The Role of Ras Gene Mutation in Gastric Cancer and Precancerous Lesions

Abnormality of ras gene family was studied in a total of 206 cases of gastric cancer and precancerous lesions by PCR-RFLP, PCR-SSCP and DNA sequencing. The results showed that mutation rate of H-ras 12 codon in metaplasia,atypical hyperplasia, early-stage cancer and advanced cancer was 16. 7%, 31. 2 %, 50. 0%, and 32. 2%, respectively. In the groups of superficial gastritis and normal controls, no mutation were detected in codon 12 of ras. Mutations of Hras 61 codon and N-ras 12 codon in various groups were the same as those in normal control. K-ras 12 codon mutation was detected in only 2 cases of gastric cancer by using PCR-SSCP, but it was not detected by DNA sequencing, which may be polymorphism. All H-ras 12 codon mutations were G→T mutation. There were significant difference between the groups of metaplasia, dysplasia, gastric carcinoma and normal control group (P<0.05, P<0.01, P<0.01,respectively). It was concluded that H-ras 12 codon mutation was an early event and may play an important role in gastric carcinogenesis. Although K-ras, N-ras mutation rates are high in colon cancer and leukemia, it seems to bear no relationship with gastric cancer.

Rapid Detection ofK-ras Gene Point Mutation at Codon 12 by PCR-SSPin Pancreatic Adenocarcinom a

To evaluate the feasibility and clinical significance of the PCR SSP technique in detecting K ras gene mutation at codon 12 in pancreatic adenocarcinoma tissues. 80 specimens of surgical resection or biopsy samples were tested at our hospital from January 1994 to September 1995. Three different special sequence primers (SSP) synthesized according to mutation styles of CGT, GTT, GAT were respectively prepared. Three amplification reactions were performed for each sample. The amplification products were analyzed by conventional polyacrylamide gel electrophoresis, stained with ethidium bromide and observed under UV transillumination. Results: All of the 34 pancreatic adenocarcinoma samples had positive PCR results with the mutation rate 100%. 7 cases were CGT mutation, 18 GGT and 17 GAT mutation, in which 2 types of mutation existed in 8 cases. No mutation appeared in 13 normal pancreatic tissues, 6 insulinomas, 6 chronic pancreatitis, 5 benign pancreatic cysts, 7 bile duct carcinoma, 5 ampulla carcinoma and 4 carcinomas of duodenal papilla. Conclusion: Pancreatic adenocarcinoma is one of the commonly encounted tumors and is still very difficult to diagnose at the early stage and to distinguish from other lesions preoperatively. Our study indicates that PCR SSP is an ideal assay in comparison with other methods to detect K ras gene mutation. It is simple, rapid, specific, sensitive and easily generalized for clinical application on preoperative diagnosis.

APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM:To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed. RESULTS: K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P 】 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P 【0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor. CONCLUSION: ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder

BACKGROUND Mutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer.Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder(UCB).AIM To determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.METHODS This study was performed to determine the mutational spectrum at the hotspot regions of H-Ras,K-Ras and N-Ras genes by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and DNA sequencing followed by their clinical impact(if any)by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.RESULTS None of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene;codon 61 of N-Ras gene and codons 12,13 of K-Ras gene by PCR-RFLP.Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras,NRas and K-Ras genes in the tumor and control bladder mucosal specimens.CONCLUSION Our findings on the lack of mutations in H-Ras,K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression,inherent genetic susceptibility,tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

RAS AND p53 EXPRESSION IN HUMAN THYROID CARCINOMA

Objective: To investigate the possible interaction between the ras and p53 genes over-expression in thyroid carcinoma, and whether there is a correlation between the ras and p53 over-expression and clinicopathological criteria. Methods: Eighty patients with thyroid lesions were examined for expression of ras and p53 genes by the labeled streptavidin biotin peroxidase (LSAB) method. Of these patients, 54 were diagnosed (average age: 39.9±15.9 years) with malignant lesions. Of those included in the study, 31 has papillary carcinoma, 13 had follicular carcinoma, 7 had medullary carcinoma, 3 had undifferentiated carcinoma and 19 were stratified to stage I, 28 to stage II, 2 to stage III and 5 to stage IV according to TNM staging system. Twenty-six benign nodular thyroid disorders were studied as control. Results: Positive immunostain results for ras and p53 genes were statistically significant between thyroid carcinomas and benign disorders (90.7% vs 23%, 55.5% vs 30.7%,P<0.05). Both p53 and ras overexpressions coexisted in 30 thyroid carcinomas, and of these, 3 died and 5 had recurrences within 4 years. Conclusions: Activation of ras gene and inactivation of p53 gene were cooperatively associated in thyroid tumorigenesis. The concurrent overexpressions of ras and p53 could result in a poor prognosis.

