The utility of the Ion Torrent PGM next generation sequencing for analysis of the most commonly mutated genes among patients with colorectal cancer in India

Background: The requirement for the mutation analysis for Kirsten rat sarcoma viral oncogene (KRAS) in colorectal cancer (CRC) is rapidly increasing as it is a predictive biomarker and also, its absence signifies response to anti?epidermal growth factor receptor (anti?EGFR) antibody treatment. The aim of our study was to investigate the pathological diagnosis and distribution of KRAS mutations in colorectal cancer with the use of next generation sequencing platform (Ion Torrent). Methods: A total of 56 CRC samples were tested to identify the genetic mutations, especially KRAS using the primers which included ~2800 COSMIC mutations of 50 oncogenes. Ion Torrent personal genome machine (semiconductor?based sequencing) was used for the sequencing and analysis. Along with KRAS, other 49 genes were also studied for COSMIC mutations. Results: KRAS mutation 25 (44.6%) had the highest frequency, followed by TP53 10 (17.9%) and PIK3CA mutation 4 (7.1%). Of all the KRAS mutations identified, mutations in codon 12 were most frequent followed by mutations in codon 13 and 61. The most frequent substitution was glycine to aspartate mutation in codon 12 (p.Gly12Asp) followed by glycine to valine (p.Gly12Val). Combinations of mutations were also studied. Our study revealed that seven cases (12.5%) had both KRAS and TP53 mutations (highest of all the combinations). Conclusion: The analysis of KRAS mutation frequency and its mutational subtype analysis in human CRCs by using semiconductor?based platform in routine clinical practices have been performed in Indian population. The findings were similar to earlier published reports from the Western literature.

Association of inflammatory markers with the disease & mutation status in pancreatic cancer

Background & objectives: Inflammation has been studied to be an important contributory factor to carcinogenesis through pro-inflammatory markers such as interleukin (IL)-6 and C-reactive protein (CRP). Furthermore, K-ras mutation is an important genetic alteration in the pathogenesis of pancreatic cancer. This study aimed to compare these inflammatory markers in pancreatic ductal adenocarcinoma (PDAC) with the diseased and healthy controls (HCs) and to check for any association between IL-6 and CRP serum levels with the disease status, survival and K-ras mutation status of PDAC patients. Methods: The study included 135 PDAC, 25 chronic pancreatitis (CP) patients and 25 HCs. The serum levels of IL-6 and CRP were detected by enzyme-linked immunosorbent assay and K-ras mutations were detected by polymerase chain reaction-restriction fragment length polymorphism technique. Results: The serum levels of both these markers were elevated in PDAC cases than that in HCs. High IL-6 levels and higher CRP levels were found to be associated with locally advanced disease, lymphatic invasion, metastasis and advanced stage of the PDAC. In patients with unresectable PDAC, higher IL-6 levels were found to be associated with the presence of K-ras mutations. Interpretation & conclusions: Higher IL-6 and CRP levels in patients with advanced PDAC suggest an important role of these inflammatory markers in tumour progression. Furthermore, the association of mutations in the K-ras gene with serum IL-6 indicates cross-talks that may contribute to the progression of the PDAC.

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer / 药物分析学报

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addi-tion,enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer.Furthermore,the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer.In this study,a novel FR-directed,macropinocytosis-enhanced,and highly cytotoxic bioconjugate folate(F)-human serum albumin(HSA)-apoprotein of lidamycin(LDP)-active enediyne(AE)derived from lidamycin was designed and prepared.F-HSA-LDP-AE consisted of four moieties:F,HSA,LDP,and AE.F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells.Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells.By in vivo optical imaging,F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice,showing clear and lasting tumor localization for 360 h.In the MTT assay,F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines.It also induced apoptosis and caused G2/M cell cycle arrest.F-HSA-LDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice.At well-tolerated doses of 0.5 and 1 mg/kg,(i.v.,twice),the inhibition rates were 91.2％and 94.8％,respectively(P＜0.01).The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Molecules linked to Ras signaling as therapeutic targets in cardiac pathologies

The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Highlights - The Ras (Rat Sarcoma) gene family is a group of small G proteins - Ras is regulated by growth factors and neurohormones affecting cardiomyocyte growth and hypertrophy - Ras directly affects cardiomyocyte physiological and pathological hypertrophy - Genetic alterations of Ras and its pathways result in various cardiac phenotypes? - Ras and its pathway are differentially regulated in acquired heart disease - Ras modulation is a promising therapeutic target in various cardiac conditions.

Expression of K-Ras, p53 and Ki-67 in Precancerous and Cancerous Lesions of Colorectum.

The immunological assessment at molecular profiling of K-Ras, p53 and Ki-67 along with EGFR, BRAF and others like BCL-2 has been the focus of several workers. The incidence of colorectal adenomatous polyp with dysplastic changes and colorectal carcinoma per se in the context of Indian population as per the ICMR registry, New Delhi, India, is significant. We wanted to study precancerous lesions (adenoma, polyp) and adenocarcinoma of colorectum for the expression of K-Ras, p53 and Ki-67 in biopsy or surgical specimen of colon by immunohistochemistry for its occurrence. We also wanted to study the intratumor heterogeneity of aforesaid expression for the purposes of prognostication and correlation with other clinicopathological parameters. MethodsThis is a cross sectional study conducted in the Surgical Pathology and Division of Immunohistochemistry, Department of Pathology, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra, India, for a duration of 2 years. Total seventy patients of all ages and genders suspected of 1) precancerous lesions of adenomas and polyps (35 cases) and 2) adenocarcinoma on colonoscopic biopsy or surgical specimens (35 cases) were included in the study. The expression of K-Ras, p53 and Ki-67 as primary objectives along with EGFR, BRAF and others like BCL-2 as secondary objectives were recorded, tabulated and compared for the purposes of predictive parameters in colorectal neoplastic lesions. ResultsMost of the cases of CRC were over the age of 50 years and Male:Female ratio was 5:3.Twenty-five cases of group 2 (CRC) showed positive IHC for K-Ras. p53 was detected in 28 cases and high Ki-67 index was observed in all 32 cases of CRC. The comparative statistics in the pilot study showed that the immunoexpression of K-Ras, p53 and Ki-67 is significantly higher in group 2 (CRC) compared to cases in group 1 (polyps) and group 3 (normal). ConclusionsExpression of K-Ras, p53 and Ki-67 is likely to be the distinguishing tissue biomarkers between benign and malignant colorectal disease process as well as have prognostic and predictive value in colorectal cancer. The addition of EGFR, BRAF and BCL-2 would help in determining the pathogenesis and offer targeted therapeutic intervention.

Differences in Microsatellite Instability, K-ras Gene Mutation and Other Clinicopathological Features Between Left and Right Hemicolon Cancer / 胃肠病学

Background: Colon cancer is one of the most common malignant tumor of digestive system. There are differences in pathogenesis, biological behavior, gene expression between left and right hemicolon cancer. Aims: To investigate the differences in clinicopathological features, microsatellite instability (MSI) and K-ras gene mutation between left and right hemicolon cancer. Methods: Data of 144 patients with colon cancer diagnosed by postoperative pathology from June 2017 to June 2018 at Qingdao Municipal Hospital were collected. MSI was assessed by immunohistochemistry, K-ras gene mutation was detected by PCR. The differences in clinicopathological features, MSI and K-ras gene mutation between the two groups were compared. Results: Right hemicolon cancer was more common in female, and left hemicolon cancer was more common in male. The incidence of lymph node metastasis, positivity rate of CEA and MSI in right hemicolon cancer were significantly higher than left hemicolon cancer (P<0.01), while the K-ras gene mutation rate in left hemicolon cancer was significantly higher than right hemicolon cancer (P<0.05). The K-ras gene mutation in left hemicolon cancer was correlated with gender, lymph node metastasis and positivity rate of CEA (P<0.05). MSI in right hemicolon cancer was correlated with gender, age, and lymph node metastasis (P<0.05). Conclusions: There are differences in the MSI and K-ras gene mutation between left hemicolon cancer and right hemicolon cancer, which can be used as the reference for diagnosis, individualized treatment and prognosis of colon cancer.

