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.     

微小RNA-223及其标靶基因c-myc在肝癌发病中的作用

目的 探讨微小RNA-223(miR-223)对原癌基因c-myc的调控及其在肝癌发病中的作用.方法 通过实时定量聚合酶链反应和Western blot检测正常肝脏组织、癌旁组织、肝癌组织及肝癌HepG2细胞、胎肝L02细胞中miR-223和c-myc的mRNA和蛋白质表达水平.构建miR-223模拟物(mimics)上调肝癌细胞HepG2中miR-223表达后,实时定量聚合酶链反应和Western blot 检测HepG2细胞中c-myc表达水平的变化.分别用独立样本t检验和单因素方差分析进行两组及多组数据间的比较,P＜0.05为差异有统计学意义.结果 miR-223在正常肝组织、癌旁组织和肝癌组织中的相对表达量分别为0.055±0.015、0.030±0.008和0.020±0.016,肝癌组织低于正常肝组织(t=-0.031,P＜0.05).miR-223在HepG2细胞中的表达较L02细胞下调(0.005±0.003比0.011±0.006,t=12.74,P＜0.01).c-myc mRNA在正常肝组织、癌旁组织和肝癌组织中的相对表达量分别为0.029±0.023、0.136±0.071和0.425±0.026,肝癌组织高于正常肝组织(t=-0.317,P＜0.05);c-myc蛋白在正常肝组织、癌旁组织和肝癌组织中的相对表达量分别为0.137±O.015、0.299±0.033和0.439±0.027,差异有统计学意义(F=103.35,P＜0.01).miR-223 mimics转染HepG2细胞后,c-myc蛋白在空白组、转染组和阴性对照组的相对表达量分别为0.423±0.041、0.116±0.015和0.432±0.034,转染组较其他两组明显降低(F=94.93,P＜0.05).结论 miR-223在肝癌组织中表达下调,丧失其对c-myc表达的抑制而导致c-myc异常高表达可能是肝癌发生的重要机制.

Abstract：

Objective To investigate the regulatory role of microRNA-223 (miR-223) on c-myc and its role in hepatocarcinogenesis.Method miR-223 and c-myc mRNA expressions in normal tissue,paraneoplastic tissue,liver cancer tissue and liver cancer cells were tested with microRNA microarray and quantitative real-time PCR (qRT-PCR).C-myc protein expression was detected by Western blot.MiR-223mimic was transfected into HepG2 cells and the expression changes of c-myc mRNA and protein were tested with qRT-PCR and Western blot respectively.Results MiR-223 was down-regulated by 61.53% and 30.77% respectively in hepatocellular carcinoma and adjacent tissues as compared to normal liver tissues and the expression of miR-223 was also decreased in HepG2 cell as compared to fetal liver cells L02,whereas the expressions of c-myc mRNA and protein increased in paraneoplastic and HCC tissues compared with normal liver tissues.It prompts that the expressions of miR-223 and c-myc are negatively correlated.No obvious difference found among c-myc mRNA expressions after miR-223 mimics transfection.Conclusions The cmyc abnormal high-expression may play a dynamic role in hepatocarcinogenesis due to the miR-223 downregulation.     

磷脂酰肌醇蛋白聚糖-3的表达特征及其在肝癌诊断与鉴别诊断中的价值

目的 研究磷脂酰肌醇蛋白聚糖-3(GPC-3)的表达特征及其在肝癌诊断与鉴别诊断中的临床价值.方法 制作鼠肝癌模型,并按病理组织检查结果分为正常组、肝细胞变性组(变性组)、癌前病变组(癌前组)和肝细胞癌组(癌变组).以Western blot和逆转录-聚合酶链反应分别观察GPC-3蛋白质及mRNA的动态表达;以自身配对法收集术后肝癌组织,根据其组织学类型分为肝癌组、癌旁组、远癌组,以免疫组织化学法分析GPC-3表达与病理学特征的关系;以酶联免疫吸附法定量分析肝病患者外周血GPC-3表达水平,并评价其诊断效率.多个样本均数比较用单因素方差分析,组间GPC-3的表达及病理学特征比较用单因素秩和检验,血清GPC-3比较用秩和检验,率的比较采用x2检验或Fisher's exact分析,以受试者工作特征曲线下面积比较GPC-3诊断肝癌的敏感性、特异性及诊断效率.结果 在鼠肝癌形成过程中,正常、变性、癌前和癌变组GPC-3阳性率分别为0(0/6)、83.3%(15/18)、100.0%(9/9)和100.0%(9/9).人肝癌组织GPC-3阳性呈棕黄色颗粒状染色,定位于胞质和细胞膜,肝癌、癌旁和远癌组的阳性率分别为80.6%、41.7%和0,肝癌组明显高于远癌组(x2=48.56,P＜0.01)和癌旁组(x2=11.455,P＜0.01);癌旁组明显高于远癌组(x2=18.94,P＜0.01).GPC-3表达与肿瘤分化程度和数目间未见明显相关,与瘤体大小有关(Z=2.941,P＜0.01).肝病患者血清GPC-3异常主要见于肝癌(52.8%,65/123),且在不同性别、年龄、甲胎蛋白水平、肿瘤数目、Child分级和肝外转移者间未见明显差异;但肝癌＜3.0cm组明显高于≥3.0cm组(x 2=6.318,P＜0.05); HBsAg阳性组明显高于阴性组(x 2=23.362,P＜0.01).GPC-3与甲胎蛋白(＞20 μg/L)联合诊断肝癌的敏感度、特异度、准确度、阳性预测值和阴性预测值分别为87.00%、79.66%、82.17%、69.03%和92.16%.结论 GPC-3表达与肝癌密切相关,其表达的检测有助于肝癌早期诊断和鉴别诊断.

Abstract：

Objective To investigate the expression features of glypican-3 (GPC-3)and its diagnostic and differential values in hepatocellular carcinoma (HCC). Methods Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases. Results The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precanceration and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule-like staining localized in membrane and cytoplasm in human HCC.The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P＜0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of minor except of rumor size (Z=2.941, P＜0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, mm or number, Child classification or extrahepatic metastasis except of rumor size (x2 = 6.318, P＜0.05) and HBV infection (x2 = 23.362,P＜0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. Conclusions GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.     

PIWI蛋白在人类结肠癌组织中的表达及临床意义

目的 分析PIWIL1、PIWIL3和PIWIL4三种蛋白在结肠癌组织和癌旁组织中的表达与临床意义.方法 选用106例结肠癌患者的癌组织和癌旁组织标本,分别制成62点和150点2份组织芯片,采用免疫组织化学方法检测结肠组织芯片中PIWIL1、PIWIL3和PIWIL4的表达.采用SPSS 17.0统计学软件,不同组织间PIWI蛋白表达差异采用配对t检验或配对Wilcoxon检验,相关性采用Pearson或Spearman等级相关分析.结果 PIWIL1、PIWIL3、PIWIL4在结肠癌组织中的表达均显著高于癌旁组织(P＜0.01).PIWIL1、PIWIL3、PIWIL4在结肠癌和癌旁组织中的表达均呈现两两显著正相关(均P＜0.01).癌组织PIWIL1在低分化组的表达显著高于高分化组(t=-2.840,P＜0.01).癌组织PIWIL3表达随着临床分期的升级呈现显著升高(F=3.112,P＜0.05).癌组织PIWIL3和PIWIL4在有远处转移组的表达显著高于无远处转移组(t=-3.349,P＜0.01;t=-2.168,P＜0.05).癌组织PIWIL3和PIWIL4的表达均与结肠癌的发生显著相关(P值均＜0.01).结论 PIWIL1、PIWIL3、PIWIL4在结肠癌组织中的表达与结肠癌的分化、临床分期、有无远处转移等临床指标显著相关,且PIWIL3和PIWIL4的表达是结肠癌发生的独立相关因素,有望进一步研究探讨其是否能作为结肠癌早期诊断的分子标志物及分子治疗靶点.