Preliminary Investigation on Regulating Effects of Different TCM Treatments on Transcription of the Correlated Genes of Liver Cancer in Rats

The regulating effects of TCM treatments including clearing away heat and toxic materials,promoting blood circulation and removing blood stasis,and strengthening the spleen and regulating qi on the oncogene transcription were observed in the liver cancer model rats.The preliminary results indicated that the mRNA levels of H-ras N-ras and K-ras,and signal molecules correlated with the ras/MAPK signal transduction pathway were down-regulated by the different TCM treatments in varying degrees.Also,the regulating effects of the treatments on differently-displayed genes were discrepant.It is suggested that the molecular mechanisms of the TCM treatments for liver cancer was complex with different target genes.

Detecting K-ras and p53 gene mutation from stool and pancreatic juice for diagnosis of early pancreatic cancer

OBJECTIVE: To explore new methods for the early diagnosis of pancreatic cancer through detection of K-ras and p53 mutations in pancreatic juice and stool. METHODS: 201 patients in PUMC Hospital from 1994 - 2000 and 60 control individuals were enrolled in this study. K-ras point mutation was detected by PCR-RFLP while p53 mutation was detected by PCR-SSCP. RESULTS: K-ras mutation was found in pancreatic juice in 87.8% (36/41) of pancreatic cancer patients and 23.5% (4/17) of benign pancreatic disease patients. In 261 stool specimens, amplification found mutations successfully in 235 patients (90%). K-ras mutation was found in stool in 88% (66/75) of pancreatic cancer patients, 51.1% (24/47) of benign pancreatic disease patients and 19.6% (9/46) of normal individuals. p53 mutation was found in pancreatic juice in 47.4% (18/38) of pancreatic cancer patients and 12.5% (2/16) of benign pancreatic disease patients. p53 mutation was found in stool in 37.1% (23/62) and 19.1% (4/21) of chronic pancreatitis patients. CONCLUSION: K-ras mutation in pancreatic juice has higher diagnosis sensitivity and specificity, and therefore may be used as a supplement in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can aid in the screening of pancreatic cancer.

Zebularine对人鼻咽癌细胞CNE-2Z增殖的抑制作用

某些抑癌基因转录起始区域的启动子发生异常超甲基化，会导致抑癌基因表达失活引发肿瘤，因此有关去甲基化药物的研究正在成为肿瘤治疗领域的热点。在国外研究中已证实抗肿瘤药——Zebularine（Zeb）可以抑制多种肿瘤细胞生长，并上调因甲基化失活的相应抑癌基因表达。但Zeb对鼻咽癌细胞的影响在国内、外还未见报道，鉴于鼻咽癌在我国华南地区的高发率，我们对此进行初步探讨，以期为鼻咽癌的综合治疗提供新的思路。

Intestinal dysbiosis in pediatric Crohn's disease patients with IL10RA mutations

BACKGROUND Several studies have employed animal models to explore the association between microbiota and interleukin(IL) 10 signaling;however,limited information is available about the human microbiome.AIM To characterize the microbiome in patients with IL10 RA mutations and to explore the association between gut dysbiosis and disease severity.METHODS Fecal samples were collected from patients who were diagnosed with loss-offunction mutations in the IL10 RA gene between January 2017 and July 2018 at the Children’s Hospital of Fudan University.Age-matched volunteer children were recruited as healthy controls.Patients with Crohn’s disease(CD) were used as disease controls to standardize the antibiotic exposure.Microbial DNA was extracted from the fecal samples.All analyses were based on the 16 S rRNA gene sequencing data.RESULTS Seventeen patients with IL10 RA mutations(IL10 RA group),17 patients with pediatric CD, and 26 healthy children were included.Both patients with IL10 RA mutations and those with CD exhibited a reduced diversity of gut microbiome with increased variability.The relative abundance of Firmicutes was substantially increased in the IL10 RA group(P=0.02).On further comparison of the relative abundance of taxa between patients with IL10 RA mutations and healthy children,13 taxa showed significant differences.The IL10 RA-specific dysbiosis indices exhibited a significant positive correlation with weighted pediatric CD activity index and simple endoscopic score for CD.CONCLUSION In patients with IL10 RA mutations and early onset inflammatory bowel disease,gut dysbiosis shows a moderate association with disease severity.