Expression of Fascin-1 and β-catenin protein in colorectal adenocarcinoma and its relevance with K-ras gene mutations / 临床与实验病理学杂志

Purpose To examine the expression of Fascin-1 and β-catenin protein and K-ras gene mutation in colorectal adenocarcinoma, and to explore their role in progression of colorectal neoplasm and their relevance. Methods Fascin-1 and β-catenin were analyzed by use of immunohistochemistry En Vision two-step. K-ras gene mutation was detected by ARMS method.Relationship between overexpression of Fascin-1, the nuclear expression of β-catenin, and the mutations of K-ras gene and clinicopathologic parameters was analyzed, the correlation between them was also analyzed. Results In 112 colorectal adenocarcinoma samples, the overexpression rate of Fascin-1 protein was 27.7％ (31/112), significantly higher than non-neoplastic mucosa (P < 0.01). The high nuclear expression rate of β-catenin was 29.5％ (33/112) in adenocarcinoma and non-neoplastic mucosa respectively with a significant difference between two groups (P < 0.01). High expression rate of Fascin-1 protein and β-catenin were correlated significantly with lymph node metastasis (P = 0.022, P = 0.027), and TNM staging (P =0.042, P = 0.019) in colorectal adenocarcinoma. The overexpression of Fascin-1 protein was correlated with tumor location (P = 0.004). The mutation rate of K-ras gene was 34.8％ (39/112), which showed no correlation with age, gender, tumor size, grade of differentiation, lymph node metastasis and TNM staging (P> 0.05). There was a correlation between the overexpressison of Fascin-1 protein, the nuclear expression of β-catenin and the mutation of K-ras gene (rs= 0.252, rs= 0.258, P < 0.05). The overexpression of Fascin-1 protein positively correlated with the nuclear expression of β-catenin (rs= 0.213, P < 0.05). Conclusion Fascin-1 protein and β-catenin protein are involved in invasion and metastasis of colorectal cancer and are associated with K-ras gene mutation. K-ras may promote the overexpression of Fascin-1 by virtue of nuclear expression ofβ-catenin, which provided a new research direction on the treatment of K-ras gene mutated colorectal adenocarcinoma.

Correlation between expression of β2 adrenergic receptors and HER2, K-ras genes in breast cancer / 中华内分泌外科杂志

Objective To investigate the expression of β2 adrenergic receptor (β2 AR) in invasive ductal carcinoma of the breast,and their correlation with HER2 and K-ras genes in breast cancer.Methods The expression of β2 AR,HER2 and K-ras gene in 10 cases of normal breast tissues and 33 cases of invasive ductal carcinoma of the breast were detected by immunohistochemical staining of streptomycin antibiotin protein and peroxidase assay (SP).Such statistical methods as SPSS17.0 statistical software,Mann-Whitney test and Spearman rank correlation test were applied to process and analyze the experimental data.Results β2 AR was expressed in all breast cancer cells,with an overexpression rate of 21/33.The positive expression rate of HER2 in breast cancer cells was 23/33,with an overexpression rate of 20/33.K-ras was positively expressed in breast cancer tissue(28/33),with an overexpression rate of 11/33.All of these three were significantly higher than those in the normal breast tissues.Z value of β-2 AR expression was 4.876,P value was 0.000.Z value of HER2 expression was 2.752,and P value was 0.006.Z value was 3126,and P value was 0.020 for K-ras expression.In the study of the correlation of β2 AR expression with HER2 and K-ras,the expression of β2 AR and HER2 and the expression of β2 AR and K-ras were correlated.The difference in expressions of β2 AR was statistically significant only in groups with or without lymph node metastasis,but not in groups varied in age,tumor size,clinical stage,pathological grading or hormone level.Conclusion The high expression of β2 AR,HER2,and K-ras in breast cancer tissues is associated with the occurrence and development of breast cancer.There is also a greater risk of potential lymph node metastasis in breast cancer with high expression of β-2 AR.β-2 AR may play a role in pathogenesis of breast cancer through HER2 and K-ras.

