mi R-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1

AIM To evaluate the levels of mi R-192-5 p in non-alcoholic fatty liver disease(NAFLD) models and demonstrate the role of mi R-192-5 p in lipid accumulation. METHODS Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet(HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic mi R-192-5 p and stearoyl-Co A desaturase 1(SCD-1) levels were measured. Mi R-192-5 p mimic and inhibitor and SCD-1 si RNA were transfected into Huh7 cells exposed to palmitic acid(PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays.RESULTS The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic mi R-192-5 p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection(P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of mi R-192-5 p and SCD-1 protein levels, respectively(P < 0.01). Transfection with mi R-192-5 p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively(P < 0.01). Luciferase activity was suppressed and enhanced by mi R-192-5 p mimic and inhibitor, respectively, in wild-type SCD-1(P < 0.01) but not in mutant SCD-1. Mi R-192-5 p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 si RNA transfection abrogated the lipid deposition aggravated by mi R-192-5 p inhibitor(P < 0.01).CONCLUSION This study demonstrates that mi R-192-5 p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.

Roles of the canonical myomiRs mi R-1,-133 and-206 in cell development and disease

Micro RNAs are small non-coding RNAs that participate in different biological processes, providing subtle combinational regulation of cellular pathways, often by regulating components of signalling pathways. Aberrant expression of mi RNAs is an important factor in the development and progression of disease. The canonical myomi Rs(mi R-1,-133 and-206) are central to the development and health of mammalian skeletal and cardiac muscles, but new findings show they have regulatory roles in the development of other mammalian non-muscle tissues, including nerve, brain structures, adipose and some specialised immunological cells. Moreover, the deregulation of myomi R expression is associated with a variety of different cancers, where typically they have tumor suppressor functions, although examples of an oncogenic role illustrate their diverse function in different cell environments. This review examines the involvement of the related myomi Rs at the crossroads between cell development/tissue regeneration/tissue inflammation responses, and cancer development.

Hp阳性慢性萎缩性胃炎患者miR-92a-1-5p和FOXD1表达特征及其与预后的相关性

目的 探讨幽门螺杆菌(Hp)阳性慢性萎缩性胃炎(CAG)患者胃粘膜组织中微小RNA-92a-1-5p(mi R-92a-1-5p)和人转录因子叉头框1(FOXD1)表达特征及其与预后的相关性。方法 选取2017年1月至2021年10月四川省妇幼保健院收治的Hp阳性CAG患者124例作为研究对象,所有患者均在接受胃镜检查时取萎缩胃粘膜活检组织与周边正常形态粘膜活检组织,置于液氮内储存待检,测定mi R-92a-1-5p、FOXD1相对表达水平,并根据患者病情及病理评估结果给予针对性治疗。比较研究对象病变粘膜组织与正常粘膜组织mi R-92a-1-5p、FOXD1表达水平和不同病情程度患者粘膜组织mi R-92a-1-5p、FOXD1表达水平,并比较不同预后Hp阳性CAG患者mi R-92a-1-5p、FOXD1表达水平。统计分析mi R-92a-1-5p、FOXD1表达水平与疾病病情程度、Hp阳性CAG预后的相关性。结果(1)病变粘膜组织mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平高于正常粘膜组织(P<0.05)。(2)不同病情程度的Hp阳性CAG患者粘膜组织mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平差异有统计学意义(P<0.05),且随着病情程度的增加,Hp阳性CAG患者粘膜组织mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平呈持续增高趋势(P<0.05)。(3)预后不良患者mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平高于预后良好患者(P<0.05)。(4)经Pearson检验可知,mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平与Hp阳性CAG病情程度呈正相关(P<0.05),mi R-92a-1-5p、FOXD1mRNA及蛋白表达水平与Hp阳性CAG预后呈负相关(P<0.05)。结论 Hp阳性CAG患者胃粘膜组织中mi R-92a-1-5p、FOXD1表达较正常水平异常增高,且其增高幅度在不同病情程度患者中具有明显差异,其表达水平与疾病病情、预后均具有密切相关性,可评估病情、预测预后。

miR-21对1型糖尿病患者外周血单个核细胞分泌IFN-γ与IL-17的影响

目的:探讨mi R-21在1型糖尿病患者外周血单个核细胞(PBMC)分泌干扰素-γ(IFN-γ)与白介素-17(IL-17)的影响。方法:采集60例1型糖尿病患者及60例正常健康人外周血,分离外周血单个核细胞(PBMC),用q RT-PCR检测两组PBMC中mi R-21的表达;向PBMC中转染mi R-21,ELISA检测PBMC分泌IFN-γ与IL-17情况。结果:1型糖尿病患者PBMC中mi R-21表达低于对照组,差异有统计学意义(P<0.01);增加1型糖尿病患者PBMC中mi R-21的表达,IFN-γ与IL-17的分泌均减少,差异有统计学意义(P值分别<0.05或<0.01)。结论:mi R-21在1型糖尿病患者PBMC中的表达下降,并可抑制PBMC中IFN-γ与IL-17的分泌。

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Hepatitis C virus core protein-induced miR-93-5p upregulation inhibits interferon signaling pathway by targeting IFNAR1

AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.

