The Relationship Of CYP2W1 In The Proliferation And Prognosis Of Gastric Cancer

Gastric cancer is one of the mostcommonmalignanttumors, the highest malignant neoplasms of digestive tract tumors. The number of new case was about 1,000,000 annually worldwide, which was 8%,4th of malignanttumors. Almost 700,000 people die of gastric cancer every year, ranking mortality rate of 2nd, and no gender difference. China is one of the highest morbidity and mortality countries for gastric cancer, where new case account for 30 percent the global total each year, the morbidity and mortality rate was double of the world average. The screening rate of early gastric cancer was low in our country, only about 10%, many patients was advanced gastriccarcinoma during treatment. For early gastric cancers in Japan, no symptoms 47.6%, indeterminate gastrointestinal tract symptoms 31.2%, peptic ulcer symptoms 17.9%. Among early gastric cancers,91.3 percent were treated emergently because the symptom of indeterminate gastrointestinal tract, no symptoms only 2.72%. At present in China, the clinical status of gastric cancer have the character "three high and three low". The "three low" means that early diagnosis (<10%), excision rate (<70%) and survival rate in 5 years was low. The "three high" means high in incidence, postoperative recurrence, metastatic rate and mortality, and tends to be younger. The genesis, development of gastric carcinoma is a multi-factor, multistep progressive process, its mechanisms present in many ways, are not fully understood. For prognosis, NCCH concluded that factors affecting the prognosis of gastric cancer are the depth of tumor invasion, followed by the lymph node metastasis, distant metastasis, histological types and tumor size with a multi-factor analysis of the clinical records. The invasion and metastasis of gastric carcinoma is a continuous and complicated active process with many stages, steps and many factors to participate in. Therefore, the research has the vital clinical significance to early diagnosis, treatment and prognostic assessment, which on genetic and molecular markers for gastric carcinoma about its infiltration and diversion.CYP2W1 is a member of cytochrome P450 superfamily, the study shows that the enzyme endogenously expressed in fetus’s colon, and stilled because of methylation after birth, expressed only in fetal rat colon and human tumor tissues. CYP2W1 are highly expressed in gastroenteric tumor, particularly in colon, rectal cancers and colorectal neoplasms specimen. The CYP2W1 genic segment was firstly found in cDNA storage of HepG2 in liver cancer cell lines. The human’s CYP2W1 gene is on the chromosome of 7p22.3, containing 1473 nucleotides,9 exons and 490 amino acids. Although the specific functions of CYP2W1 in its exogenous and endogenous material metabolism and its beginning and development of tumor have not yet clarified, there has already had study showed that the CYP2W1 expression, related to prognosis of patients and being the independent prognostic indicator of colon cancer, has tumor specificness, especially on colon tumor and adrenal tumor, which has aroused researchers’wide concern. The researches with regard to CYP2W1 expression in gastric cancer and relations of emergence, infiltration, transfer and prognosis with stomach cancer have still not been reported at home and abroad.Only existing in cell nucleus of proliferation cell, Ki-67 is a relatively positive landmark of nuclear proliferation related to cell cycle. Its coded gene is on the tenth chromosome, and its expression will add as the cell cycle progresses. In the cell division cycle, Ki-67 doesn’t express in G0, but begins to express in the later period of G1. In period of S and G2, the expression will gradually rise and last to its climax in M period and suddenly decrease and vanish after the cell division. Therefore, Ki-67 is regarded as the important indicator for the cell proliferation activity and widely used in the proliferation assessment of tumor cell. Study shows that Ki-67expresses in lung cancer, liver cancer, breast cancer, endometrium cancer and many other cancers and it is related to tumor’s emergence and development, infiltration and transfer, and prognosis. The extent of expression of Ki-67 in the tumor structure plays an important role in the assessment of tumor cell proliferation activity, in the research of biological behavior and prognosis of tumor. Studying the expression of Ki-67 in gastric cancer structure makes sense for assessing the proliferation activity of stomach cancer cell and judging prognosis.This study firstly adopts IHC technology to test the expression of CYP2W1 and Ki-67 in adenocarcinoma of gastric structure and its corresponding normal gastric mucosa structure, to analyze and compare the relation between expression of CYP2W1 and Ki-67 and clinicopathological parameter of gastric cancer, and to analyze the relation between CYP2W1 and Ki-67 and gastric cancer prognosis. The expression and correlation of CYP2W1 and Ki-67, mRNA and protein in gastric cancer structure will be tested by applying RT-PCR and Western blot. The adenocarcinoma of gastric and normal gastric mucosa cell line will be constructed. The expression quantity of CYP2W1 and Ki-67 in every group of cell line will be tested, and its correlation be analyzed. The adenocarcinoma of gastric cell line with relevant high CYP2W1 expression quantity will be chosen as the research target to conduct the cell sorting and purity identification. Growth curve graph by applying the MTT experiment method will be drawn and the influence of CYP2W1 expression towards the external proliferation growth capacity of gastric cancer cell will be further researched by applying flat cloning experiment. The influence of CYP2W1 expression towards the external motive invasion and metastasis capacity of gastric cancer cell will be researched with scratch healing test and Transwell cell experiment.All in all, regarding the CYP2W1 as the target molecule, the experiment with two parts will discuss the function of CYP2W1 in the gastric cancer cell proliferation and prognosis.Part I CYP2W1 Expression and Prognosis Analysis in gastric Cancer StructureMethods1. The expressions of CYP2W1 and Ki-67 protein in 326 