Relevant research of PD-1, PD-L1 protein expression and K-RAS gene mutations in lung adenocarcinoma / 临床与实验病理学杂志

Purpose To investigate the association of the PD-1 and PD-L1 protein expression with K-RAS gene mutations in lung adenocarcinoma.Methods The protein expression of PD-1 and PD-L1 was detected by immunohistochemical EnVision two-step staining,the K-RAS mutation was examined by realtime fluorescent quantitative PCR.Results The positive rate of PD-1 and PD-L1 was higher in lung adenocarcinoma than benign lung disease (P ＜ 0.01).There was no relationship between PD-1 and PD-L1 protein expression with the gender,age,smoking condition,differentiation,lymph node metastasis and TNM stages (P ＞ 0.05).The K-RAS gene mutations were detectable in 8 patients (22.2％) among 36 lung adenocarcinoma,there was no association between K-RAS gene mutation with the gender,age,smoking condition,differentiation,lymph node metastasis and TNM stages (P ＞ 0.05).The correction analysis showed that there was no relationship between PD-1 and PD-L1 protein expression with K-RAS mutation (P ＞ 0.05).Conclusion The positive rate of PD-1 and PD-L1 is higher in lung adenocarcinoma than benign lung disease,but there is no relationship among PD-1 and PD-L1 protein expression with its clinal pathological characteristics and K-RAS mutation in lung adenocarcinoma.

Regulatory mechanisms of K-ras to girdin in COS7 cells and expression of K-ras and girdin in colorectal carcinoma / 中国病理生理杂志

AIM:To investigate molecular regulatory mechanism of K-ras to girdin protein in COS7 cells and expression of K-ras and girdin in colorectal carcinoma tissues .METHODS:The lentiviral vector carrying K-ras gene was constructed and transfected in the COS 7 cells.The expression of K-ras, girdin proteins and other related proteins in COS 7 cells and colorectal carcinoma tissues was observed by Western blot .RESULTS:The COS7 cells with K-ras over-expres-sion showed an irregular cell morphology .The results of Western blot indicated that the downstream signal protein levels of p-ERK1/2, p-Stat3 and girdin were significantly increased in the COS 7 cells with K-ras over-expression.Transfection with the K-ras siRNA into the COS7 cells significantly reduced the protein levels of p-ERK1/2, p-Stat3 and girdin.In the color-ectal carcinoma tissues (7 cases), 5 cases had higher expression of K-ras and girdin compared with pericarcinous tissues . CONCLUSION:K-ras regulates girdin expression through the signal pathway of K-ras-ERK1/2-Stat3-girdin.

Significance of K-ras gene status and ras protein expression in immunophenotypic classification of gastric signet ring cell carcinoma / 重庆医学

Objective To investigate the significance of K-ras gene status and ras protein expression in immunophenotypic classification of gastric signet ring cell carcinoma.Methods The expression of ras protein in 180 cases of gastric signet-ring cell carcinoma was detected by tissue microarray immunohistochemistry.Meanwhile,the mutation in codon 12,13 of K-ras gene was determined by using PCR-based DNA direct sequencing analysis.Results The rate of ras protein expression was 27.8％.The rate of ras protein expression in intestinal phenotype was significantly higher than those in gastric and gastrointestinal phenotypes(P<0.05).The rate of ras protein expression in cases with lymph node metastasis was significantly higher than those in cases without nodal involvement(P<0.05).The rate of ras protein expression was significantly higher in cases with deeper invasion(P<0.05).The frequency of K-ras gene mutation was 22(12.2％).All of them were found in codon 12.The types of mutation included GGT→AGT(1 case),GGT→TGT(1 case),GGT→GCT(2 cases),GGT→GTT(8 cases)and GGT→GAT(10 cases).K-ras mutation was significantly associated with intestinal phenotype(P<0.05).The rates of ras protein expression in cases with mutational type of K-ras gene was higher than those in cases with wild type(P<0.05).The ras protein expression was positively associated with K-ras gene mutation(r=0.61,P<0.05).Conclusion The ras protein expression is correlated with nodal involvement and invasion.K-ras gene mutation and expression of ras protein is related to phenotypic classification,and they might influence the phenotypic transformation in gastric signet ring cell carcinoma.

