Inhibition effect of miR-577 on hepatocellular carcinoma cell growth via targeting β-catenin

Objective:To investigate the expression and the regulation effect of cell growth of microRNA-577 in hepatocellular carcinoma(HCC).Methods:qRT-PCR was applied to detect the relative expression of miR-577 in 70 paired HCC and matched tumor adjacent tissues collecting from resection between March 2011 and March 2014.Pearson chi-square test was used to analyze the relationship between the miR-577 expression and clinical features.The miR-577 mimics were transfected into HepG2 cells:cell cycles were detected by flow cytometry,cell proliferation was measured by MTT assay and BrdU incorporation assay,and cell apoptosis was determined by flow cytometry and Caspase 3/7 activity analysis.The expressions ofβ-catenin were measured by immunohistochemistry.Spearman correlation analysis was used to analyze the relationship between miR-577 and p-catenin.qRT-PCR and westernblot were used to detect the expression of p-catenin in transfected HepG2 cells.Results:The relative expressions of miR-577 was significantly lower in HCC tissues compared to the matched normal tumor-adjacent tissues(P0.05).Low expression of miR-577 was significantly associated with large tumor size(≥5 cm,P0.05) and advanced tumor node metastasis stage(Ⅲ+Ⅳ,P0.05).Transfection of miR-577 mimics could inhibit repress cell proliferation,enhance cell apoptosis and block the cell cycles in G_1/G_1 phase(P0.05).miR-577 in HCC group had a significant negative correlation relationship with the expression of downstream target of β-catenin(P0.05).Both the mRNA and protein expression in HepG2 cells were down-regulated after transfection(P0.05).Conclusions:Low expression of miR-577 is related to the malignant clinicopathological features in HCC tissues,and miR-577 may suppress HCC growth through down-regulating p-catenin.     

干扰肝细胞核因子-1α对HepG2细胞载脂蛋白M及胆固醇相关代谢酶表达的影响

目的 检测肝细胞癌和癌旁组织中肝细胞核因子(HNF)-1α、肝受体同系物(LRH)-1与载脂蛋白(apo)M表达的相关性,探讨HNF-1α对apoM、apoA-Ⅰ和胆固醇合成与转化关键酶表达的影响.方法 RT-PCR检测人肝细胞癌组织和癌旁组织中HNF-1α、LRH-1、apoM的表达水平.采用小干扰RNA(SiRNA)在人肝癌细胞株HepG2细胞中干扰HNF-1 α表达后,RT-PCR检测apoM、apoA-Ⅰ及羟甲基戊二酰辅酶A还原酶(HMGCR)、胆固醇7 α-羟化酶1(CYP7A1)、法尼醇X受体(FXR)、小异源二聚体伴侣(SHP)-1的变化情况;Western blot检测apoM在蛋白水平的变化情况.对不同数据进行单因素方差分析、配对t检验或直线相关分析.结果 HNF-1 α、LRH-1、apoM mRNA在人肝细胞癌组织中明显高于癌旁组织(t值分别为-7.075,-8.803,-7.167,P值均＜0.01);HNF-1α和LRH-1与apoM的mRNA表达水平呈正相关(r=0.353,P＜0.01;r=0.523,P＜0.01).干扰HNF-1α后,与阴性对照相比,apoM、FXR、SHP-1 mRNA水平分别下降47.4%(F=77.627,P＜0.01)、47.9%(F=76.363,P＜0.01)、65.2%(F=365.772,P＜0.01),apoM蛋白水平下调54.3%(F=43.482,P＜0.01);HMGCR、CYP7A1 mRNA水平分别升高101.1%(F=218.666,P＜0.01)、138.5%(F=171.926,P＜0.01);而对apoA-Ⅰ无明显影响(F=0.170,P＞0.05).结论 HNF-1 α可提高apoM的表达水平,并通过促进胆固醇代谢关键酶的表达和抑制胆固醇代谢相关抑制因子的生成而促进胆固醇的转化,对心血管疾病具有保护作用.

