在结肠癌细胞中RNA干扰TGFBR2慢病毒载体的构建及其功能的初步研究

目的:构建TGFBR2基因的RNA干扰慢病毒载体,为研究该基因在结肠癌细胞中的作用奠定基础.方法:合成TGFBR2基因特异性RNAi靶序列,与pcDNATM6.2-GW/EmGFP-miR载体连接,构建干扰载体并鉴定.与TGFBR2质粒共转染HEK293细胞,qRT-PCR和Western blot方法筛选最佳干扰序列,感染SW480细胞.10ng/ml TGF-β1和20ng/ml TNF-a共作用TGFBR2干扰后的SW480细胞和未干扰组细胞72h后,通过细胞侵袭试验计算细胞侵袭数目.结果:成功构建TGFBR2的RNA干扰载体,第4组序列效果最佳,感染SW480后,TGFBR2基因的mRNA表达抑制率为78.45％.TNF-a和TGF-β1处理干扰前后的SW480细胞,通过细胞侵袭试验显示各组细胞迁移的数目:干扰组为89.3±7.9,未干扰组为15.1±8.2,干扰组细胞明显多于对照组(P＜0.01).结论:成功构建人TGFBR2基因特异性的慢病毒干扰载体,并建立稳定的细胞株,为预防和干预结肠癌的早期转移奠定基础.初步提示在TGF-β1和TNF-a因子存在的炎症微环境中,TGFBR2基因突变的结肠癌细胞更具有侵袭性.     

MicroRNA‐3191 promotes migration and invasion by downregulating TGFBR2 in colorectal cancer

Mutations in transforming growth factor beta receptor II (TGFBR2) are detected in up to 30% of overall colorectal cancer (CRC). Dysregulation of some microRNAs participated in the CRC pathogenesis. In this study, we used the gene ontology analyses, the Kyoto Encyclopedia of Genes and Genomes pathway analyses and gene set enrichment analysis to indicate that miR‐3191 was involved in the regulation of transforming growth factor beta (TGF‐BETA) signal pathway in CRC. These bioinformatics results were supported by data obtained from CRC samples and experiments in vitro. The luciferase reporter assay was used to confirm that miR‐3191 modulates TGF‐BETA signal pathway by targeting TGFBR2. By transwell migration and invasion assays, we showed that miR‐3191 promoted CRC cell migration and invasion by downregulating TGFBR2. And it may serve as a novel therapeutic strategy for treating CRC patients.     

Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF,TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.     

Relative strength of 5’ splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5’ splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5’ splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5’ splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5’ splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5’ splice-site or higher strength of the other 5’ splice-site strength limited the role of SRSF2 and SRSF6 in 5’ splice-site activation.     

Mad2beta, an alternative variant of Mad2 reducing mitotic arrest and apoptosis induced by adriamycin in gastric cancer cells

Mad2beta is an alternative splicing variant of spindle checkpoint gene mad2, which was previously found by us and was related to the drug resistance in gastric cancer cells. In this paper, we explored the molecular mechanisms that Mad2beta variant promoted the formation of multidrug resistance in gastric cancer cells. We found that Mad2beta variant was detected only in the two human drug resistant gastric cancer cell sublines SGC7901/VCR and SGC7901/ADR, and it did not appear in its parental cell line SGC7901 and other detected gastric cancer cell lines. Expressions of Mad2 mRNA and protein in SGC7901 cells transfected with Mad2beta, SGC7901/VCR and SGC7901/ADR were significantly lower than that in SGC7901 cells. Moreover, SGC7901 cells overexpressing Mad2beta variant became more resistant to adriamycin, vincristine and mitomycin by abrogating mitotic arrest and apoptosis. This suggests that expression of Mad2beta variant decreases the relative expression of efficient MAD2, which may help gastric cancer cells to develop the phenotype of multidrug resistance.     