MicroRNA-4279通过靶向核心启动子区域上调Notch-1基因表达进而抑制细胞凋亡

【目的】寻找与Notch-1基因核心启动子结合的micro RNA(miRNA)并阐明其调控功能,研究免疫细胞肿瘤的发生发展机制。【方法】首先使用生物信息学手段筛选与Notch-1基因核心启动子区域结合的miRNA,进而利用荧光素酶报告系统对其调控作用进行实验验证;通过突变核心启动子上的结合位点进一步确定该miRNA特异性靶向基因核心启动子区域。检测转染该miRNA后H9细胞中Notch-1的mRNA与内源蛋白表达水平。另外通过转染该miRNA并用H2O2诱导凋亡,研究该miRNA对H9细胞凋亡的影响。进一步在上述实验条件下,使用siRNA降低靶基因(Notch-1)的表达,然后检测miRNA对H9细胞系的凋亡的影响。【结果】首先通过软件预测,我们发现了miR-4279可以与Notch-1的核心启动子序列结合。荧光素酶报告实验表明miR-4279可以增强Notch-1启动子的转录活性;而在miR-4279的靶位点引入突变后,该增强作用消失。RT-qPCR和Western Blot实验表明miR-4279能够显著增强内源Notch-1 mRNA与蛋白的表达。H2O2诱导凋亡实验表明,miR-4279可以抑制H9细胞的凋亡。siRNA干扰实验表明,当Notch-1通路被抑制后,miR-4279导致的细胞凋亡抑制的效果消失。【结论】miR-4279通过靶向Notch-1基因核心启动子区域,特异性增加Notch-1基因的表达,进而增强H9细胞对H2O2诱导的凋亡的抗性。

m icroRNA-449b对高迁移率族蛋白B1介导树突状细胞功能的影响

目的:探讨mi R-449b在高迁移率族蛋白B1(HMGB1)刺激树突状细胞(DC)中的变化及其对DC免疫功能的影响。方法:分别以10、100、1 000 ng/ml剂量的HMGB1刺激小鼠脾脏来源的CD11c+DC,于48后检测DC表面分子CD80、CD86、MHC-II及细胞内mi R-449b改变;流式细胞仪分析不同剂量HMGB1刺激对DC凋亡的影响。DC转染mi R-449b模拟物(mi R-449b)、抑制物(In-mi R-449b)及相应对照(mi R-NC、In-mi RNC)后,HMGB1(100 ng/ml)刺激48 h收取细胞,检测DC表面分子、凋亡改变以及与T细胞混合培养后T细胞增殖、分化功能的变化。结果:HMGB1刺激DC后,其表面分子随着剂量的增加表达上调,其中以100 ng/ml组上调最为明显,而大剂量(1 000 ng/ml)时表达有所下调;mi R-449b在48 h 100及1 000 ng/ml HMBG1组表达明显上调(P<0.05);随着HMGB1剂量的增加,DC在48 h凋亡逐渐增加(P<0.05)。转染mi R-449b可上调其在细胞内表达,而抑制物可抑制其表达;上调mi R-449b后HMGB1(100 ng/ml)刺激48 h DC表面分子CD86表达显著上调(P<0.05)、凋亡增加(P<0.01)、对T细胞的共刺激增殖效应减弱、混合性淋巴细胞反应中IL-4水平增多(P<0.01)、IFN-γ水平下降(P<0.05)。结论:mi R-449b可负性调控HMGB1介导DC免疫功能及促进其凋亡,进而在机体免疫反应障碍过程中发挥调控作用。

土贝母苷甲经miR-21/PDCD4途径诱导胶质瘤U251细胞凋亡

目的:研究土贝母苷甲(TBMS I)对胶质瘤U251细胞的抗肿瘤作用以及探究土贝母苷甲对miR-21及其靶基因PDCD4基因表达的影响。方法:U251细胞在体外进行培养,四甲基偶氮唑盐(MTT)法检测不同浓度的土贝母苷甲对细胞增殖的影响;Hoechst33258染色观察细胞核形态的变化;实时荧光定量PCR检测miR-21和PDCD4基因的表达情况;Western blot检测PDCD4蛋白的表达情况。结果:土贝母苷甲能够显著抑制U251细胞的增殖,其抑制作用呈剂量和时间依赖性。Hoechst33258染色观察到土贝母苷甲处理组细胞的细胞核形态表现出典型的凋亡特征。PCR结果显示:随着土贝母苷甲浓度增加,miR-21的表达逐渐降低(P<0.05),PDCD4基因表达显著增加(P<0.05)。Western blot结果提示:与阴性对照组相比,15、30μg/mL土贝母苷甲显著上调了PDCD4蛋白的表达(P<0.05)。结论:土贝母苷甲能够显著抑制人胶质瘤U251细胞的增殖并诱导细胞发生凋亡,其机制可能与下调miR-21的表达和上调PDCD4的表达有关。