Abstract：

Objective To determine wether there were connections among hepatocyte nuclear factor1 alfa (HNF-1α),liver receptor homolog-1 (LRH-1),apolipoprotein M (apoM) and to investigate the effects of HNF-1 αt in HepG2 on the expressions of apoM,apolipoprotein A-Ⅰ (apoA-Ⅰ) and the key enzymes in cholesterol metabolism and biotransformation.Methods The mRNA expressions of apoM,LRH- 1 and HNF- 1 α were detected by RT-PCR.HNF- 1 α was interfered and RT-PCR was used to detect the changes of apo M,apo A-Ⅰ,Cyp7Al,farnesoid X receptor (FXR) and small heterodimer partner-1 (SHP-1).Western blot was used to detect the change of apo M protein.Results The expressions of apoM,LRH-1 and HNF-1αmRNA were obviously higher in HCC tissue than that in para-cancer tissue (the vaule of tis -7.167,-7.075,-8.803,P ＜0.01 respectively).HNF-1 α and LRH-1 positively correlated with the expression of apoM (r = 0.353,P ＜0.01;r = 0.523,P ＜ 0.01 respectively);RT-PCR and western blot results showed that the expressions of apoM,FXR and SHP-1 mRNA,could be obviously suppressed by HNF-1 α interfering as compared to the negative controls by 47.4%,47.9%and 65.2% (P ＜ 0.01) respectively,and the expression of apoM protein also decreased by 54.3% (F = 43.482,P ＜ 0.01).The expressions of HMGCR and CYP7A mRNA increased by 101.1% and 138.5% (P ＜ 0.01) respectively as compared to the negative control.But there is no effect on expression of apoA-Ⅰ mRNA (F = 0.170,P ＞ 0.05).Conclusions HNF-1 α could promote cholesterol biotransformation by increasing the expression of apoM and the key enzymes in cholesterol metabolism and decreasing inhibiting factor.So HNF-1 α provided protection against cardiovascular disease.     

miR-221/222在肝癌细胞抵抗内质网应激凋亡中的作用

目的 研究内质网应激对miR-221/222的调控及其在肝癌细胞抵抗内质网应激诱导细胞凋亡中作用.方法 采用miR-221/222抑制物和miR-221/222类似物分别阻断或模拟内源性miR221/222的功能,并利用Western blot和流式细胞技术分析内质网应激条件下miR-221/222对肝癌细胞周期和凋亡的调控作用.结果 内质网应激诱导miR-221/222表达下调,miR-221/222类似物和抑制物分别抑制和促进内质网应激诱导的p27Kip1表达上调,干扰p27Kip1不仅抑制了内质网应激诱导的肝癌细胞G0/G1期阻滞,也促进了内质网应激介导的肝癌细胞凋亡.结论 内质网应激诱导miR-221/222下调能够通过促进p27Kipl表达对内质网应激条件下肝癌细胞周期和凋亡起重要调控作用.

Abstract：

Objective To investigate the role of miR-221/222 in inhibiting endoplasmic reticulum stress-induced human hepatocarcinoma cells apoptosis. Method miR-221/222 mimics and inhibitors were used to mimic or block the function of endogenous miR-221/222 respectively. Western blot and flow cytometry were used to test the effects of miR-221/222 on cell cycle and apoptosis under endoplasmic reticulum stress in human hepatocellular carcinoma cells. Results Endoplasmic reticulum stress resulted in miR-221/222down-regulation in human hepatocellular carcinoma cells. miR-221/222 mimics and inhibitors inhibited and promoted respectively endoplasmic reticulum stress-mediated p27Kip1 induction. Moreover, p27Kip1 suppression not only resulted in reduction in the fraction of G1 phase cells, but also promoted the endoplasmic reticulum stress-mediated apoptosis in human hepatocellular carcinoma cells. Conclusions miR-221/222 were downregulated by endoplasmic reticulum stress in human hepatocellular carcinoma cells, which subsequently protected human hepatocellular carcinoma cells against endoplasmic reticulum stress-induced apoptosis through p27KiP1 regulation.     

尿激酶型纤溶酶原激活物基因转染骨髓源性肝干细胞移植对肝纤维化大鼠肝细胞再生的影响

目的 探讨尿激酶型纤溶酶原激活物(uPA)基因修饰骨髓源性肝干细胞(BDLSC)移植对四氯化碳(CCl4)诱导肝纤维化大鼠肝细胞再生的影响.方法 纯系Fisher 344雄性大鼠10只,为BDLSC供体大鼠.体外将AduPA转染雄性大鼠BDLSC.纯系Fisher 344雌性大鼠36只,均分为正常组(皮下注射橄榄油)、模型组(CCl4造模,尾静脉注射0.9%氯化钠)、BDLSC组(CCl4造模,尾静脉输入BDLSC)、转基因组(CCl4造模,尾静脉输入转基因BDLSC).检测大鼠肝功能和肝组织胶原面积.半定量RT-PCR方法检测大鼠肝组织肝细胞生长因子(HGF)及其受体c-met mRNA表达水平.免疫组织化学法检测大鼠肝组织增殖细胞核抗原(PCNA)蛋白表达.结果 正常组、模型组、BDLSC组、转基因组肝组织胶原染色面积分别为0.12%±0.03%、14.49%±1.40%、8.25%±0.82%、5.12%±0.40%,组间差异均有统计学意义(P值均<0.05).与模型组和BDLSC组相比,转基因组大鼠肝功能明显改善,血清透明质酸(HA)、血清Ⅲ型前胶原(PCⅢ)、肝组织羟脯氨酸含量明显降低,肝组织HGF和c-met mRNA水平均显著上调,PCNA蛋白表达显著增加.结论 uPA基因修饰BDLSC移植可能诱导肝细胞增殖,从而改善CCl4诱导肝纤维化大鼠的肝功能.

Abstract：

Objective To explore the effects of urokinase type plasminogen activator (uPA) gene-modified bone marrow-derived liver stem cells ( BDLSC) transplantation on hepatocyte regeneration in CCl4-induced liver fibrosis rats. Methods Ten male Fisher 344 rats were donor rats of BDLSC. The BDLSC of male rat was transfected with AduPA. Thirty-six female Fisher 344 rats were equally divided into normal group (injected subcutaneously with olive oil) , model group (CCl4 induced the model, injected through tail vein with 0. 9% sodium chloride), BDLSC group (CCl4 induced the model, injected through tail vein with BDLSC) and gene transfected group (CCl4 induced the model,injected through tail vein with gene transfected BDLSC). Liver function and area of collagen were observed. The expression of hepatic growth factor ( HGF) and its receptor c-met mRNA in rats' liver tissues were tested by semiquantitative RT-PCR. The expressions of proliferating cell nuclear antigen (PCNA) in rats' liver tissues were determined by immunohistochemistry staining. Results The areas of collagen in normal group, model group, BDLSC group and gene transfected group was 0. 12% ± 0.03%, 14. 49%±1.40%, 8. 25%±0. 82% and 5. 12%±0. 40% accordingly, there were significant differences between groups (P<0. 05). Compared with model group and BDLSC group, the liver function of gene transfected group significantly improved, the serum levels of hyaluronic acid (HA),procollagen Ⅲ (PCⅢ) and the content of hydroxyproline in liver tissues decreased dramatically. The expression of HGF and c-met at mRNA levels were up-regulated significantly, and the expression of PCNA protein in liver tissues increased obviously. Conclusion uPA gene-modified BDLSC transplantation may induce proliferation of hepatocytes, and then improve the liver functions of fibrotic rats induced by CCl4.     