Abstract：

Objective To study the expression of PIWIL1, PIWIL3 and PIWIL4 in human colon cancer and its clinical significance. Methods We collected cancerous tissues and its adjacent tissues of 106 patients with colon cancer, two tissue microarrays were constructed, with 62 and 150 points respectively. We studied the expression of PIWIL1, PIWIL3 and PIWIL4 through immunohistochemistry. Results The expression of PIWIL1, PIWIL3 and PIWIL4 were significantly higher in cancerous tissues than those in adjacent tissues (P＜0. 01). In cancerous tissues and its adjacent tissues, postive correlation were seen among PIWIL1, PIWIL3 and PIWIL4 expression (P＜0. 01). PIWIL1 expression was significant higher in low differentiation group than that in high differentiation group (t =- 2. 840, P＜0.01 ). PIWIL3 expression was higher in high clinical stage than that in low clinical stage (F= 3. 112, P＜0.05). The expression of PIWIL3 and PIWIL4 were significantly higher in patients with colon cancer with distant metastasis than those without distant metastasis (t= -3. 349, P＜0.01 ; t = - 2. 168, P＜0. 05). PIWIL3 and PIWIL4 expression were correlated with occurring of colon cancer (P＜0. 01). Conclusions The expressions of PIWIL1,PIWIL3 and PIWIL4 in colon cancer were correlated with the differentiation, clinical stage and distant metastasis of colon cancer. PIWIL3 and PIWIL4 expression were two independent related factors of occurring of colon cancer, which would be furtherly investigated to be served as novel markers for early diagnosis and promising molecular targets for colon cancer therapy.     

Predictive value of quantitative contrast-enhanced ultrasound in hepatocellular carcinoma recurrence after ablation

AIM: To investigate the relationship between contrastenhanced ultrasound(CEUS),basic fibroblast growth factor(b FGF),endothelin-1(ET-1),and hepatocellular carcinoma(HCC) recurrence after ablation. METHODS: A total of 51 HCC patients(38 males and 13 females) who received radiofrequency ablation in our hospital from June 2012 to July 2014 were enrolled in this study. The patients were divided into two groups: recurrence group and non-recurrence group. Routine abdominal examination was first performed in the horizontal position. Then the patients underwent CEUS and immunohistochemical staining before receiving radiofrequency ablation. All patients were followed-up every three months for one year.The results of CEUS and serum tumor marker levels were evaluated and combined together to estimate HCC recurrence and metastasis. Patients were divided into two groups: recurrence group and non-recurrence group. Quantitative parameters of CEUS and tumor expression levels of b FGF and ET-1 were compared between the two groups,respectively. Binary logistic regression analysis was used to analyze the relationship between CEUS quantitative parameters,expression levels of ET-1 and b FGF,and HCC recurrence after ablation. RESULTS: Based on the quantitative parameters of CEUS before patients received radiofrequency ablation,the levels of tumor rise time(t RT),tumor time to peak(t TTP),tumor peak intensity(t PI) and tumorparenchymal peak intensity(t-p PI) in the recurrence group were significantly lower than those in the nonrecurrence group(16.6 ± 6.1 vs 23.2 ± 7.0,P = 0.000; 41.2 ± 10.2 vs 59.6 ± 14.2,P = 0.000; 23.8 ± 6.7 vs 31.4 ± 6.4,P = 0.000; 7.1 ± 3.4 vs 14.6 ± 7.4,P = 0.000; respectively). The expression levels of b FGF in the recurrence group were significantly higher than those in the non-recurrence group(P 0.05). Levels of t TTP showed a significant inverse correlation with the level of b FGF in tumors(r =-0.312,P = 0.037). The Binary logistic regression analysis results revealed that the levels of t RT,t TTP,t PI and the level of b FGF were associated with HCC recurrence after radiofrequency ablation(P 0.05). CONCLUSION: CEUS is a noninvasive and effective method for evaluating the angiogenesis of HCC,and predicting its recurrence and prognosis.     

