-122 mimic and inhibitor were transfected to cardiomyocytes and verified by qRT-PCR. Cell viability and apoptosis post-transfection were assessed by MTT assay and flow cytometry, respectively. Changes in expression of caspase-8 were quantified by qRT-PCR and western blot. Results showed that miR-122 mimic and inhibitor successfully induced changes in miR-122 levels in cultured cardiomyocytes (P<0.01). MiR-122 overexpression suppressed viability and promoted apoptosis of cardiomyocytes (P<0.05), and miR-122 knockdown promoted cell viability and inhibited apoptosis (P<0.05). The mRNA and protein levels of caspase-8 were elevated by miR-122 overexpression (P<0.01) and reduced by miR-122 knockdown (P<0.001). These results suggest an inductive role of miR-122 in cardiomyocyte apoptosis, which may be related to its regulation on caspase-8.

Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent

OBJECTIVE@#To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells.@*METHODS@#Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively.@*RESULTS@#The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = -4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05).@*CONCLUSIONS@#A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.

Studies About the Relationship between miR-1 2 2 and Hepatitis C Virus and Hepatocellular Carcinoma / 现代检验医学杂志

Hepatitis C virus (HCV)was easily developed into hepatitis,cirrhosis and liver cancer after infecting human.Cur-rently,the traditional method of treatment of HCV was pegylated interferon and ribavirin program,which was ineffective and had poor side effects,while the new direct antiviral drugs were expensive.Therefore,looking for cheap and non-toxic side effects of treatment was the focus of current research.More and more evidence indicate that micro-Ribose Nucleic Acid (miRNAs)play an important role in the development of liver disease,regeneration and functional regulation.This review studies the relationship between miR-122 and Hepatitis C virus and hepatocellular carcinoma.

The effects of vinyl chloride monomer on the expression of mir-122 in the liver tissue and serum of male rat / 中国职业医学

OBJECTIVE: To explore the effects of vinyl chloride monomer( VCM) on the expression of mir-122 in the liver tissue and serum of rat. METHODS: Specific pathogen free adult male SD rats were randomly divided into 4 groups with 24 rats in each group: the control group,the low-,medium- and high-dose groups. The 4 groups were intraperitoneally injected with VCM exposure levels at 0,5,25 and 125 mg / kg body weight 3 times per week,respectively. Eight rats per group were randomly selected and sacrificed after 6,8 and 12 weeks. Small RNA( < 200 nt) in the liver tissue and total RNA in the serum were isolated and purified. Real-time fluorescent quantitative polymease chain reaction technique was used to detect their expressions of mir-122. RESULTS: Compared with the control group and the low-dose group at the same time points,the relative expressions of mir-122 in the liver tissue of rats in the medium- and high-dose groups were significantly increased after exposed to VCM for 6 weeks( P < 0. 05),and then were significantly decreased after exposed to VCM for 12 weeks( P < 0. 05). The relative expressions of mir-122 in the liver tissue of rats in the medium- and highdose groups were decreased with VCM exposure time( P < 0. 05). Compared with the control group at the same time points,the relative expressions of mir-122 in serum of rats in the three dose groups were significantly increased after being exposed to VCM for 8 weeks( P < 0. 05),and then were significantly decreased after being exposed to VCM for 12 weeks(P < 0. 05). Among the three dose groups,the relative expressions of mir-122 in serum of rats after 12-week exposure were lower than those after 6-week and 8-week exposure in the same group respectively( P < 0. 05). CONCLUSION:s The VCM exposure significantly changes the expression of mir-122 in the liver tissue and serum of rat,which suggested that mir-122 may be one of the effect biomarkers for VCM exposure.

Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent

Objective To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells. Methods Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. Results The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = −4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05). Conclusions A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage, inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.

Role of miR-122 in bone marrow derived stem cell therapy for acute liver injury in rats / 吉林大学学报(医学版)

Objective To investigatet the effects of miR-122 in the therapy of bone marrow derived stem cells (BMSCs)for acute liver injury in the rats,and to clarify the mechanism.Methods The BMSCs were isolated from the bone marrow of male rats by density gradient centrifugation.The BMSCs were divided into transfection group and control group.The BMSCs in transfection group were transfected with miR-122 mimics by liposome,while the BMSCs in control group were not.60 SD rats with acute liver injury induced by 10%CCl4 were randomly divided into control group (the saline was injected through mainline),normal treatment group (the normal BMSCs were injected through mainline) and experimental therapy group (the BMSCs transfected with miR-122 mimics by liposome were injected through mainline)(n=20).The liver function and tissue pathology were examined at 1 d, 7 d and 14 d after transplantation.Results The expression level of ALB in BMSCs was up-regulated,while the AFP expression level was down-regulated after the transfection of miR-122 mimics.At 1 d after transfection of BMSCs,the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities had no significant difference between normal treatment group and experimental therapy group.At 7 d and 14 d after transfection of BMSCs,the serum ALT and AST activities in experimental therapy group were obviously lower than those in normal treatment group (P <0.05).The liver congestion,cytoplasm degeneration and liver cell necrosis in experimental therapy group were improved compared with normal treatment group.Conclusion The up-regulation of miR-122 expression in BMSCs would promote its differentiation into hepatocyte like cells,which plays a role in promoting the recovery of liver injury.

Detection of Serum miR-122 and miR-22 in Patients with Chronic Hepatitis B by Using of a Quantitative Real-time PCR / 医学研究杂志

Objective To establish a real -time quantitative PCR ( RT-PCR) assay for detecting serum miR -122, miR-22, and evaluate the clinical significance of miR -122 and miR-22 in patients with chronic hepatitis B ( CHB) by using of this assay .Meth-ods The mature miRNAs were reversely transcripted by using of stem -loop primers .SYBR GreenⅠquantitative real-time PCR ( qRT-PCR) was used for quantification of the miRNAs .The sensitivity of this assay was evaluated by using of the 10-fold-diluted miRNA-122 cDNA standards and the specificity was verified by using of melting curve assay .The accuracy was assessed by intra -assay coeffi-cient of variation (CV) of threshold cycle (Ct value), which were calculated from a 20-times-repeat detection of the miR -122 cDNA (2 ×105 , 2 ×106 , 2 ×107 copies/μl) standards.Using the established qRT -PCR assay, we detected the expression of serum miR -122 and miR-22 in the patients with CHB and healthy controls .Results The qRT-PCR assay exhibited good performances in the linear range, sensitivity and reproducibility while detecting miR -122 and miR-22.The relative level of miR -122 and miR-22 was 17.88 vs 5.35 in the CHB patients and 1.80 vs 1.67 in the controls (P=0.000).Conclusion Using of stem-loop primers, we established a qRT-PCR assay for detection of serum miR -122 and miR-22.Serum miR-122 and miR-22 increased significantly in the CHB pa-tients.

GATA4 Expression During Induction of the Cardiomyocyte-Like Cells Differentiation Regulated by miR-122 / 天津医药

Objective To identify the miR-122 which regulateing GATA4 expression during the induction of rat bone marrow mesenchymal stem cells (bMSCs) differentiating into cardiomyocyte-like cells. Methods BMSCs were isolat-ed from bone marrow and induced to differentiate into cardiomyocyte-like cells using 5-azacytidine. The miR-122 which may regulate expression of GATA4 were predicted using miRanda and TargetScan softwares and identified by dual luciferase report system. The expressions of miR-122 and GATA4 were determined using q-PCR during the differentiation of bMSCs into cardiomyocyte-like cells. Results The induced cells were completely in contacted with adjoining cells and uniform in shape and aligned parallelly. Cardiac troponin I (cTnI) expression was detected by immunofluorescence cytochemistry. Using dual luciferase reporter system in vitro, miR-122 were proved to be able to effectively inhibit GATA4 expression by binding the 3′UTR of GATA4 mRNA. q-PCR results showed that the expression of miR-122 is negatively correlated with that of GATA4 mRNA transcription. Conclusion These results indicated that miR-122 regulate the expression of GATA4 during the induction of cardiomyocyte-like cells.