Effects of miR-133a-5p on the proliferation and adhesion of gastric cardia adenocarcinoma cells and the polarization of M1 macrophages / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究miR-133a-5p调控血清外泌体源性纤连蛋白1（fibronectin1，FN1）的表达对胃贲门腺癌（gastric cardia adenocarcinoma，GCA）细胞增殖、黏附和M1型巨噬细胞极化的影响。方法：借助GEO数据库分析GCA组织中的差异表达基因，进行功能富集分析。采用qPCR检测FN1在GCA组织、血清、血清外泌体和细胞中的表达。向GCA患者血清外泌体中转染FN1的过表达载体及其对照质粒，向HGC-27细胞中转染miR-133a-5p模拟物及其对照，将转染后的HGC-27细胞和外泌体共培养，再将此细胞与THP-1细胞共培养。采用CCK-8和细胞黏附实验分别检测各组HGC-27细胞的增殖和黏附情况，WB法、ELISA分别检测细胞中CD86、iNOS的水平以及对巨噬细胞分泌IL-6、IL-1β的影响。采用双荧光素酶报告基因实验验证FN1 mRNA和miR-133a-5p之间的相互作用关系。结果：与健康人对照组相比，GCA组织、血清、血清外泌体和细胞中的FN1表达水平显著上调（均P<0.05），FN1高表达的血清外泌体与GCA患者的TNM分期（P=0.032 9）和淋巴结转移有关联（P=0.012 7）。富含FN1的血清外泌体能够被GCA细胞内化，与过表达FN1的外泌体共培养能够提高HGC-27细胞的增殖和黏附能力，抑制THP-1细胞中IL-6、IL-1β、CD86和iNOS的表达，抑制M1型巨噬细胞极化（（P<0.05或P<0.01）。miR-133a-5p在GCA组织和细胞中较对照组显著降低，可负调控FN1的表达，过表达miR-133a-5p能够通过降低GCA细胞增殖和黏附能力，促进IL-6、IL-1β、CD86和iNOS的表达，部分逆转FN1对GCA细胞恶性行为的促进作用（P<0.05或P<0.01）。结论：miR-133a-5p可通过抑制血清外泌体分泌FN1对GCA细胞的恶性行为起抑制作用，对M1型巨噬细胞极化起促进作用。

Effect of Qigesan on Proliferation， Apoptosis and miR-133a/Akt/mTOR Signaling Pathway of Esophageal Cancer EC9706 Cells / 中国实验方剂学杂志

Objective:To observe the effect of Qigesan on the proliferation and apoptosis of the human esophageal cancer cell EC9706， and the effect on miR-133a/protein kinase B（Akt）/mammalian target of rapamycin （mTOR） signaling pathway. Method:The effective constituent of Qigesan was extracted by ethyl acetate. Thiazolyl blue tetrazolium bromide（MTT） colorimetric assay was used to determine the dosage of Qigesan on cells and to detect the effect of Qigesan on the proliferation of EC9706 cells. The effect of Qigesan on apoptosis of EC9706 cells was detected by flow cytometry. The effect of Qigesan on miR-133a and insulin-like growth factor 1 receptor（IGF-1R） mRNA expression was detected by Real-time quantitative polymerase chain reaction （Real-time PCR） . The protein expression of Akt and mTOR in EC9706 cells was detected by Western blot. Result:Qigesan can inhibit the proliferation of EC9706 cells in a dose-dependent manner（<italic>P</italic><0.01）. Inhibitory concentrations 30% inhibition concentration（IC₃₀） 40 mg·L^-1 and median inhibition concentration（IC₅₀） 80 mg·L^-1 were selected for follow-up experiments. Compared with the blank group， both the inhibitor group and the combination drug group can inhibit the proliferation of EC9706 cells （<italic>P</italic><0.01）. The inhibitor at 0.25 μmol·L^-1 was selected for subsequent experiments. Compared with the blank group， Qigesan 80 mg·L^-1 dose group could significantly promote the late apoptosis rate and total apoptosis rate of EC9706 cells（<italic>P</italic><0.05）， and the 40 mg·L^-1 dose group could significantly promote the late apoptosis rate of EC9706 cells（<italic>P</italic><0.05）， which shows synergistic effect after concomitant use with Akt/mTOR inhibitor（<italic>P</italic><0.05）. Compared with the blank control group， each group can effectively increase expression of miR-133a（<italic>P</italic><0.05）. The combination of inhibitor and traditional Chinese medicine（TCM） has obvious promotion effect. Compared with blank control group， the expressions of Akt and mTOR were significantly decreased in each group（<italic>P</italic><0.05）. Compared with single medication， the expressions of Akt and mTOR were decreased in combination of inhibitor and TCM group. Conclusion:Qigesan can inhibit the growth of EC9706 cells and promote apoptosis， and its inhibitory mechanism may be related to the Akt/mTOR signaling pathway by regulating the expression of miR-133a.

Role of miR-133a in radiation-induced cardiac injury in mice / 中华放射肿瘤学杂志

Objective:To investigate the regulatory role of microRNA in radiation-induced heart disease (RIHD) in mice and provide a new strategy for its treatment.Methods:Based on the Gene Expression Omnibus database (GSE147241), which includes normal heart tissue and irradiation heart tissue, we conducted bioinformatics research and analysis to determine the differentially-expressed genes. Then, thirty male C57/BL6 mice were randomly divided into the control group, irradiation group and miR-133a overexpression intervention group. The heart received single dose of X-ray 20 Gy in the irradiation group and miR-133a overexpression intervention group, but not in the control group, and then fed for 16 weeks. Cardiac function was assessed by echocardiography. Myocardial fibrosis was detected by Masson staining. The expression levels of miR-133a, CTGF, COL-1 and COL-3 mRNA were detected by qRT-PCR. The expression levels of CTGF, COL-1 and COL-3 proteins were detected by western blot.Results:miR-133a was the differentially-expressed gene between the irradiation and control groups. Overexpression of miR-133a could mitigate the decrease in cardiac function and increase in myocardial collagen content ( P<0.01). Meantime, overexpression of miR-133a could down-regulate the expression levels of CTGF, COL-1, COL-3 mRNA and protein ( P<0.01). Conclusions:Radiation increases the synthesis of collagen and leads to myocardial fibrosis remodeling. Overexpression of miR-133a can alleviate the radiation-induced myocardial fibrosis.

Early diagnosis and prognostic evaluation value of miR-133a in patients with acute myocardial infarction / 上海交通大学学报（医学版）

Objective: To investigate the value of serum miR-133a in early diagnosis and the assessment of 30-day incidence of cardiovascular adverse events in patients with acute myocardial infarction (AMI). Methods: Ninety patients with acute chest pain within 6 h were included, and 63 cases of AMI, 13 cases of unstable angina pectoris (UAP) and 14 cases of control (chest pain of other causes) were finally diagnosed. The levels of troponin I (cTnI), creatine kinase MB (CK-MB) and myoglobin (Mb) were measured by electrochemical fluorescence. Quantitative real-time PCR (qPCR) was used to detect the expression of miR-133a in the serum of patients immediately after admission and 24 h after percutaneous coronary intervention (PCI). The Gensini score of patients who underwent coronary angiography was recorded. The incidence of cardiovascular adverse events was observed within 30 days. Spearman correlation analysis, multivariate Logistic regression analysis and receiver operator characteristic curve (ROC curve) were used to analyze the corresponding data. Results: The expression of miR-133a in the AMI group was significantly higher than that in the UAP group and the control group (both P<0.05). Spearman correlation analysis showed that the expression of miR-133a was positively correlated with cTnI, CK-MB, MB level and Gensini scores (all P<0.05). Multivariate Logistic regression analysis showed that miR-133a and the history of coronary heart disease were independent risk factors for AMI. ROC curve showed that the area under the curve (AUC) of miR-133a in the diagnosis of AMI was 0.816 (95% CI 0.716-0.917), and the AUC of 30 days cardiovascular adverse event was 0.700 (95% CI 0.535-0.865). Conclusion: The expression of miR-133a in patients with AMI is significantly increased, which is expected to be a biomarker for early diagnosis of AMI. The expression level of miR-133a in serum may be related to the severity of coronary artery disease and short-term prognosis.

miR-133a mediates Bcl-xl expression to regulate endothelial cell apoptosis / 医学研究生学报

ObjectiveThe specific mechanism of microRNA-133a (miR-133a) involved in the pathological process of atherosclerosis (As) remains an open question. This study aims to explore the role of miR-133a in the regulation of endothelial cell apoptosis.MethodsCultured human coronary endothelial cells (HCAECs) were treated with oxidized low density lipoprotein (ox-LDL). The cell viability was detected by MTT assay. The mRNA levels of Bcl-xl and miRNA (miR-133a, etc) were detected by qRT-PCR method. The expression of anti-apoptotic protein Bcl-xl and cleaved-caspase3 was detected by Western blotting, and the apoptosis rate was detected by flow cytometry. The transient transfection technique was used to observe the effect of overexpression and silencing of miR-133a, on the expression of target gene Bcl-xl protein and endothelial cell apoptosis.Results Ox-LDL was observed to decrease the viability of HCAECs cells and induce HCAECs apoptosis; miR-133a increased abnormally in the apoptosis model; after silencing miR-133a, the decrease of Bcl-xl and the increase of apoptosis rate induced by ox-LDL were partially reversed; the overexpression of miR-133a, Bcl-xl decreased and the apoptosis rate increased, and the difference was statistically significant.Conclusion miR-133a might target and regulate the anti-apoptotic protein Bcl-xl, to induce endothelial cell apoptosis and promote the formation of AS.