Advances in research on the modulation of autophagy by Ras/Raf/MEK/ERK signaling pathway / 中国药科大学学报

@#Autophagy is a conserved self-defense mechanism of organism, degrading the necrotic organelles and excess protein into small molecules for recycling. Autophagy plays a role in both physiological and pathological condition, influencing the expression of intracellular substance through multiple signaling pathways. Although it has been demonstrated that Ras/Raf/MEK/ERK signaling pathway was not only extensively involved in the regulation of cell growth, proliferation, differentiation and apoptosis, but was also implicated in autophagy and autophagic cell death, though its detailed mechanisms involved in regulation of autophagy has not been fully elucidated yet. This review focused on the advances of autophagy induced by Ras/Raf/MEK/ERK signaling pathway, to better understand the role of Ras/Raf/MEK/ERK signaling pathway in regulation of autophagy.

The Influence of Biomarker Mutations and Systemic Treatment on Cerebral Metastases from NSCLC Treated with Radiosurgery

OBJECTIVE: The purpose of this study was to analyze outcomes and identify prognostic factors in patients with cerebral metastases from non-small cell lung cancer (NSCLC) treated with gamma knife radiosurgery (GKS) particularly, focusing on associations of biomarkers and systemic treatments. METHODS: We retrospectively reviewed the medical records of 134 patients who underwent GKS for brain metastases due to NSCLC between January 2002 and December 2012. Representative biomarkers including epidermal growth factor receptor (EGFR) mutation, K-ras mutation, and anaplastic lymphoma kinase (ALK) mutation status were investigated. RESULTS: The median overall survival after GKS was 22.0 months (95% confidence interval [CI], 8.8–35.1 months). During follow-up, 63 patients underwent salvage treatment after GKS. The median salvage treatment-free survival was 7.9 months (95% CI, 5.2–10.6 months). Multivariate analysis revealed that lower recursive partition analysis (RPA) class, small number of brain lesions, EGFR mutation (+), and ALK mutation (+) were independent positive prognostic factors associated with longer overall survival. Patients who received target agents 30 days after GKS experienced significant improvements in overall survival and salvage treatment-free survival than patients who never received target agents and patients who received target agents before GKS or within 30 days (median overall survival: 5.0 months vs. 18.2 months, and 48.0 months with p-value=0.026; median salvage treatment-free survival: 4.3 months vs. 6.1 months and 16.6 months with p-value=0.006, respectively). To assess the influence of target agents on the pattern of progression, cases that showed local recurrence and new lesion formation were analyzed according to target agents, but no significant effects were identified. CONCLUSION: The prognosis of patients with brain metastases of NSCLC after GKS significantly differed according to specific biomarkers (EGFR and ALK mutations). Our results show that target agents combined with GKS was related to significantly longer overall survival, and salvage treatment-free survival. However, target agents were not specifically associated with improved local control of the lesion treated by GKS either development of new lesions. Therefore, it seems that currently popular target agents do not affect brain lesions themselves, and can prolong survival by controlling systemic disease status.

Cáncer Colorectal: características de la expresión del oncogen K-ras / Colorectal cancer: characteristics of the expression of K-ras oncogene

Introducción: El cáncer de colon ocupa el cuarto lugar como causa de muerte por cáncer en adultos. Objetivo: Describir características de la expresión del oncogen K-ras en pacientes con cáncer colorectal (CCR) que acudieron a consulta de Gastroenterología del Hospital Universitario de Caracas en el período enero-julio 2014. Metodología: Estudio de corte transversal, descriptivo y prospectivo. La población de estudio estuvo conformada por pacientes con diagnóstico de CCR por colonoscopia e histología con evaluación molecular del K-ras. Resultados: de 35 pacientes 57,14% fueron del sexo masculino y 42,86% del femenino con edad media de 57±17años; el 100% de la muestra por histología correspondió a ADC predominando el tipo moderadamente diferenciado (40,00%). 28 pacientes (80%) no presentó mutación del K-ras mientras que 7 (20,00%) sí, de los cuales 6 (85,71%) reportó mutación en el codón 12 y 1 en el 13 (14,28%); en 4 de los 7 pacientes (57,14%) la mutación estuvo en el colon izquierdo. Conclusiones: la mutación del K-ras predomina en el sexo masculino con edad media de 57años estando presente en 20% de la población; la mutación en el codón 12 es más frecuente asociada al colon izquierdo y el CCR más común es el ADC bien diferenciado.

