Study On The Influence Of KRAS Gene Mutation On The Related Proteins In Colorectal Cancer

Background and purposeColorectal cancer is the most common malignant tumor of the digestive system,with a high incidence in developed countries such as Europe and the United States.The incidence of male and female in China ranks fourth and fifth respectively.And with the economic development and improvement of living standards in our country,the incidence and mortality of colorectal cancer have significantly increased.The occurrence and development of colorectal cancer is a complex process involving multiple genes,multiple steps and multiple signal transduction pathways.The main treatment is surgery,chemotherapy and radiotherapy.In recent years,some new drugs,especially targeted drug development and application,the prognosis of patients greatly improved in colorectal cancers,especially in patients with advanced colorectal cancer prognosis.However,the drug resistance of targeted drugs is gradually increasing,and understanding the signal transduction pathways in colorectal cancer will help to find the mechanism of drug resistance.The RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways all belong to mitogen-activated protein kinase(MAPK)signaling pathways,which are closely related to the proliferation,differentiation,apoptosis invasiveness and metastasis of tumor cells.The abnormal activation also found in colorectal cancer cells.Mutations in any one of the upstream genes(such as the RAS gene or the BRAF gene)may be transmitted to the protein through transcription or translation,resulting in abnormal activation of the signaling pathway.This article studies the relationship between the KRAS gene mutation and the clinicopathological features and prognosis of colorectal cancer and the effect of KRAS gene mutation in colorectal cancer on related proteins in RAS signaling pathway.Tofurther understand the cause of tumor progression and drug resistance caused by mutation of KRAS gene.Part one: Relation among KARS gene mutation status with clinicopathological features and prognosis in colorectal carcinomaObjectiveTo investigate the KRAS gene mutation rate and mutation types and the relationship among KRAS gene mutations with clinicopathological features and prognosis in colorectal carcinoma.MethodsFrom Jan 2012 to Dec 2012,146 cases of colorectal carcinoma samples in The Affiliated Cancer Hospital of Zhengzhou University were collected.By Real-time Quantitative Polymerase Chain Reaction(qPCR)method,we detected the KRAS gene mutation status in 146 specimens of colorectal carcinoma;the Chi-squared Test was used to evaluate the relation between KRAS gene mutation status and clinicopathological features in colorectal carcinoma;while the Kaplan-Meier survival analysis and Log-rank Test were used to analyze the relevance between KRAS gene mutation status and prognosis.Results:47of the 146 patients with colorectal cancer had KRAS gene mutations in codons12,13 of exon 2,and The mutation rate of KRAS gene in colorectal carcinoma was32.2%,40 of 47 KRAS gene mutation patients were found in codon 12,and the other7 of 47 were found in codon 13,The types of mutation in codon 12 included GGT→GTT(18),GGT→GAT(16),GGT→GCT(5),GGT→TGT(1),While only 7 of 47 patients had GGC→GAC in codon 13.Based on the analysis of correlation with the clinicopathological features,it was found that k-ras mutations was not associated with age,sex,the situs and size,grade of differentiation,depth of infiltration,lymph node metastasis and distant metastasis of the carcinoma(P>0.05),but the rate of KRAS mutation in Mucinous colorectal adenocarcinomas(MCAs)was higher than nonmucinous colorectal cancers,The difference was statistically significant(P < 0.05).The Kaplan-Mmeier survival analysis and Log-rank Test reveal that thecumulative progression-free survival(PFS)rate in cases with mutational type of KRAS gene was 68.09%,which was shorter than the cases with wild type(77.78%),the cumulative overall survival(OS)was74.47%.which was shorter than the cases with wild type(81.82%),but There was no significant difference respectively(P=0.258;P=0.339).Conclusions:1.The mutation rate of KRAS gene in colorectal carcinoma was 32.2%,and the most frequently seen mutation locus is GGT>GTT lotus and GGT>GAT lotus in the codon 12.2.The KRAS gene mutational type in colorectal carcinoma was significantly correlated with the mucinous colorectal adenocarcinomas(MCAs),the MCAs show higher mutation rate.3.The patients with KRAS gene mutational type show poor prognosis,the KRAS gene mutation in clinical detection may be taken as the prognostic index to evaluate colorectal carcinoma.Part Two: Effect of KRAS gene mutation on KRAS,NRAS,BRAF,MEK,ERK and PI3 K protein in colorectal cancerObjectives:To analyze the influence of KRAS gene mutation on the positive expression of PI3 K protein,KRAS,NRAS,BRAF,MEK,ERK,p MEK1,p MEK2 and perk1/2proteins,as well as the expression levels of PI3 K,KRAS,NRAS,BRAF,MEK and ERK protein,as well as the mutual influence of each protein.To understand whether mutations in the KRAS gene cause the increase of protein expression in the RAS signaling pathway