The clinical significance and prognostic value of Xenopus kinesin-like protein 2 expressions in human tumors: A systematic review and meta-analysis

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that plays a pivotal part in the formation of spindles. There is accumulating evidence that the expression of TPX2 is upregulated in many kinds of human cancers and that this protein is involved in the occurrence and progression of tumors. The purpose of this meta-analysis was to investigate the relationship between the overexpression of TPX2 and poor prognosis in cancer patients. A total of 18 eligible studies encompassing 3115 patients were included by searching relevant databases. Hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were pooled under random-/fixed-effect models. After calculation, the results showed that patients with increased TPX2 expression had a significantly shorter overall survival (HR = 2.21; 95% CI: 1.70–2.86), and disease-free survival (HR = 2.10; 95% CI: 1.67–2.64). In addition, it was found that increased TPX2 expression was significantly associated with TNM stage (OR = 2.17; 95% CI:1.42–3.32), lymph node metastasis (OR = 2.98; 95% CI: 2.28–3.89), distant metastasis (OR = 2.25; 95% CI:1.03–4.92), and vascular invasion (OR = 2.22; 95% CI:1.26–3.91). Nevertheless, there was no significant correlation between increased expression of TPX2 and either gender, tumor differentiation, or tumor size. Thus, we can come to the conclusion that the overexpression of TPX2 is related to poor clinical outcomes and can be used as a biomarker for the prognosis of patients with cancer.     

The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.     

The prognostic significance of immune changes in patients with renal cancer,melanoma and colorectal cancer,treated with interferon α2b

 In the present study we evaluated the response rate and the immunorestorative properties of interferon α2b (IFnα2b) administered to patients with advanced renal cell carcinoma (RCC), melanoma (MEL) or colorectal cancer (CC). We studied the immune status and correlated it with clinical responses. Thirty-five patients with advanced RCC, and 14 with MEL were treated with recombinant INFα2b. The dose was increased progressively from 5×10⁶ IU/day in the first week (three times every week) to 10×10⁶ IU/day in the second week and thereafter to 15×10⁶ IU/day subcutaneously. In patients with CC INFα2b was given at 5×10⁶ IU/day every other day (three times every week); these patients also received (together with INF) leucovorin 200 mg m^–2 day^–1 in a 1-h i. v. infusion every week, and mid-infusion 400 mg/m² 5-FU was administered as an intravenous bolus every week. The response rate was as follows: for RCC, 6 patients achieved partial response (PR), 10 stable disease (SD), and 21 progressed (PD); for MEL, 5 patients achieved PR and 9 PD; for CC, 6 achieved PR, 5 SD, and 9 PD. In all patients blood was withdrawn prior to INFα2b treatment and then monthly. T lymphocytes, after isolation from peripheral blood, were tested for proliferation in the autologous mixed-lymphocyte reaction and allogeneic mixed-lymphocyte reaction, interleukin-2 (IL-2) production, expression of IL-2 receptors during the allogeneic-mixed-lymphocyte reaction, and the production of IL-1 by peripheral blood monocytes. Striking increases were demonstrated in all parameters 2 months after treatment with INFα2b. In comparison to normal controls, all patients with the malignant neoplasms presented decreased (>45%) mean values of the immunological parameters under investigation (P 0.0001). Responders (patients with RCC, MEL, and PR) presented lower mean values of all the parameters studied than did non-responders (P 0.0001). Patients with CC presented the lowest mean values of the parameters than did the other patients (RCC, MEL) (P 0.0001). After therapy with INFα2b, patients with RCC experiencing PR showed a mean increase of more than 30% (P 0.0001). Patients with SD showed a mean increase of about 20% (P 0.0001), and those with PD showed a 6% increase in the immunological parameters under investigation. Patients with MEL experiencing PR showed a mean increase of more than 30% and patients with PD a decrease of more than 10% (P 0.0001). All patients, regardless of the clinical response, achieved an increase of more than 60% (P 0.0001). Administration of IFNα2b resulted in a marked potentiation of a deficient cellular immune response in vitro in those patients with RCC and MEL who responded to the treatment. On the other hand, non-responders demonstrated a decrease in the examined parameters and, in some, deterioration of the already depressed immunological functions was observed. This observation can have prognostic significance regarding clinical response of INF. In contrast, our findings show that the immune stimulation associated with INFα treatment in all our CC patients did not predict an improved clinical outcome. There are several theoretical explanations for this discrepancy. Received: 30 November 1996 / Accepted: 25 June 1996     

MAT2A与NDRG2基因在结直肠癌中表达情况及临床意义的研究

目的 研究MAT2A（甲硫氨酸腺苷转移酶2A）和NDRG2（N-Myc下游调节基因-2）基因在结直肠癌组织中的表达及两者的相关性,为结直肠癌的研究及治疗提供重要参考。方法 选取68例术中切除结直肠癌组织及相应的癌旁组织标本,应用RT-PCR（逆转录‒聚合酶链式反应）和Western blotting（蛋白质印迹法）检测结直肠癌中MAT2A和NDRG2表达水平。结果 结直肠癌中MAT2A和NDRG2 mRNA表达情况：MAT2A阳性表达率（66.2%,45/68）高于癌旁组织（7.4%,5/68）;NDRG2阳性表达率（36.8%,25/68）低于癌旁组织（91.2%,62/68）;两者的表达与患者年龄、性别、肿瘤生长位置等因素均无关,与癌肿临床分期、分化水平及淋巴结转移密切相关。癌肿组织中MAT2A蛋白表达量相较于癌旁组织明显增多（t=39.1152,P=0.0000）,而癌肿组织中NDRG2蛋白表达量较癌旁组织明显降低（t=46.5103,P=0.0000）;且在结直肠癌组织中两基因的表达可能有相关性（χ2=7.41,P=0.009）。结论 在结直肠癌组织中MAT2A表达增高,NDRG2表达低下,两者与肿瘤的发生发展具有一定相关性,推测可能与DNA甲基化异常有关,但具体机制仍待进一步研究。     

COX-2、Ki-67和结核菌L型感染在前列腺癌中的表达

目的探讨环氧合酶-2（COX-2）和Ki-67在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化、原位杂交和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benign prostatic hyperplasia,BPH）中的COX-2、Ki-67蛋白及mRNA的表达,以及结核菌L型的检出率;并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果COX-2、Ki-67蛋白及mRNA阳性表达和结核菌L型检出率,前列腺癌明显高于前列腺增生（P〈0.001～0.05）。COX-2、Ki-67蛋白及mRNA阳性表达和结核菌L型检出率与前列腺癌的临床分期、病理分级有明显差异（P〈0.01～0.05）。淋巴结转移组中COX-2、Ki-67蛋白及mRNA的阳性表达率明显高于非转移组（P〈0.01）。结核菌L型检出率淋巴结转移组明显高于非转移组（P〈0.05）。结论COX-2、Ki-67蛋白及mRNA在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移呈正相关,提示2种基因均可作为判断前列腺癌生物学行为及患者预后参考指标。结核菌L型感染极有可能导致基因的变异或过表达,成为诱发肿瘤因素之一,它们可能有协同致瘤作用。     

食管鳞癌组织中CDC2、CLDN5蛋白表达及其临床病理意义的研究

目的探讨CDC2及CLDN5在食管鳞癌中表达及其临床病理特征的关系。方法应用免疫组化Elivision法检测90例食管鳞癌组织、28例正常食管黏膜组织及16例重度不典型增生组织中CDC2和CLDN5的蛋白表达情况。结果在食管鳞癌和正常食管黏膜组织中CDC2和CI。DN5的阳性表达率分别为88．89％（80／90）、85．56％（77／90）和48．86％（12／28）、25．00％（7／28）,两者差异有统计学意义（P〈O．05）。CDC2蛋白表达在低分化食管鳞癌中明显高于高分化食管鳞癌;临床分期Ⅲ＋Ⅳ期组的CDC2蛋白的表达显著高于I期、Ⅱ期组（P〈O．05）。CDC2和CLDN5在食管鳞癌中表达呈正相关（r=0．537,P〈o．05）。结论CDC2和CLDN5在食管鳞癌的发生、发展过程中可能发挥重要作用,可能作为食管癌临床早期诊断的重要指标。     

Clinical and prognostic effects of CDKN2A,CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Abstract

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.

Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.

Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).

Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.     

Clinical significance of the NKG2D ligands,MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis

Objective  To investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer. Methods  Eighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured. Results  Expression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands. Conclusions  High expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from Grant-in-Aid for Scientific Research (19390426, 19591932, 18209052 and 19659421) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.