Expression of miR-133a-3p in gastric cancer tissues and plasma and its effect on proliferation of gastric cancercells / 中国肿瘤生物治疗杂志

@#Objective: To detect the expression of miR-133a-3p in gastric cancer (GC) tissues and plasma of GC patients, and to investigate its effect on the proliferation of GC cells as well as its correlation toprognosis of GC patients. Methods: 52 cases of cancertissues (non-necrosis part) and corresponding adjacent tissues as well as the pre-operative peripheral blood samples from GC patients, who underwent surgery at Department of General Surgery, the Forth Hospital of Hebei Medical University(Shijiazhuang, China) between May 2012 and May 2013, were collected for this study. The plasma sample (n=35) from healthy donors were obtained during their physical examination. RT-qPCR was adopted to detect the expression of miR-133a-3p in gastric cancer tissues, adjacent tissuesand plasma samples of GC patients and healthy volunteers. The relationships between miR-133a-3p expression and the median DFS as well as clinicopathological parameters were also analyzed. CCK-8 assay was adopted to detect the effect of miR-133a-3p silence or over-expression on proliferation of gastric cancer SGC7901 cells. Results: miR-133a-3p was dramatically decreased in gastric cancer tissues (P<0.01), and its expression was associated with TNM stage, tumor infiltration (T), lynphonode metastasis (N), and vascular tumor thrombus (all P<0.01); miR-133a-3p was significantly increased in the plasma of GC patients (P<0.01), and its expression was associated with TNM stage, lynphonode metastasis (N), and vascular tumor thrombus (all P<0.05). miR-133a-3p expression was positively correlated with serum CA199 level of GC patients (P<0.01). The median DFS of patients with high miR-133a-3pexpression in cancer tissues was significantly longer than that of the patients with low expression(20.8 vs 14.8 months, P<0.05); The median DFS of patients with high plasma miR-133a-3p expression was significantly shorter than that of the patients with low expression (14.4 vs 20.3 months, P<0.05). Over-expression of miR-133a-3p could significantly inhibit the proliferation of gastric cancer SGC7901 cells, while miR-133a-3p silence could significantly promote the proliferation (all P<0.05). Conclusion: miR-133a-3p could significantlyinhibit the proliferation of SGC7901 cells; miR-133a-3p aberrantlyexpressed in gastric cancer tissues and plasma, and obviously correlated with prognosis of gastric cancer patients, which may be used as a potential clinical bio-maker for early diagnosis and treatment as well as the prognosis prediction of gastric cancer.

Regulation of miR-1 and miR-133 a on L-type calcium channel Cavβ2 and α1C subunits in rat cardiomyocyte hypertrophy / 基础医学与临床

Objective To investigate the regulation of miR-1 and miR-133 a on L-type calcium channel β2 subunit ( Cavβ2 ) and α1C subunit during rat cardiomyocyte hypertrophy .Methods Cardiomyocyte hypertrophy was in-duced by isoproterenol (ISO, 10μmol/L).The targets of miR-1 and miR-133a were predicted by online database microCosm and Targetscan , respectively .The 3′untranslated region sequences of Cavβ2 andα1C were respectively cloned into reporter vector and then transiently transfected into HEK 293 cells.The luciferase activities of samples were measured for demonstrating the expression of luciferase reporter vector .The protein expression of Cavβ2 andα1C were evaluated by Western blot .The expression levels of Cavβ2 andα1C were inhibited by RNAi to determine theeffectsofCavβ2andα1Concardiomyocytehypertrophy.Results 1)Cavβ2wasoneofpotentialtargetsof miR-1,α1C was the one of potential targets of miR-133a.2) The luciferase activities of HEK293 cells with the plasmid containing widetype Cavβ2 3′UTR sequence or α1 C significantly decreased ( P <0.05 , P <0.01 ) . 3 ) Upregulation of the miR-1 and miR-133 a by miR-1 mimic and miR-133 a mimic transfection suppressed pro-tein expression of Cavβ2 and α1C, respectively(P<0.01, P<0.05).4)Downregulation of Cavβ2 andα1C by RNAi could markedly inhibit the increase of cell surface area ( P<0.01 ) , mRNA expression of ANP andβ-MHC (P<0.05).Conclusions Cavβ2 is the target gene of miR-1 and α1C is the target gene of miR-133a.miR-1 and miR-133a can negatively regulate the expression of L-type calcium channel Cavβ2 andα1C subunit, inhibi-ting cardiomyocyte hypertrophy.