and a the influence of the increase of upstream protein expression on downstream protein.Methods:Immunohistochemistry was used to detect the positive expression of PI3 K,KRAS,NRAS,BRAF,MEK,ERK,p MEK1,p MEK2 and p ERK1/2 proteins in colorectal cancer tissues.Chi-square test was used to analyze the relationship between PI3 K,KRAS,NRAS,BRAF,MEK,ERK and clinicopathological indicators of colorectal cancer,as well as the correlation between the positive expression rate of each protein.The independent sample T test was used to analyze the difference in the expression levels of PI3 K,KRAS,NRAS,BRAF,ERK and MEK proteins in mutant and wild-type patients with KRAS gene.Comparison of 4-year PFS and OS differences between PI3 K,NRAS,KRAS,BRAF,ERK and MEK protein positive and negative patients in phase II and III tumor tissues by Kaplan-Meier Log-rank test and Cox multivariate regression analysis.Results:The positive rates of PI3 K,KRAS,NRAS,BRAF,ERK,MEK,p MEK1,p MEK2 and p ERK1/2 proteins in colorectal cancer tissues were 72.4%,71.4%,68.2%,78.8%,64.3%,and 50.8%,respectively.42.0%,67.2%,44.5%(53/119).The positive expression rates of KRAS and ERK proteins were significantly higher in the poorly differentiated cancer group than in the highly differentiated group.The positive expression rate of KRAS protein in the median age group of less than 56 years was significantly higher than that of the group over 56 years old.The positive expression rate of PI3 K protein was significantly higher in the lymph node metastasis group than in the no lymph node metastasis group.The positive expression rate of MEK protein was significantly higher in the distant metastasis group than in the distant no metastasis group(P<0.05).There was no significant correlation between the positive expression rate of other indicators and the clinicopathological features(P>0.05).H-score method was used for semi-quantitative determination of KRAS and PI3 K protein and it was found that there was no statistical difference in their protein expression in colorectal cancer in phase I,II,III and IV(P>0.05).There was no statistical difference in the expression of KRAS protein in the cell membrane and cytoplasm between the mutant-type and wild-type of KRAS gene (P>0.05),and there was no statistical difference in the expression of PI3 K,NRAS,BRAF,MEK and ERK protein(P>0.05).The positive rate of BRAF protein was not significantly different between KRAS and NRAS protein positive group and negative group(P>0.05).The positive rate of MEK,p MEK2 and ERK protein was higher in the KRAS protein positive group than in the negative group(P <0.05).The positive rates of MEK and ERK protein were higher in the NRAS protein positive group than in the negative group(P<0.05).There was no significant difference in the positive rate of MEK and ERK protein between BRAF protein positive group and negative group(P>0.05).The positive rate of p MEK1 and p MEK2 protein in BRAF protein positive group and negative group were statistically significant(P< 0.05).The positive rates of ERK and p ERK1/2 protein in MEK protein positive group and negative group were statistically significant(P<0.05).In patients with stage II and stage III,the 4-year recurrence-free survival rate of KRAS protein-positive patients in tumor tissues was significantly higher than that of negative patients(P<0.05),but the overall survival rate was not statistically different(P>0.05).Between positive and negative groups of NRAS,BRAF,MEK,ERK and PI3 K protein,there was no statistical difference of the 4-year recurrence-free survival rate and overall survival rate(P>0.05).Multivariate analysis showed that only KRAS protein-positive was risk factors for tumor recurrence(P<0.05).Conclusions:1.The expression levels of KRAS,NRAS,BRAF,MEK,ERK and PI3 K in colorectal cancer tissues can be determined by semi-quantitative analysis of immunohistochemical h-score.2.KRAS gene mutation has no effect on the expression level of KRAS protein in cell membrane and cytoplasm.The activation of KRAS protein and RAS signaling system has little relationship with the amount of KRAS protein and the location and distribution of KRAS protein in the cell.3.In the colorectal cancer tissues with KRAS gene mutation,the expression levels of BRAF,MEK and ERK in the downstream proteins of RAS/RAF/MEK/ERK signaling pathway were not increased.4.In the KRAS protein positive group,the positive expression rates of MEK,p MEK2 protein and ERK were increased.In the NRAS protein positive group,the positive expression rates of downstream proteins MEK and ERK were increased.In the BRAF protein positive group,the positive expression rates of downstream proteins p MEK1 and p MEK2 were increased.In the p MEK2 protein positive group,the downstream protein p ERK1/2 was positively elevated.5.In patients with stage II and III colorectal cancer,the 4-year relapse-free survival rate in the KRAS protein-negative group was significantly higher than that in the positive group.Multivariate analysis of KRAS,NRAS,BRAF,MEK,ERK and PI3 K protein expression in colorectal cancer tissues showed that only KRAS protein expression in tumor tissues was a risk factor for tumor recurrence.