adenocarcinoma of gastric structures and in the corresponding 326 normal gastric mucosa structures will be tested by applying Immunohistochemical Technique. The relation between expression of CYP2W1 and Ki-67 and clinicopathological parameter of gastric cancer will be analyzed and compared.2. The expressions of CYP2W1 and Ki-67 mRNA or protein in 10 random chosen examples of adenocarcinoma of gastric structures and its corresponding normal gastric mucosa structures will be tested and it will be hopefully further verified the experimental results of Immunohistochemical Technique.3. The Kaplan-Meier survival curve graph will be made and the relation between the expression of CYP2W1 and Ki-67 protein and the stomach cancer patients’ survival time will be analyzed. The correlation of CYP2W1 and Ki-67 will be analyzed by adopting Spearson Correlation;4. The experimental data will be statistically processed by using SPSS 17.0 software. The immunohistochemical results will be statistically processed by using the χ2 test. The relation between the expression of CYP2W1 and Ki-67 protein and the stomach cancer patients’prognosis will be analyzed by applying Kaplan-Meier survival curve graph. Whether CYP2W1 is the independent prognostic factor or not will be analyzed with COX proportional hazard model. The expression correlation of CYP2W1 and Ki-67 will be analyzed with Spearson Correlation. Other data will be analyzed by using single factor variance. The difference of P<0.05 has statistical significance.Results1. CYP2W1 protein are mainly located in the cytoplasm of gastric cancer structure and they basically don’t express in the normal gastric mucosa structure; CYP2W1 protein and CYP2W1 mRNA in the gastric cancer structure clearly is more than that in normal gastric mucosa structure(P<0.05).2. The expression of CYP2W1 doesn’t show clear correlation with gastric cancer patient’s gender, age and differentiated degree(P>0.05). However, it is closely associated with the patient’s TNM by stages and lymph gland transfer (P<0.05).3. The gastric cancer patient’s survival rate with high expression of CYP2W1 is dramatically lower than that with low expression of CYP2W1. The expression of CYP2W1 is closely related to stomach cancer patient’s prognosis. The result of COX proportional hazard model shows that CYP2W1 is the independent prognostic factor of stomach cancer patient.4. The result of correlation analysis shows that the expressions of CYP2W1 and Ki-67 share the prominent correlation(P<0.05).Part II Influence of Up-Regulation of CYP2W1 ExpressionTowards gastric Cancer Cell Biological BehaviorMethods1.4 groups of adenocarcinoma of gastric structure and normal gastric mucosa cell line will be constructed with the technology of QRT-PCR and Western blotting. The expression quantity of CYP2W1 in every group of cell line will be tested and its correlation will be analyzed.2. Growth curve graph of CYP2W1 positive stomach cancer cell will be drawn by applying the MTT experiment method and the change of tumor-making capacity for each gastric cancer cell will be observed by applying flat cloning experiment.3. The influence of CYP2W1 expression towards the external motive invasion and metastasis capacity of gastric cancer cell will be researched with scratch healing test and Transwell cell experiment.4. Statistics Process:The experimental data will be statistically processed with SPSS 17.0 software. With the data indicated by x±s statistical analysis of two groups of statistical data will apply t to test, and statistical analysis of three groups or more than three groups of statistical data will apply One-way ANOVA to statistically analyze. The difference of P<0.05 has statistical significance.Results1. The result of QRT-PCR and Western blotting shows that the expression quantity of CYP2W1mRNA and protein in the 4 groups of gastric cancer cell lines(BGC-823、SGC-7901、MKN-28、MGC-803) is obviously higher than that in normal gastric mucosa cell lines. Among the 4 groups of gastric cancer cell lines, the expression quantity of CYP2W1 mRNA and protein in the BGC-823 cell line is much higher than that in SGC-7901、MKN-28 and MGC-803 cell lines. Moreover, the difference also has statistical significance (P<0.05).2. The results of MTT growth curve graph, flat cloning formation show that the growth capacity of gastric cancer cell in experimental group is much stronger than that in control group; The result of flat cloning formation shows that the cloning-making quantities of gastric cancer cell in the experimental group are clearly higher than those in control group, and the difference has statistical significance (P<0.05).3. The result of Transwell cell invasiveness experiment shows that the cell numbers that gastric cancer cell invades and passes through Transwell cell matrigel in the experimental group for 24 hours are clearly more than those in the control group. The comparative difference between them has statistical significance (P<0.05).4. The result of scratch healing experiment shows that the scratch repair rate of gastric cancer cell after scratching for 24hours/48hours in experimental group is much higher than that in control group. The scratch healing proportion of gastric cancer cell in experimental group is much larger than that in control group. The comparative difference between them has statistical significance (P<0.05).Conclusions1. CYP2W1 only expresses in the gastric cancer structure. However, it doesn’t express in the normal gastric mucosa structure; The expression of CYP2W1 doesn’t show clear correlation with gastric cancer patient’s gender, age and differentiated degree.However, it is closely associated with the patient’s TNM by stages and lymph gland transfer.2. The expression of CYP2W1 and Ki-67 in gastric cancer shows positive correlation; The expression of CYP2W1 is closely related to the prognosis; CYP2W1 is the independent prognostic factor of gastric cancer.3. CYP2W1 is closely tied up with the growth multiplication capacity and motive invasion and metastasis capacity.4. The high expression of CYP2W1 that may well be a new drug target spot of tumor treatment may play an important role in the beginning and development of stomach cancer and open up a new thought for targeted therapy of stomach cancer.