Relationship between the renin angiotensin system genes and diabetic nephropathy in the Chinese

To clarify whether genetic variants of the renin angiotensin system (RAS) contribute to the development of diabetic nephropathy (DN) in the Chinese Methods Totally 173 Chinese subjects of Han nationality from Shanghai were classified into!control, DN ( ) and DN (+) groups The latter was subdivided according to diabetic duration at the onset of DN and the stage of DN Genotyping of five polymorphic sites in four key genes of the RAS: the AGT T174M, AGT M235T and AGTR1 genotypes were determined by PCR/restriction enzyme digestion The insertion/deletion (I/D) and [ACAC]n STR microsatellite polymorphic markers were used for ACE and REN genotyping, respectively Statistical analysis showed comparisons of gene frequencies between any two groups were made with Fisher's exact test or Chi square test Logistic regression analysis was performed to identify predictors of DN Results The frequencies of ACE DD genotype and ACE D allele were much higher in DN(+) group than in DN( ) group (0 25 vs 0 05, 0 47 vs 0 29, respectively), so were the frequencies of TT genotype and T allele in AGT M235T (0 73 vs 0 54, 0 85 vs 0 68, respectively) DN (+) DUR<5 years group had greatly increased frequencies of AGT M235T allele and ACE DD genotype in comparison with DN( ) group (0 92 vs 0 68 and 0 28 vs 0 05, respectively) Logistic regression analysis further identified these two genes as contributing factors to DN Although AGTR1 and AGT T174M genotyping analysis revealed differences in frequency distribution between DN (+) and DN ( ) or control groups, logistic regression analysis failed to implicate them in the development of DN Conclusions Our study revealed RAS genes, ACE and AGT M235T but not AGT T174M, AGTR1 or REN genotypes, as contributing factors for DN in type 2 diabetes mellitus in Chinese

Effect of blocking Ras signaling pathway with K-Ras siRNA on apoptosis in esophageal squamous carcinoma cells

OBJECTIVE： To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （no transfection）, nega- tive control group （transfection no-carrier）, trans- fection group （transfected with pSilencer-siK-ras）, taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and west- ern blotting were used to analyze the expression of MAPK1 （mitogen-activated protein kinases 1） and cyclin D1 in response to siRNA （small interfering RNA） transfection and taxol treatment. RESULTS： K-Ras （K-Ras gene） siRNA transfection of EC9706 esophageal squamous carcinoma cells de- creased the expression of K-Ras, MAPK1 and cyclinD1 at the mRNA and protein level. Reverse tran- scription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group （P〈0.05）. Western blotting showed that 72 h after EC9706 cell trans- fection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the ex- pression levels in the transfection group were sig- nificantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as com- pared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. CONCLUSION： Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-me- diated inhibition of Ras signaling, with the most ob- vious increase observed in the transfection plus tax- ol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeu- tic agent, taxol. Ras knockdown also down-regulat- ed the expression of the downstream genes, MAPKI and cyclin DI, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.

Frequent 3p21 Allelic Loss and Methylation-Associated RASSF1A Inactivation in Non-Small Cell Lung Cancer and Its Clinical Implication

A total of 110 primary NSCLCs （non-small cell lung cancers） were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation-specific PCR （MSPCR） as well as bisulfite genomic sequencing （BGS）, and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of （41.2±3.7）%. Significant associations between 3p21 LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyses shows significant correlations from one another. Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significant correlation was also observed in extent between methylation and RASSF1A expression, illustrating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions.