Syndecan-1 and -4 differentially regulate oncogenic K-ras dependent cell invasion into collagen through α2β1 integrin and MT1-MMP

Syndecans function as co-receptors for integrins on different matrixes. Recently, syndecan-1 has been shown to be important for α2β1 integrin-mediated adhesion to collagen in tumor cells by regulating cell adhesion and migration on two-dimensional collagen. However, the function of syndecans in supporting α2β1 integrin interactions with three-dimensional (3D) collagen is less well studied. Using loss-of-function and overexpression experiments we show that in 3D collagen syndecan-4 supports α2β1-mediated collagen matrix contraction. Cell invasion through type I collagen containing 3D extracellular matrix (ECM) is driven by α2β1 integrin and membrane type-1 matrix metalloproteinase (MT1-MMP). Here we show that mutational activation of K-ras correlates with increased expression of α2β1 integrin, MT1-MMP, syndecan-1, and syndecan-4. While K-ras-induced α2β1 integrin and MT1-MMP are positive regulators of invasion, silencing and overexpression of syndecans demonstrate that these proteins inhibit cell invasion into collagen. Taken together, these data demonstrate the existence of a complex interplay between integrin α2β1, MT1-MMP, and syndecans in the invasion of K-ras mutant cells in 3D collagen that may represent a mechanism by which tumor cells become more invasive and metastatic.     

应用寡核苷酸微阵列检测肺癌样品中的P53和K-ras基因点突变

对影响寡核苷酸微阵列检测点突变的敏感性和特异性的各种因素,如杂交液,杂交温度,标记引物浓度及其比例等,进行了研究,采用不对称PCR扩增有利于敏感性提高,多重不对称PCR不影响杂交的特异性,且敏感性有所增加,对30例肺癌标本进行寡核苷酸微阵列检测,发现12例标本发生了P53基因来点突变,K-ras突变有5例,与测序结果相比,P53基因突变符合率达到80％,由于检测样本较少且检测位点不完全,因而未得到K－ras和P53基因突变与肿瘤的种类,病期及吸烟之间的明显相关性。     

电化学发光PCR方法检测结肠癌中K-ras癌基因点突变

发展了一种可用于快速检测K-ras癌基因点突变的电化学发光PCR(ECL-PCR)分析方法,该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;随后,采用限制性内切酶MvaI对扩增产物进行酶切,由于突变导致酶切位点的丢失,所以只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到反应池中进行电化学发光检测。采用该法对20例结肠癌组织中的K-ras癌基因第12位密码子进行点突变分析,得出其中有9例存在点突变,点突变率为45%。该方法操作简便、安全、快速、灵敏,可用于快速检测K-ras癌基因点突变。     

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3′-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.     

K-ras基因突变及表达与胰腺癌的关系

目的:探讨K-ras基因突变及蛋白表达对胰腺癌的诊断价值。方法:采用突变等位基因特异性扩增(PCR-MASA)检测31例胰腺癌和22例非胰腺癌石蜡包埋组织中K-ras基因12密码子点突变情况,并通过免疫组化法检测其K-ras蛋白的表达。结果:胰腺癌患者K-ras基因12密码子点突变率为80.6%(25/31),K-ras蛋白表达阳性率为87.1%(27/31);而非胰腺癌患者K-ras基因12密码子点突变率为27.3%(6/22),K-ras蛋白表达阳性率31.8%(7/22)。胰腺癌患者K-ras基因突变率及K-ras蛋白表达阳性率均明显高于非胰腺癌患者(P均0.05)。K-ras基因突变与其蛋白表达呈正相关(r=0.542,P0.05),而与胰腺癌患者的性别、年龄、肿瘤部位、肿瘤大小、临床分期及分化程度等临床病理特征均无显著相关性(P均0.05)。结论:胰腺癌K-ras基因突变的发生率升高,且K-ras蛋白表达异常上调,二者可能有助于胰腺癌的诊断。     

胃癌中Fascin 表达与K-ras 突变情况及两者相关性的研究

目的：观察胃癌组织中Fascin 表达和K-ras 突变情况,并分析其与临床病理特征的关系,研究Fascin表达和K-ras突变之间 的相关性,探讨两者在胃癌浸润转移过程中的作用。方法：在108 例胃癌组织中采用免疫组织化学法检测Fascin 表达,采用DNA 测序法检测K-ras突变。结果：108 例胃癌组织中Fascin 表达率为25.00 %,K-ras突变率为11.11 %;胃癌组织中Fascin 阳性表达, 发生淋巴结转移组多于未发生淋巴结转移组(P＜0.05),发生远处转移组多于未发生远处转移组（P＜0.01）;K-ras 突变,发生远处 转移组多于未发生远处转移组（P＜0.01）;胃癌组织中K-ras 突变和Fascin 表达之间存在相关性,Fascin阳性比Fascin阴性的胃癌 组织更倾向于发生K-ras突变（rp=0.378,P＜0.01）。结论：在胃癌组织中K-ras 基因突变和Fascin蛋白阳性表达存在相关性,且与 肿瘤的转移有关。     

甲醛致大鼠鼻腔癌与K-ras癌基因活化相关

为探讨甲醛致大鼠鼻腔癌的分子机制,对甲醛诱发的大鼠鼻腔癌细胞系FAT7中的转化序列进行检测.采用的实验方法,包括肿瘤细胞DNA与选择标志基因(neo)共转染、裸鼠成瘤性分析、Southern杂交、聚合酶链反应(PCR)和序列分析等.结果发现:在第二轮裸鼠肿瘤DNA中含有大鼠源性的K-ras基因序列,而无大鼠源性的H-ras、N-ras和p53基因序列.这表明甲醛诱发的大鼠鼻腔癌细胞系FAT7中所含的转化序列与H-ras、N-ras及p53基因无关,K-ras癌基因的活化可能参与甲醛致大鼠鼻腔癌.     

超声内镜引导下细针穿刺活检联合基因检测对胰腺癌的诊断价值

目的:研究超声内镜引导下细针穿刺活检(EUS-FNA)联合K-ras基因检测对胰腺癌的诊断价值,为临床诊疗提供依据。方法:选取2013年11月到2015年11月我院收治的胰腺占位病变患者90例,患者入院次日行EUS-FNA,检测患者血清及活检物中K-ras基因阳性率,比较EUS-FNA单独与EUS-FNA联合K-ras基因检测对胰腺癌诊断的准确率与敏感性。结果:90例胰腺占位病变者中,经病理证实胰腺癌56例,EUS-FNA单独与EUS-FNA联合K-ras基因分别检出胰腺癌50例、53例,准确率分别为89.29%、94.64%,敏感性分别为92.59%、98.15%,两组比较差异均有统计学意义(P0.05)。胰腺癌患者活检物中K-ras阳性率为83.93%,明显高于血清中的41.07%(P0.05)。结论:EUS-FNA联合K-ras基因检测可提高对胰腺癌诊断的准确率与敏感性。