Expression of IGF-Ⅱ in early experimental hepatocellular carcinomas and its significance in early diagnosis

AIM: To investigate the serum level and expression of insulingrowth factor Ⅱ (IGF-Ⅱ) in liver tissues of rats with earlyexperimental hepatocellular carcinomas (HCC) and itssignificance in early diagnosis.METHODS: Early experim ental hepatocellular carcinomaswere induced by diethylnitrosamine (DENA) in 180 male SDrats. Another 20 male SD rats served as control. The IGF-Ⅱserum level was measured by ELISA. Immunohistochemistryand electron microscopic immunohistochemistry were usedto observe the expression of IGF-Ⅱ in normal and tumorliver tissues and its ultrastructural location in malignanthepatocytes. The expressions of IGF-Ⅱ in human hepatomacell lines HepG2, SMMC7721 and human embryonic livercell line L-02 were measured by immunocytochemistry. IGF-Ⅱ mRNA level was studied by in situ hybridization.RESULTS: IGF-II was expressed in the cytoplasm of bothsinusoidal cells in paracancerous cirrhotic liver tissue andmalignant hepatocytes in early experimental HCC tissues.Gold particles were seen on the rough endoplasmic reticulumand the mitochondrion in malignant hepatocytes. IGF-Ⅱ wasexpressed in the human hepatoma cell lines. The mRNAlevel of IGF-Ⅱ was higher in rat liver tumor tissue than innormal rat liver tissue. The serum IGF-Ⅱ level of the earlyexperimental HCC group was 34.67±10.53 ng@ml-1 and thatof the control group was 11.75±5.84 ng@ml-1. The rank sumtest was used for statistical analysis. There was a significantdifference between the two groups (P＜0.01).CONCLUSION: During the induction of early experimentalHCC by DENA, IGF-Ⅱ may promote hepatocytic proliferationvia a paracrine mechanism in the pre-cancerous stage. Whenhepatocytes are transformed into malignant cells, they maysecrete IGF-Ⅱ and promote malignant cell proliferation byan autocrine mechanism. IGF-Ⅱ may be a possible biologicalmarker in the early diagnosis of HCC.     

miRNA沉默缺氧诱导因子1α基因对HepG2细胞增殖的抑制作用

目的 探讨以miRNA沉默缺氧诱导因子1 α(HIF-1 α)基因,观察其对肝癌细胞株(HepG2细胞)增殖的抑制作用.方法 构建靶向HIF-1 α基因miRNA干扰载体,转染HepG2细胞,以定量PCR和Western blot分析靶基因和蛋白的表达;构建含缺氧反应元件双荧光素酶报告基因检测转染后相对光单位值;酶联免疫吸附法定量检测血管内皮生长因子和血管生成素2的表达;细胞凋亡及增殖周期以流式细胞仪和膜联蛋白/碘化丙啶(V-FITC/PI)双重染色法分析.采用t检验和两因素方差分析及q或q'检验分析计量资料.结果 HepG2细胞在转染HIF-1 αmiRNA后72 h,HIF-1 α在转录和翻译水平上分别下降87%和56%双荧光素酶报告基因减少46%,血管内皮生长因子和血管生成素2分别减少54%和36%;癌细胞凋亡率为22.46%±0.61%(P＜0.01),G1和S期比例分别为61.49%±1.12%和22.40%±0.58%;联合阿霉素处理后,凋亡率增至36.99%±0.88%,G1、S期比例分别为65.68%±0.91%和19.47%±1.34%.结论 HIF-1 α miRNA可抑制HIF-1 α基因表达,联合阿霉素有效调控肝癌细胞周期,促进凋亡、抑制增殖.

Abstract：

Objective To investigate the effect of miRNA silencing HIF-1 α gene on the proliferation of HepG2 cells. Methods The eukaryotic expression plasmids of HIF- 1α miRNA and report gene containing hypoxia-reponse element were constructed and trasnfected into HepG2 cells. The expressions of HIF-1 α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF1 α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay. Results 72 h after transfection the downregulations of HIF-1 α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46 ± 0.61%(P ＜ 0.01). The cell cycle changed greatly at the ratio of G1 (61.49 ± 1.12%) and S (22.40 ± 0.58%, P ＜0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99 ± 0.88% and the ratios of G1 and S phases were upregulated to 65.68 ± 0.91% and 19.47 ± 1.34% respectively. Conclusion HIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptotosis and inhibit the cell proliferation.     

AXIN2基因在肝细胞癌中的表达及甲基化研究

目的 观察肝细胞癌(HCC)中AXIN2 mRNA表达水平,分析AXIN2基因甲基化水平对mRNA表达的影响及在HCC发生、发展中的意义.方法 收集手术切除的53例HCC和配对癌旁组织标本、7例正常肝组织标本和5种人肝癌细胞系.应用荧光定量PCR方法检测AXIN2mRNA水平的表达和AXIN2基因启动子区甲基化状态.结果 AXIN2在癌组织中的mRNA表达水平(0.1629±0.0679)低于癌旁组织(0.4155±0.2330),差异有统计学意义(Z=-2.567,P=0.010).HCC和癌旁组织中AXIN2基因甲基化水平(39.77%±3.89%和36.92%±2.81%)均高于正常肝组织(7.38%±2.40%,t 值分别=-3.663和-4.591,P值分别=0.009和0.007).5种人肝癌细胞系中AXIN2基因均呈高甲基化状态.AXIN2 mRNA表达水平与甲基化程度呈负相关(r=-0.458,P=0.032).TNMⅢ期HCC患者的AXIN2甲基化水平高于TNM Ⅰ和Ⅱ期患者(P=0.008).结论 AXIN2 mRNA表达水平下降与其高甲基化状态相关,AXIN2 mRNA低表达和启动子区异常甲基化可能是HCC发生、发展的重要机制之一.

Abstract：

Objective To investigate AXIN2 mRNA expression level in hepatocellular carcinoma (HCC) , and to analyze the effect of AXIN2 gene methylation status on its mRNA expression and HCC genesis and development. Methods Fifty-three surgical excised HCC specimens and paired adjacent non-cancerous specimens, seven normal liver specimens and five HCC cell lines were collected. The expression of AXIN2 at mRNA level and the methylation status of AXIN2 gene promoter were determined by quantitative PCR. Results The expression of AXIN2 mRNA was lower in HCC tissues (0.1629 + 0.0679) than that in adjacent non-cancerous tissues (0. 4155 + 0. 2330), and there was significant difference (Z= -2. 567, P = 0. 010). The methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues (39. 77% ±3. 89%, and 36. 92% ±2. 81%) was significantly higher than that in normal liver tissues (7. 38% ±2. 40% , t=-3. 663 ,P = 0. 009;t= -4. 591 ,P = 0. 007).AXIN2 gene was hypermethylated in all five HCC cell lines. There was a negative correlation between AXIN2 mRNA expression level and the degree of methylation ( r = -0. 458, P = 0. 032). The methylation level was higher in TNM Ⅲ patients of HCC than that in TNM Ⅰ and Ⅱ patients (P =0.008). Conclusion The down-regulation of AXIN2 gene mRNA expression is correlated with its hypermethylation status. The low expression of AXIN2 mRNA and the abnormal methylation of promoter may be one of the important mechanism of HCC genesis and development.     

MicroRNA-377和组蛋白甲基转移酶SMYD3在肝细胞癌中的表达及两者的相关性

目的:探讨microRNA-377(miR-377)与组蛋白甲基转移酶SMYD3在肝癌中的表达规律及与肝癌的相关性.方法:利用实时定量PCR分别检测不同肝组织及肝细胞系中miR-377表达水平, 应用实时定量PCR和Western blot分别检测不同肝组织及肝细胞系中SMYD3 mRNA和蛋白水平的表达情况. 通过转染miR-377模拟物上调其在肝癌细胞株HepG2中表达后, 应用实时定量PCR、Western blot分别检测转染前后HepG2中SMYD3 mRNA和蛋白表达的变化.结果:MiR-377 mRNA在人肝癌旁组织和肝癌组织中的表达较正常肝脏明显降低(0.331±0.059, 0.139±0.064 vs 0.874±0.178, 均P <0.05);在HepG2中的表达较L-02明显降低(0.145±0.021 vs 0.868±0.194, P <0.05).SMYD3 mRNA和蛋白质在人肝癌旁组织和肝癌组织中的表达较正常肝脏明显升高(mRNA: 3.836±0.137, 5.836±0.965 vs 1.235±0.332;蛋白: 0.381±0.020, 0.484±0.030 vs 0.252±0.015;均P <0.05). SMYD3 mRNA和蛋白质在肝癌细胞系HepG2中的表达较正常肝细胞系L-02明显升高(mRNA: 0.845±0.047vs 0.348±0.134;蛋白: 0.575±0.008 vs 0.259±0.007, 均P <0.05). 转染miRNA-377模拟物上调HepG2中miR-377表达后转染组SMYD3mRNA和蛋白表达较空白组和阴性对照组均明显下降(mRNA: 0.125±0.010 vs 0.857±0.163, 0.779±0.167;蛋白: 0.092±0.026 vs0.347±0.040, 0.383±0.054, 均P <0.05).结论:miRNA-377在肝癌中表达明显下调,其靶基因SMY D3表达上调;表达下调的miRNA-377丧失对SMYD3表达的抑制可能是肝癌发生的重要机制.     

miR-155在肝细胞癌中的表达及对肝癌细胞增殖的影响

目的:检测微小RNA-155(miR-155)在肝癌组织中的表达并分析其对肝癌细胞增殖和细胞凋亡的影响.方法:采用TagMan MGB探针法荧光定量P C R分析42例原发性肝癌及对应的癌旁组织miR-155的表达;利用miR-155反义寡核苷酸(ASO-miR-155)降低肝癌细胞HepG2和SMMC7721中miR-155的表达;利用MTT比色法检测肝癌细胞增殖的变化,并通过流式细胞技术检测肝癌细胞早期凋亡情况.结果:42例肝癌及癌旁组织标本中,miR-155在52%(22/42)肝癌组织中的表达明显高于癌旁组织(P<0.05);利用脂质体将ASO-miR-155转染肝癌细胞HepG2和SMMC7721后,miR-155的表达明显降低,肝癌细胞HepG2和SMMC7721生长受到明显抑制;并且细胞的早期凋亡明显增加.结论:miR-155在肝癌组织中过表达,降低其表达能明显抑制肝癌细胞的生长并诱导细胞早期凋亡,miR-155有可能成为肝癌治疗的新靶点.     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.     

PeroxiredoxinⅡ基因在肝癌组织中的表达及意义

目的观察peroxiredoxinⅡ（PrxⅡ）mRNA和蛋白在黄曲霉毒素B1（AFB1）诱发树鼩HCC形成过程中的表达变化,并在人肝癌癌旁组织和正常肝组织中进行验证,以探讨PrxⅡ在肝癌形成过程中的作用。方法应用RT-PCR和Western blot方法,检测6例AFB1诱发的树鼩肝癌、癌旁组织和这些动物肝癌发生前的活检肝组织,以及6例对照组动物相应时期的肝活检组织PrxⅡ在mRNA水平和蛋白水平的表达情况,并在18例人肝癌及其相应癌旁组织以及17名正常人肝组织进行验证。结果PrxⅡ在树鼩肝癌组织中的mRNA和蛋白质表达水平均明显高于相应的癌旁和癌前组织,也高于对照组同期活检肝组织（P〈0．05）;人肝癌组织中PrxⅡ的mRNA和蛋白表达改变和树鼩相符,即肝癌中的表达高于癌旁组织和正常肝组织（P〈0．05）。结论PrxⅡ基因可能与肝癌的发生发展有关,并有可能成为防治肝癌的分子靶标。     

The influence of fibrate initiation on INR and warfarin dose in patients receiving chronic warfarin therapy