miR-142通过靶向HMGB1对宫颈癌细胞生物学行为影响的实验研究

目的:通过实验探究miR-142靶向高迁移率族蛋白1(high-mobility group box 1 protein,HMGB1)对宫颈癌(cervical cancer,CC)细胞生物学行为的影响及其潜在的作用机制。方法:采用实时荧光定量PCR(RT-PCR)和蛋白质免疫印迹法(Western Blot)检测CC组织和正常组织中miR-142和HMGB1 m RNA及蛋白表达水平,采用荧光素酶报告实验分析miR-142与HMGB1的靶向关系,CCK-8法检测CC细胞生存能力,克隆形成实验检测CC细胞增殖能力,划痕修复实验检测CC细胞迁移能力,基质胶侵袭实验检测CC细胞侵袭能力。结果:CC组miR-142 m RNA和蛋白表达水平显著低于正常组(P<0.05),HMGB1 m RNA和蛋白表达水平显著高于正常组(P<0.05),且CC癌组织中miR-142和HMGB1 m RNA和蛋白表达水平均呈显著负相关(r=-0.399,P=0.002;r=-0.429,P=0.001);miR-142与HMGB1存在靶向关系;CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,miR-142 mimic组细胞生存、增殖、迁移和侵袭能力显著低于miR-NC组(P<0.05),miR-142 inhibitor组细胞生存、增殖、迁移和侵袭能力显著高于miR-NC组;Western Blot实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组HMGB1蛋白表达水平显著高于miR-142 mimic+control plasmid组(P<0.05),显著低于miR-NC+plasmid组(P<0.05);CCK-8法实验、克隆形成实验、划痕修复实验和基质胶侵袭实验结果显示,HMGB1过表达时miR-142 mimic+plasmid组细胞生存、增殖、迁移和侵袭能力显著高于miR-142 mimic+control plasmid组(P<0.05),显著低于miR-NC+plasmid组(P<0.05)。结论:miR-142可通过靶向负调控HMGB1表达,进而抑制CC细胞生存、增殖、迁移和侵袭。

Human cytomegalovirus miR-US5-1 inhibits viral replication by targeting Geminin mRNA

Viruses commonly create favorable cellular conditions for their survival through multiple mechanisms. Micro RNAs(mi RNAs), which function as post-transcriptional regulators, are utilized by human cytomegalovirus(HCMV) in its infection and pathogenesis. In the present study, the DNA replication inhibitor Geminin(GMNN) was identified to be a direct target of hcmv-mi R-US5-1. Overexpression of hcmv-mi R-US5-1 could block the accumulation of GMNN during HCMV infection, and the decrease of GMNN expression caused by hcmv-mi R-US5-1 or GMNN specific si RNA reduced HCMV DNA copies in U373 cells. Meanwhile, ectopic expression of hcmv-mi R-US5-1 and consequent lower expression of GMNN influenced host cell cycle and proliferation. These results imply that hcmv-mi R-US5-1 may affect viral replication and host cellular environment by regulating expression kinetics of GMNN during HCMV infection.

促心肌素C-末端肽对再灌注损伤大鼠心肌细胞凋亡的影响

目的观察促心肌素(CT-1)C-末端肽在缺血再灌注损伤前后进行干预对大鼠心肌细胞凋亡的影响。方法制备心肌缺血再灌注损伤(MI/R)模型,27只SD大鼠随机分为正常组(N,n=5)、再灌注损伤模型组(D,n=6)、MI/R后CT-1干预组(T,n=8)和MI/R前CT-1干预组(O,n=8)。检测各组血清肌酸激酶(CK)活性和丙二醛(MDA)浓度;并取缺血区及其周围心脏组织计算心肌细胞凋亡指数(AI)。结果 MI/R后,模型组SD大鼠的平均存活时间为(93.17±24.7)min,在MI/R前的CT-1干预组为(87.88±18.3)min,MI/R后的CT-1干预组为(155.5±80.13)min,明显长于模型组和MI/R前干预组(q值分别为3.171、3.716,P<0.01);模型组SD大鼠的血清CK、MDA浓度升高,梗死区周围的AI也升高(N vs D,q值分别为14.391、11.015、21.668,P<0.01);MI/R后CT-1干预组的大鼠,血清CK、MDA浓度及AI均有所降低(T vs D,q值分别为10.649、6.167、16.493,P<0.01),但仍高于正常组(q值分别为5.197、5.782、7.391,P<0.01);CT-1前干预组的动物,血清CK、MDA浓度明显高于模型组(q值分别为6.147、10.551,P<0.01),AI高于正常组(q=24.609,P<0.01),但与模型组比较无明显差异(q=1.683,P>0.05);心肌细胞的凋亡情况与心肌的损伤及氧化损伤程度,有明显的相关性(r值分别为0.9245、0.8679,P<0.01)。结论 CT-1 C-末端多肽再灌注早期短期使用能减轻心肌组织损伤及氧化损伤,以及心肌细胞凋亡的程度,使动物存活时间延长;但腹腔注射CT-1 C-末端多肽较长时间后,大鼠对MI/R的耐受力降低,组织损伤及氧化损伤的程度明显加重,且梗死周边区有相当的心肌细胞发生凋亡,动物的存活时间也较短。