The Function Of MiR-30b In Gastric Cancer And The Study Of Neoadjuvant Chemotherapy In The Treatment Of Advanced Gastric Cancer

Early diagnosis is difficult, chemotherapy resistance and lack of effective prognostic indicator are the problems of gastric cancer research and these affect the overall prognosis of gastric cancer patients. So, identification of new diagnostic markers, disclosing the mechanisms of gastric cancer development and reverse the resistance to chemotherapy were important means to improve the prognosis of gastric cancer patients.MicroRNAs (MiRNAs) are short, single-stranded RNA molecules with 22 nucleotides, which involve in tumorigenesis, cancer development, proliferation, metastasis and chemoresistance. MiR-30b was low expression in colorectal cancer, cervical cancer and breast cancer, which was involved in regulating a variety of tumor biology phenotype. However, the expression level and regulation mechanism in gastric cancer is unclear. Therefore, it is important to detect the miR-30b expression level and investigate the regulatory roles and mechanisms of miR-30b in gastric cancer and assess its diagnostic and prognostic values.Neoadjuvant chemotherapy (NAC) as a part of comprehensive cancer treatment, has gradually been applied in locally advanced gastric cancer. While a number of important findings significantly favored the neoadjuvant approach:higher complete resection rate, smaller primary tumor size, less lymph node metastases, and less frequent lymphangiosis carcinomatosa as compared with the surgery alone. Patients were in good nutrition status before the chemotherapy, so they can better tolerate the side effects of chemotherapy. In recent years, pilot studies using neo-adjuvant chemotherapy showed promising results. However, there are controversial in uniform standard of chemotherapy drugs, chemotherapy episodes and the time of NAC efficacy evaluation.Objective:(1) Our aim was to investigate the value of combined detection of serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, CA 242 and CA 50 in diagnosis, clinical pathological factors and prognosis in consecutive gastric cancer patients.(2) To explore the role of miR-30b in cell proliferation, apoptosis, invasion and migration of gastric cancer, to identify the target gene and signal pathway.(3) To evaluate the efficacy of neoadjuvant chemotherapy, the relevant factors impacting curative effect and the prognosis of gastric cancer patients who accept the neoadjuvant chemotherapy. To investigate the effects on the nutrition condition and complications of gastric cancer treated with insertion of jejunal nutrient canal after NAC and surgery.Method:(1) Clinical data of 181 gastric cancer patients including preoperative serum CEA, CA 19-9, CA 242, and CA 50 values and clinical pathological factors were collected and analyzed retrospectively. ROC curve was used to analyze the sensitivity of four tumor markers when detection with different combinations. Univariate and multivariate survival analyses were used to explore the relationship between tumor markers and survival prognosis.(2) Real-time PCR was used to detect the expression level of miR-30b, cell proliferation was assessed using the CCK8, analyses of tumor cell migration and invasion test were carried out using transwell chambers. Western blot was used to detect the protein expression and luciferase activity assay was used to validate the target gene of miR-30b.(3) We retrospectively analysis the clinical data of 115 gastric cancer patients who received the preoperative chemotherapy and surgery from February 2007 to March 2015 in Peking Union Medical College Hospital. All of these patients received the Xelox regimen which consisted of oxaliplatin at 130mg/m2 intravenously (more than 2 hours) on the first day, oral capecitabine at 1000mg/m2 in the next two weeks and have a rest in the third week. Three weeks were a cycle for the Xelox regimen. These patients received at least two cycles of neoadjuvant chemotherapy. The chemotherapy efficacy and toxicity was assessed before the surgery. Also, the relationship between neoadjuvant chemtherapy and prognosis was evaluated. By monitoring the clinical and laboratory indexes, we assess the effect of jejunal catheterization enteral nutrition on the nutritional status and complication in gastric cancer patients.Results:(1) Positive rates of tumor markers CEA, CA 19-9, CA 242 and CA 50 in the diagnosis of gastric cancer were 17.67,17.12,20.44 and 13.81%, respectively, and the positive rate for all four markers combined was 36.57%. Patients with elevated preoperative serum concentrations of CEA, CA 19-9, CA 242 and CA 50, had late clinical tumor stage and significantly poorer overall survival. Five-year survival rates in patients with elevated CEA, CA 19-9, CA 242 and CA 50 were 28.12,25.83,27.02 and 24.05%, respectively, compared with 55.01,55.37,56.36,54.51% in patients with these markers at normal levels (P<0.01). In multivariate Cox proportional hazards analyses, elevated CA 242 level was determined to be an independent prognostic marker in gastric cancer patients.(2) miR-30b was low expression in gastri cancer tissues. Low expression miR-30b was associated with lymph nodes metastasis, no association was found between miR-30b expression and age, sex, Lauren type in 23 gastric cancer patients.(3) CCK8 test showed that overexpression of miR-30b could inhibit the growth of gastric cancer cells. Instead, when trasfected miR-30b inhibitor into gastric cancer cells and reduce the miR-30b expression, then promote the growth of gastric cancer cells. Flow cytometry was used to evaluate the effect of miR-30b on cancer cell apoptosis, the results suggesting that overexpression miR-30b may induce apoptosis of gastric cancer cells. Transwell test indicated that overexpression miR-30b could inhibit cell number pass through the chamber membrane, conversely, we dected increased migration and invasion in cancer cells transfected with miR-30b inhibitor.(4) We cloned the region of the EIF5A23’UTR containg the complementary site into a luciferase reporter vector. The wild and mutant construct were transfected into HEK293T cells with miR-30b mimics or mimics control. The results showed that the luciferase activity in cotransfection mimics and mutant vector group was significantly decreased compared with the control group, indicating that EIF5A2 was the target gene of miR-30b.(5) miR-30b lowed EIF5A2 expression and promoted cancer cell metastasis through activate EMT marker such as E-cadherin and p-catenin.(6) Patients who received pre-operative chemotherapy and surgery were analyzed,2 patients had complete response (CR),55 persons had a partial responses (PR),47 had stable disease (SD), and 11 had progression of disease (PD). The response rate (CR+PR) was 49.6%, and clinical benefit response (CR+PR+SD) was 90.4%. The pre-operation tumor T stage down rate was 48.7%. The common adverse events were leukocytopenia (16.5%) and gastrointestinal (12.2%), most of toxicity were grade Ⅰ/Ⅱ. All these symptoms alleviate after the treatment. The median surviva time of all the patients was 48 months and the 5-year survival rate was 42.4%. Patients who had PR to the chemotherapy had a better survival than those with SD and PD, the difference was statistically significant (P<0.001). Patients with lower GPS score (Glasgow prognostic score) had better remission to the chemotherapy than those with higher GPS score. Multivariate regression analysis suggested that response rate (P=0.026), postoperative tumor pTNM stage (P=0.037), Lauren type (P=0.033) and GPS score (P=0.002) were independent predictors of overall survival.(7) The albumin level in the jejunostomy group was significantly higher than that in the tube-free group 3 and 7 days after the surgery. The pre-albumin level in the jejunostomy group was higher than that of in the tube-free group 3 days after the surgery. Enteral nutrition through jejunal feeding tube could promote the early anal discharge and protect the liver function. There was no significant differences in survival between the two groups.Conclusion:(1) Combined detection of four tumor markers increased the positive rate for gastric cancer diagnosis. CA 242 showed higher diagnostic value and CA 50 showed lower diagnostic value. In resectable gastric carcinoma, preoperative CA 242 level was associated with disease stage, and was found to be a significant independent prognostic marker in gastric cancer patients.(2) miR-30b was low expression in gastri cance cells, overexpression miR-30b can suppress cell proliferation, promote cell apoptosis and inhibit invasion and migration of gastric cancer cells.(3) EIF5A2 was the target gene of miR-30b, miR-30b could lower the EIF5A2 expression and activate the EMT marker E-cadherin and p-catenin, then inhibit the invasion and migration of the gastric cancer cell.(4) Neoadjuvant chemotherapy with Xelox regimen demonstrated a good response rate for the advanced gastric cancer patients, which had acceptable toxicities. Patients with clinical remission to the chemotherapy had a good prognosis. Pathological stage, the response rate, Lauren type and GPS score can be used as independent prognostic factors for patients with gastric cancer who received neoadjuvant chemotherapy and surgery.(5) It is safe and convenient to use early postoperative enteral nutrition support by fine-needle/catheter jejunostomy in gastric cancer patients receiving NAC and surgery. It is also able to improve the nutritional status of patients.