Relationship between survivin expression and recurrence, and prognosis in hepatocellular carcinoma

AIM： To study the expression of the inhibitor of apoptosis protein survivin in hepatocellular carcinoma （HCC）, and its correlation with clinicopathological factors, cell proliferation, recurrence and prognosis after hepatectomy. METHODS： Immunohistochemical staining of survivin and Ki-67 was performed by the standard streptavidin- peroxidase technique on paraffin sections of 55 cases of HCC. RESULTS： The positive rate of survivin in HCC was 52.7% （29/55）. Significant correlation was found between survivin expression with portal vein thrombi and intrahepatic matastasistic nodes （P 〈 0.05）. The recurrent rate in survivin-positive HCC was significantly higher than that in survivin-negative HCC after hepatectomy, the 1- and 3-year survival rate in patients with survivin-positive tumors was significantly lower than that in patients with survivin-negative tumors （58.62 and 10.34% vs 76.92 and 30.77%, P 〈 0.05, log-rank test）. The proliferation index （Ki-67） in survivin-positive HCC （33.83% ± 18.90%） was significantly higher than that in survivin-negative HCC （19.60% ± 19.35%） （P 〈 0.05）. CONCLUSION： Survivin may play an important role in progression of HCC by promoting cell proliferation, and may be positively correlated with high risk of disease recurrence and poor prognosis in HCC. Its expression may serve as a prognostic factor for patients with HCC after hepatectomy.     

蛋白酶激活受体在腹泻型肠易激综合征患者结肠黏膜组织中的表达及意义

目的 研究蛋白酶激活受体(PAR)家族在腹泻型肠易激综合征(D-IBS)患者结肠黏膜中的表达变化情况.方法 入选28例D-IBS患者及18名健康对照者,经结肠镜钳取乙状结肠黏膜组织,采用Western印迹法和实时荧光定量PCR法观察蛋白酶激活受体超家族的表达情况.应用SPSS 18.0软件进行统计分析,两组间差异采用Mann-Whitney U检验.结果 PAR1在D-IBS患者和对照组结肠黏膜中的表达差异无统计学意义(P=0.300).PAR2和PAR4在D-IBS患者的结肠黏膜中均表达增加,其灰度值分别为0.99±0.67和0.37±0.14,显著高于对照组的0.63±0.38和0.25±0.11(P值分别=0.038和0.013).实时荧光定量PCR显示PAR mRNA的表达与蛋白水平相一致.结论 PAR2、PAR4的表达在D-IBS患者结肠黏膜中明显增高,可能在D-IBS疾病过程中发挥某些作用.

Abstract：

Objective To investigate the expression of protease-activated receptors (PARs) in colon mucosal tissue of patients with diarrhea-predominant irritable bowel syndrome (D-IBS). Methods Colon mucosa tissues were obtained from 28 D-IBS patients and 18 normal controls by colonoscopy.The expression of PARs was detected using Western blotting and real-time PCR. Data were analysed by SPSS 18. 0 softwave, and comparisons between groups were done using Mann-Whitney U test.Results There was no difference in expression of PAR1 between D-IBS patients and normal controls (P=0. 300). The expressions of PAR2 and PAR4 were higher in D-IBS patients than those in normal controls (0.99±0.67 vs 0.63±0.38, P=0.038 and 0.37±0. 14 vs 0.25±0. 11, P=0.013,respectively). The results of real-time PCR were in accordance with those of Western blotting.Conclusion The increased expressions of PAR2 and PAR4 in colon mucosa of D-IBS patients indicate that these two PAR receptors may be involved in the process of D-IBS.     

癌组织浸润的FoxP3十调节性T淋巴细胞与CD34表达及肝癌患者的预后相关

目的 探讨FoxP3+调节性T淋巴细胞(Tregs)在肝癌患者癌及癌旁组织中的表达及其对预后的影响.方法 收集55例肝癌患者术后癌及癌旁组织标本,免疫组织化学检测肝癌及相应癌旁组织Tregs数量及癌组织CD34的表达情况.Speaman相关分析法分析Tregs与患者各种临床病理因素的相关性;据中位数将Tregs绝对值分为高低组,Kaplan-Meier分析Tregs高低组的总生存期和无瘤生存期,Log rank检验差异性;Cox回归法分析临床病理因素对预后的影响.结果 肝癌组织中每高倍视野的Tregs数量为10.8 (4.4～ 19.4)个,与相应癌旁组织1.4 (0.6～3.2)个相比,明显升高(P＜0.01).癌组织Tregs与相应癌组织血管内皮细胞标志物CD34的表达呈正相关(r=0.279,P＜0.05);癌及癌旁组织Tregs与其他临床病理因素均无相关性(P＞ 0.05).癌组织高Tregs组患者中位生存时间(18.0个月)明显短于低Tregs组(25.0个月),术后5年累积生存率及无瘤生存率较低(P值均＜0.05).Cox回归分析显示癌组织中Tregs数量是影响肝癌预后的独立因素(累积生存率:风险比=3.310,95％可信区间为1.368 ～ 8.007,P＜0.0l ;无瘤生存率:风险比=2.666,95％可信区间为1.321 ～ 6.394,P＜0.01).结论癌组织中Tregs数量可作为评估手术治疗肝癌患者预后的免疫学指标,Tregs瘤内浸润与肿瘤组织血管生成相关.     

肿瘤拒绝抗原1在肝硬化和肝癌组织中的表达及其与肝癌临床病理学特征的关系

目的 探讨肿瘤拒绝抗原1(TRA1)在肝细胞癌(HCC)和肝硬化组织中的表达水平及其与HCC临床病理学特征的关系.方法 采用RT-PCR法、Western blot和免疫组织化学法分别检测了HCC和肝硬化组织中TRA1 mRNA和蛋白的表达水平,并与HCC临床病理学特征进行相关分析.RT-PCR和Western blot结果采用单因素方差分析;免疫组织化学结果分析采用Fisher's确切概率法;相关性分析采用Spearman等级相关.结果 经对RT-PCR结果分析,HCC和肝硬化组织中TRA1 mRNA表达水平高于正常肝组织水平(F值分别为20.821和12.311,P值均＜0.05).Western blot检测结果显示,HCC和肝硬化组织中TRA1蛋白表达水平也高于正常肝组织(F值分别为21.231和20.125,P值均＜0.05).经免疫组织化学分析,TRA1蛋白在正常对照组、肝硬化组和HCC组的表达逐渐增强,阳性表达率分别为57.14%、78.95%和93.75%.其表达水平与HCC分化程度呈负相关(r=-0.4655,P＜0.01);与HCC患者TNM分期呈正相关(r=0.5157,P＜0.01).结论 TRA1在肝硬化和HCC中的过表达可能与肝癌的发生和发展有关,并有可能成为一个潜在的HCC早期诊断标志物;TRA1的检测有助于判断HCC的分化程度,与HBV的感染有一定关系,同时可作为判断预后的指标.     

Phase tissue intercellular adhesion molecule-1 expression in nude mice human liver cancer metastasis model

AIM To study the phase cancer tissue intercellular adhesion molecule-1 (ICAM-1) expression of human cancer metastasis model in nude mice, and to analyze the relationship between ICAM-1 expression and the metastasis and recurrence of hepatocellular cancinoma (HCC).METHODS HCC tissues from liver cancer metastasis model in nude mice (LCI-D20) was orthotopically implanted, and ICAM-1 expression in HCC tissues at different growing time were detected by immunodot blot. Tumor size, intrahepatic and extrahepatic metastasis foci were observed by naked eyes and under light microscope.RESULTS ICAM-1 was positively correlated to the tumor growing time (r=0.88, P＜0.01) and tumor size r=0.5, P＜0.05). It was higher in metastatic HCC than in nonmetastatic HCC (8.24±0.95 vs 3.03±0.51, P＜0.01). ICAM-1 content in cancer tissues increased suddenly after metastasis occurred and then maintained in a high level. ICAM-1 was also higher in multimetastasis group than in monometastasis group (10.05±1.17 vs 5.48±0.49, P＜0.05).CONCLUSION Tissue ICAM-1 could predict not only the metastasis of human liver cancer metastasis model in nude mice early and sensitively, but also the metastasis degree. So tissue ICAM-1 may be a potential index indicating the status of metastasis of HCC patients.     

Correlation of integrin β3 mRNA and vascular endothelial growth factor protein expression profiles with the clinicopathological features and prognosis of gastric carcinoma

AIM: To investigate integrin 133 mRNA and vascular endothelial growth factor (VEGF) protein expression in gastric carcinoma, and its correlation with microvascular density, growth-pattern, invasion, metastasis and prognosis. METHODS: In situ hybridization(ISH) of integrin β3 mRNA and immunohistochemistry of VEGF and CD34 protein were performed on samples from 118 patients with gastric cancer. RESULTS: The positive rate of integrin 133 mRNA in non- tumor gastric mucosa (20%) was significantly lower than that of the gastric cancer tissue (52.5%, x2 = 10.20, P ＜ 0.01). In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of integrin β3 mRNA were significantly higher than those in patients of expanding type (P ＜ 0.01), stage T1-T2 (P ＜ 0.01), non-vessel invasion (P ＜ 0.01), without lymphatic metastasis (P ＜ 0.01), without hepatic and peritoneal metastasis (P ＜ 0.01), respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of VEGF protein were significantly higher than those in patients of expanding type (P ＜ 0.01), stage T1-T2 (P ＜ 0.01), non-vessel invasion (P ＜ 0.01), without lymphatic metastasis (P ＜ 0.01), without hepatic and peritoneal metastasis (P ＜ 0.01), respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the mean MVD were significantly higher than those in patients of expanding type (P ＜ 0.01), stage T1-T2 (P ＜ 0.01), non-vessel invasion (P ＜ 0.01), without lymphatic metastasis (P ＜ 0.01), without hepatic and peritoneal metastasis (P ＜ 0.01), respectively. It was found that the positive expression rate of integrin β3 mRNA was positively related to that of VEGF protein (P ＜ 0.01) and MVD (P ＜ 0.05), meanwhile the positive expression rate of VEGF protein was positively related to NVD (P ＜ 0.05). The mean survival period in patients with positive expression of integrin β3 mRNA and VEGF, and MVD ≥ 54.9/mm2 was significantly shorter than that in patients with negative expression of integrin β3 mRNA (P ＜ 0.05) and VEGF (P ＜ 0.01), and MVD ＜ 54.9/mm2 (P ＜ 0.01). Five-year survival rate in patients with positive expression of integrin β3 mRNA and VEGF, and MVD ≥ 54.9/mm2 was significantly lower than those with negative expression of integrin β3 mRNA (P ＜ 0.05), VEGF (P ＜ 0.05), and NVD ＜ 54.9/mm2 (P ＜ 0.01). CONCLUSION: Integrin β3 and VEGF expression can synergistically enhance tumor angiogenesis, and may play a crucial role in invasion and metastasis of gastric carcinoma. Therefore, they may be prognostic biomarkers and novel molecular therapeutic targets.     

小干扰RNA沉默巨噬细胞移动抑制因子基因对肝癌细胞p27表达的影响

目的 观察巨噬细胞移动抑制因子(MIF)和细胞周期调控因子p27在肝细胞癌中表达的相互关系,探讨小干扰RNA(siRNA)沉默MIF基因对肝癌细胞p27表达的影响.方法 免疫组织化学法和荧光定量PCR法检测MIF、p27的蛋白和mRNA在肝癌及其癌旁组织中的表达情况.化学合成MIF siRNA和对照siRNA,脂质体法转染肝癌细胞PLC和Hep3B,荧光定量PCR法检测MIF和p27 mRNA在实验组及对照组中的表达情况.根据不同资料分别采用X2检验、Logistic回归分析或单因素方差分析.结果 MIF蛋白及其mRNA在肝癌组织中过表达,在癌旁组织中低表达;p27蛋白及其mRNA在癌组织中低表达,在癌旁组织中高表达.Logistic回归分析提示MIF为肝癌发生的危险因素,p27为保护因素.MIF mRNA在肝癌细胞株中过表达(F=61.036,P＜0.01),p27 mRNA在正常肝细胞L02中高表达(F=529.853,P＜0.01).经MIFsiRNA转染后,MIF mRNA在PLC及Hep3B中的表达水平降低,并且呈剂量依赖关系(F值分别为320.1和201.2,P值均＜0.01);p27 mRNA伴随MIF mRNA的降低而增加(F值分别为419.4和459.9,P值均＜0.01).结论 MIF在肝细胞癌中过表达,MIF siRNA能特异性抑制其在肝癌细胞中的表达;MIF可能参与了p27基因表达的调控.     

Cloning and expression of MXR7 gene in human HCC tissue

AIM To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using 32P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively.The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC ＜5cm was 90% (9/10) and 83.3% (5/6),respectively.CONCLUSION MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.     

HNF4a基因在肝细胞癌中的表达及其与预后的相关性

目的探讨肝细胞癌患者组织中HNF4a蛋白表达与无病生存期和总生存期的关系。方法免疫组化检测肝癌组织中HNF4a蛋白,观察172例患者肝细胞癌组织中HNF4a的表达情况;HNF4a蛋白表达水平与各临床病例参数间的关系、患者无病生存期和总生存期的关系,建立回归模型。结果 HNF4a阳性率为81.98%(141/172),HNF4a基因表达与患者性别、年龄、HBsAg、肝硬化、肿瘤大小、TNM分期以及转移复发无相关性(P0.05),与AFP、肿瘤病理分级存在相关性(P0.05)。HNF4a蛋白高表达患者中无病生存期为22个月,HNF4a低表达患者无病生存期为4个月,差异有统计学意义(P0.05)。总生存期分析显示,HNF4a蛋白表达是其风险因素(P0.05)。结论 HNF4a蛋白低表达肝癌患者生存期降低的风险显著升高,HNF4a表达与肝细胞发生发展密切相关,有可能是肝细胞癌转移复发以及预后的参考指标。     

Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy

AIM To investigate the predictors for recurrence or metastasis of HCC, and to evaluate the effect of antiangiogenic therapy on the growth of transplantable human HCC in nude mice. METHODS RT-PCR was used to measure the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in 56 pairs of nontumorous liver and tumor samples. Sixty blood samples from human HCC were examined by nested RT-PCR to find out AFP mRNA. Recombinant human endostatin and polyclonal antibody against VEGF were administered to treat human HCC transplanted in nude mice. RESULTS Thirty of 56 HCC samples showed stronger expression of MMP-9 in tumorous tissues than in nontumorous tissues. Fifteen of the 26 patients with relative expression level of MMP-9 more than 0.34 developed tumor recurrence or metastasis, whereas only 7 of 30 patients with relative expression level less than 0.34 developed tumor recurrence (P＜0.05).There was no significant difference in the relative expression level of VEGF between patients with postoperative recurrence or metastasis and those without recurrence. AFP mRNA was detectable in 53.3% of patients with HCC. The sensitivity and specificity of AFP mRNA as a marker to detect hematogenous dissemination of HCC cells was 81.8% and 84.4%, respectively. Recombinant human endostatin and polyclonal antibody against VEGF inhibited the growth of transplantable HCC in nude mice by 52.2% and 45.7%, respectively.CONCLUSION MMP-9 expression in HCC correlates with the postoperative recurrence or metastasis of HCC. Patients with high level of MMP-9 expression in HCC are susceptible to metastasis. AFP mRNA could serve as an indicator of hematogenous spreading of HCC cells in circulation and a predictor of recurrence or metastasis of HCC. Antiangiogenesis may be an adjuvant therapy for HCC.