Introduction: Colon cancer ranks fourth leading cause of cancer death in adults. Objective: To describe characteristics of the expression of K-ras oncogene in patients with colorectal cancer (CRC) who attended the outpatient Gastroenterology of the University Hospital of Caracas (HUC) in the period from january-july 2014. Methodology: Transversal, descriptive and prospective court. The study population consisted of patients diagnosed with CRC by colonoscopy and histology with molecular evaluation of K-ras. Results: Of the 35 patients 57,14% were male and 42,86% female with a mean age of 57±17 years old; 100% of the sample corresponded to ADC histology predominating moderately differentiated rate (40.00%). 28 patients (80%) had no mutation of K-ras while 7 (20,00%) did, of which 6 (85,71%) reported mutation at codon 12 and 1 in the 13 (14,28%); in 4 of 7 patients (57,14%) mutation was in the left colon. Conclusions: K-ras mutation predominates in males with an average age of 57 years old and is present in 20% of the population; mutation in codon 12 is most frequently associated with the left colon and the most commom type of CRC by histology is the well differentiated ADC.

Inhibitory Effect of Matrine on K-ras Gene Mutation Colon Cancer and Its Anti-tumor Mechanism / 广州中医药大学学报

Objective To observe the inhibitory effect of matrine on K-ras gene mutation colon cancer, and to clarify the inhibitory mechanism. Methods SW480 cells were treated with different concentrations of matrine. MTS method was used to detect the proliferation of SW480 cell lines. The apoptosis of SW480 cells was measured by flow cytometry. The migration of SW480 cells was examined by the scratch test. The expression of MEK1/2 protein was detected by Western blotting method. Results Compared with the blank control group, matrine (0.125-1 mg/mL) could inhibit the growth and proliferation of human colorectal cancer SW480 cell lines, promote the apoptosis, restrain the migration of SW480 cells, and inhibit the expression of MEK1/2 protein(P < 0.05), the effect showing a dose-dependent trend. Conclusion Matrine can effectively inhibit the proliferation and migration of SW480 cells, and promote SW480 cell apoptosis through the down-regulation of MEK1/2 protein expression in MAPK signal pathway system.

Expression of microRNA-Let-7a in patients of Non-small cell lung cancer and anti-tumor mechanism / 中国免疫学杂志

Objective:To investigate the expression of microRNA-Let-7a in the serum and tumor tissue of non-small cell lung cancer( NSCLC) patients and the effects of cancer cell migration and proliferation.Methods: 50 cases of NSCLC patients in our hospital as the study group,50 healthy volunteers were used as control group,we used Real-time RCR to detect the expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients.Using microRNA let-7a mimics transfected into A549,the level of cancer cell migration was observed by transwell,the level of cell proliferation was observed by CCK-8,the level of k-Ras was observed by Real-time RCR and Western blot.Results: The expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients was significantly higher than the control group,the difference was statistically significant (P<0.05).After microRNA let-7a transfected into A549,the levels of cancer cell migration and proliferation were significantly decreased,the difference was statistically significant (P<0.05),the mRNA and protein levels of k-Ras were reduced inA549 cells,the difference was statistically significant (P<0.05). Conclusion:The expression of microRNA let-7a is low in the serum and tumor tissue of NSCLC patients,and may weaken the levels of cancer cell migration and proliferation through the Ras signaling pathway.

Analysis of K-ras gene mutation in 358 cases of lung cancer / 国际检验医学杂志

Objective To analyze the mutation situation of K‐ras gene in 358 cases of lung cancer to provide the guidance for the personalized therapy of lung cancer .Methods The nested and low denatured temperature compound PCR (COLD‐PCR) method was used to analyze the K‐ras mutations situation in 358 patients with lung cancer .Results In 358 lung cancer patients ,the total mutation frequency of K‐ras gene was 8 .10% .The mutation rates were 2 .13% in 94 plasma samples ,9 .60% in 250 tumor tissue samples and 7 .14% in 14 pleural and ascites samples .The mutation types included G12C ,G12D ,G12A ,G12V ,G13D and Q61H ,the mutation rate had no statistical difference among 3 kinds of samples (P= 0 .064 8) ;the mutation rate was 6 .96% in male patients and 8 .59% in female patients ,the difference was not statistically significant (P= 0 .574 0) ;the mutation rates were 4 .76% in the patients aged 30 - < 45 years old ,5 .34% in the patients aged 45 - < 60 years old and 9 .22% in the patients aged ≥ 60 years old , the differences were not statistically significant (P = 0 .250 3) .Conclusion The mutation types of K‐ras gene are mainly G12C , G12D ,G12V and G13D .The mutation rate of K‐ras gene has no significant differences among different types of sample ,different genders and different ages .

Application of quantum dots immune fluorescent labelling in colorectal cancer tissues / 国际检验医学杂志

Objective To investigate different antigens detected by a novel labelled reagent‐quantum dots(QDs) in the colorectal cancer tissues microarray(TMA) .Methods Depend on QDs streptavidin conjugate(QDs‐SA) combined specially with biotinylation IgG ,immune of luorescent histochemistry was utilized to examine expression of K‐ras ,matrix‐remodeling associated 5(MXRA5) proteins in the colorectal cancer TMA ,where the protein accurate location was observed .Results K‐ras ,matrix‐remodeling associ‐ated 5(MXRA5) proteins were high expressed in colorectal cancer tissue and located accurately in the cell membrane and nucleus of colorectal cancer cells ,respectively .Conclusion QDs exhibit excellent photostability ,broad emission spectrum and long fluorescence lifetime .Modified with streptavidin could accurately detect different protein locations in the colorectal cancer TMA .This is a novel approach for studying targeted imaging of colorectal cancer in vivo and vitro clinical diagnosis .

Analysis of K-ras gene mutation status in 560 Chinese colorectal cancer patients / 国际检验医学杂志

Objective To analyze the mutation status of K‐ras gene in colorectal cancer patients ,further more ,to provide guid‐ance for personalized therapy for colorectal cancer .Methods Nested and COLD‐PCR were used to detect the K‐ras mutations in 560 patients with colorectal cancer .Results In 560 colorectal cancer patients ,the total positive rate of K‐ras gene mutations was 27 .08% ,the mutation rate was 0 in 128 plasma samples and it was 27 .08% in 432 tissue samples .The mutate sites were G12S , G12C ,G12D ,G12A ,G12V ,G13R ,G13C ,G13D ,Q61K ,Q61L ,there were significant differences existed in different samples (P <0 .000 1) ;the mutation rate of 362 male patients was 20 .44% and the types of mutation include G12S ,G12C ,G12D ,G12V ,G13R , G13C ,G13D ,Q61K and Q61L .The mutation frequency was 21 .72% in 198 female patients ,the mutation points were G12S ,G12C , G12D ,G12A ,G12V ,G13R and G13D .There were no significant difference between different sex (P= 0 .722 7) ;the mutation fre‐quency was 20% in 80 youth patients including G12S ,G12C ,G12D ,G12V ,G13D and the mutation rate was 33 .07% in 127 middle age patients ,the points of mutation were G12S ,G12D ,G12A ,G12V ,G13R ,G13C ,G13D ,Q61K ,Q61L ,the mutation frequency was 16 .71% in 353 old age patients ,the types of mutation include G12C ,G12D ,G12V ,G13R ,G13D ,the difference was significant a‐mong different age patients (P= 0 .000 5) .Conclusion The total rate of mutations is 27 .08% in 560 colorectal cancer patients ,and the main points of mutation is G12D ,G12V ,G13D .There are significant differences in different type of samples as well as in differ‐ent ages ,but no statistical significance in different sex patients .

Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human KRAS gene.

There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T>A, rs12226937: G>A, and rs61761074: T>G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G>A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T>G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.