This study was aim to investigate the specific mechanisms of miR-411 in vein wall fibrosis remodeling. Vein wall fibrosis injury-induced deep venous thrombosis (DVT) rat model was well established. The expression of miR-411 at mRNA levels and Collagen I, hypoxia-inducible factor (HIF)-1α together with matrix metalloproteinase (MMP)-2 at protein levels in vein wall tissues and vascular smooth muscle cells (VSMCs) following transfection were determined using quantitative real-time PCR (qRT-PCR) and western blotting, respectively. Luciferase reporter assay was used to confirm the potential target of miR-411. MiR-411 mimic injected into rat model of DVT was to verify the role of miR-411 in vein wall fibrosis in vivo. MiR-411 was downregulated while Collagen I, HIF-1α and MMP-2 was upregulated in vein wall tissues and VSMCs obtained from rat model of DVT. MiR-411 overexpression in VSMCs separated from rats of vascular remodeling group (VR-VSMCs) upregulated miR-411, HIF-1α and inhibited cell proliferation and Collagen I expression, while miR-411 knockdown in VSMCs isolated from healthy rats (Control-VSMCs) reversed the effects. Furthermore, luciferase reporter assay demonstrated that HIF-1α was a target of miR-411. In addition, overexpression of miR-411 and HIF-1α in VR-VSMCs promoted HIF-1α, Collagen I expression and cell proliferation, however, tissue inhibitor of metalloproteinase (TIMP)-2 treatment led to adverse trends. MiR-411 mimic injected into rat model of DVT could suppress vein wall fibrosis in vivo. MiR-411 inhibited vein wall fibrosis by downregulating MMP-2 mediated by HIF-1α.     

miR-383靶向Notch1对结肠癌HCT116细胞增殖和迁移的影响

目的 分析miR-383和Notch1在结肠癌中的作用及机制.方法 选择2017年4月至2020年3月在河北北方学院附属第一医院经病理检查确诊为结肠癌的74例患者,收集其经手术切除的肿瘤组织及癌旁正常组织(距肿瘤边缘>3 cm).采用TargetScan预测miR-383的潜在靶基因,采用双荧光素酶报告基因实验、逆转录...     

Expression of oncogenes in human liver disease

To elucidate the role of oncogene expression in hepatocarcinogenesis, we examined the expression of 8 cellular oncogenes by dot blot and/or northern blot analysis in neoplastic, cirrhotic and non-cirrhotic human liver tissues obtained at surgery. Significantly higher levels of c-myc gene expression were observed in tissues of hepatocellular carcinoma (HCC) and adjacent cirrhotic tissues than in apparently normal liver tissues or those of chronic hepatitis (normal-chronic hepatitis). There was a tendency to higher c-myc mRNA levels in HCC than in liver cirrhosis. However, when tumorous and adjacent cirrhotic tissues from the same patient were compared, c-myc mRNA levels were not consistently higher in HCC. No significant differences in mRNA levels of c-fos, N-myc, N-ras, Ha-ras, c-erbA, c-erbB and c-abl were observed among the HCC, cirrhosis and normal-chronic hepatitis groups. Although the significance of increased c-myc gene expression in liver cirrhosis and HCC is still not known, it is conceivable that the persistent elevation of c-myc gene expression in cirrhosis contributes to the development of HCC.     

MiR-503 promotes wound healing of diabetic foot ulcer by targeting FBN1

Objective: To highlight the relationship between miR-503 and wound healing of diabetic foot ulcer(DFU). Methods: Microarray analysis was used to detect the dysregulated miRNAs between the DFU tissues and normal tissues. The expression of miR-503 in tissues and serum of patients with DFU was detected by qRT-PCR technique. Then, CCK-8 assay was applied to determine the cell proliferation. TUNEL assay was used for assessing the apoptosis of cells after treatment with miR-503. Possible correlation between miR-503 and fibillinl(FBN1)was predicted according to data accessed on RNA22 website online, and was detected for confirmation by luciferase reporter assay. Results: Microarray analysis showed that miR-503 was significantly decreased in the DFU tissues compared with normal tissues. While marked increase in the expression of miR-503 in tissues and scrum of patients with DFU was confirmed by qRT-PCR technique. Then, CCK-8 assay indicated that transfection of miR-503 mimic obviously accelerated the cell proliferation. However, TUNEL assays suggested that miR-503 mimic inhibited the apoptosis of cells to improve the survival of fibroblasts.Besides. miR-503 AMO played a role in fibroblasts of DFU tissues exactly countering to miR-503 mimic treatment. It was predicted that MiR-503 is a complementary to the FBN1 by RNA22. Besides, SiRNA-FBN1 promoted the proliferation, but brought down the apoptosis of fibroblasts. Conclusions: MiR-503 regulates the function of fibroblasts and wound healing of patients with DFU by targeting FBN I directly which provids a novel and critical target for diagnosis and treatment of DFU.     

MiR-122调控肝癌遗传印记基因PEG10的实验研究

目的 探讨遗传印记基因PEG10功能的异常与miRNA失调控的相关性.方法 通过生物信息学网站TargetScan预测可能参与PEG10调控的miRNA分子,筛选到miR-122.通过基于taqman探针的实时定量逆转录聚合酶链反应,比较miR-122在原代正常肝细胞和3株肝癌细胞株(Huh7,Hep3B,HepG2)中的表达差异.将miR-122的分子前体转染HepG2细胞,观察转染前后PEG10在mRNA和蛋白水平的表达变化.多组均数问比较采用秩和检验,配对样本组间差异比较采用t检验.结果 生物信息学预测结果显示,miR-122可能参与了PEG10的调控.实时定量逆转录聚合酶链反应结果显示,PHHC、Huh7,HepG2、Hep3B细胞miR-122的表达量(2~(-△△Ct)值)分别为1.0578±0.0975、0.5600±0.0632、0.0068±0.0012、0.0058±0.0008,H=9.667,P＜0.05.与原代正常肝细胞相比,miR-122在肝癌细胞株中表现为完全(Hep3B、HepG2细胞)或部分缺失(Huh7细胞),其表达水平与PEG10呈负相关.将miR-122分子前体转入HepG2细胞后,PEG10在mRNA水平并未显示出明显的下调,但Western blot结果提示miR-122分子前体明显抑制了PEG10蛋白的表达.结论 miR-122参与了遗传印记基因PEG10的调控,其调控主要发生在翻译阶段,即蛋白质水平.miRNA的失调控与肝癌的发生密切相关,其功能的异常可能早于受其调控的癌基因或抑癌基因的改变,是肝癌发生的早期事件,这为肝癌的早期预警和基因治疗提供了新思路.     

微小RNA miR-199a／a^＊模拟物转染肝癌细胞HepG2中Met原癌基因的表达

目的探讨在肝癌细胞系HepG2中,Met是否为miR-199a／a^＊的靶基因。方法将miR-199a／a^＊的模拟物及阴性对照物分别转染至HepG2细胞中,转染后48h及72h分别提取RNA及蛋白行RT—PCR及Western Blot检测Met的表达。结果与阴性对照相比,转染Up-miR-199a^＊组的MetmRNA（P〈0．05）和蛋白（P〈0．001）表达水平均显著下调;而Up—miR-199a组与阴性对照组相比无统计学差异。结论肝癌细胞HepG2中miR-199a^＊负调控Met的mRNA及蛋白表达。     

微RNA 18与B细胞易位基因2在肝癌细胞中差异表达相关性的研究

目的 研究微RNA 18(miR-18)对肝癌细胞HepG2基因表达谱的影响,预测其靶基因,初步探讨miR-18与抗增殖基因B细胞易位基因2(BTG2)两者在肝癌发生过程中差异表达的相关性.方法 利用基因表达谱芯片技术筛选HepG2的微RNA差异表达谱,应用生物信息学方法预测表达明显上调的miR-18的靶基因,初步筛选出直接调控的靶基因为抗增殖基因BTG2;应用RT-PCR和Northern blot法分析BTG2正常与肝癌组织中的表达情况.结果 生物信息学分析结果显示miR-18分子调控的下游靶基因有609个,涉及细胞增殖、分化和凋亡,转录调节等众多生理和病理过程;抗增殖基因BTG2在肝癌组织和细胞中呈明显低表达.结论 miR-18在肝癌细胞中表达明显上调,并可能负性调控抗增殖基因BTG2在肝癌细胞中的表达,两者共同在肝癌细胞增殖方面发挥着重要作用.