Evaluation Of Neoadjuvant Chemotherapy And The Functions Of MicroRNAs And Their Target Genes In Gastric Cancer

Background:Gastric cancer is one of the most common malignant tumors causing death worldwide. Neoadjuvant chemotherapy is an important part of the comprehensive management strategy, which has significantly improved the survival and prognosis, but there is still no reliable method to evaluate the response to neoadjuvant chemotherapy. Tumor progression caused by chemotherapy resistance is the main reason for the poor prognosis of advanced gastric cancer. So, it is necessary to identify sensitive markers to evaluate the therapeutic effect. MiRNA (microRNA) is a class of endogenous non-encoding RNA which bind to the target mRNAs through the 3’untranslated regions (3’UTRs) and inhibit the target gene expression. The alterations in the expression level of miRNAs have been reported to be closely related to the tumorigenesis of many malignant carcinomas. There are also miRNAs in peripheral blood, which can reflect the tumor size and depth of invasion. At the same time, there are usually up or down regulation of specific miRNA expression in tumor tissue. Therefore, miRNA can be used as a target for tumor diagnosis and treatment.MiR-301a is highly expressed in colorectal cancer, pancreatic cancer and is closely related with various malignant biological behaviors and prognosis. Studies have shown that the expression of miR-301a in gastric cancer tissues increased significantly compared with matched adjacent tissues, and is closely associated with the degree of tumor differentiation, proliferation, invasion, metastasis, playing an important role in the occurrence and development of gastric cancer. However, the role and mechanism of miR-301a in the development of gastric cancer is not clear, and its correlation with gastric cancer neoadjuvant chemotherapy is not clear. Further research on the mechanism of miR-301a is of great significance for exploring the evaluation of the efficacy of neoadjuvant chemotherapy in gastric cancer.There are many factors involved in the tumorigenesis and development of gastric cancer, and microRNA has also played an important role. MiR-32 is oncogene in many malignant tumors, but the role in gastric cancer is not clear. We found that mir-32 showed abnormal high expression in gastric cancer patients’peripheral blood and tumor tissue, which may promote the occurrence and development of gastric cancer. MiR-32 promotes proliferation, invasion and migration of human gastric cancer cells by regulating its target gene Kruppel like factor-4 (KLF-4). Exploring the mechanism of miR-32 in the occurrence and development of gastric cancer may provide a new theoretical basis and potential target for clinical diagnosis and treatment of gastric cancer.Gastric signet ring cell carcinoma (GSRCC) is a special pathological type of gastric carcinoma that is with low early stage diagnosis rate, poor response to treatment and prone to invade to the gastric wall. At present, unified comprehensive plan is usually used for the treatment of GSRCC. MicroRNA arrays were applied to screen the differentiated expression between GSRCC and non-GSRCC. It was found that miR-935 expression was lower in GSRCC than in non-GSRCC. MiR-935 might play as a tumor suppressor gene by targeting Notch1 3’UTR in GSRCC. Studying the mechanism of miR-935 in GSRCC is helpful to understand the specific types of gastric cancer and provide a potential diagnostic marker and therapeutic target for GSRCC.Objectives:(1) To study the significance of neoadjuvant chemotherapy and nutritional support in the treatment of patients with advanced gastric cancer.(2) To study the significance of miR-301a in evaluating the response of neoadjuvant chemotherapy for advanced GC, and to clarify the mechanism of miR-301a and its target gene in the neoadjuvant chemotherapy of GC.(3) To study the mechanism of miR-32 and its target gene in the tumorigenesis and development of GC.(4) To study the mechanism of miR-935 and its target gene in GSRCCMethods:(1) Collecting the clinical and pathological data of 147 cases of patients with gastric cancer who received neoadjuvant chemotherapy and surgical treatment in our hospital, and analyzing the tumor remission and prognosis after receiving neoadjuvant chemotherapy and nutritional support.(2) qPCR was used to detect miR-301a expression in GC tissue and plasma from GC patients with neoadjuvant chemotherapy. The expression of CA199, CEA, CA724 and the remission of the tumor were compared before and after neoadjuvant chemotherapy. Enhanced expression of miR-301a attenuated the apoptosis induced by cisplatin or 5-Fu in MGC8-03 and AGS. Glutamate-ammonia ligase(GLUL) was identified as a direct target of miR-301a. Detecting the expression of GLUL mRNA in GC by qPCR and analyzing the correlation between miR-301a and GLUL mRNA expression.(3) qPCR was used to detect miR-32 expression in GC tissue and paired adjacent normal tissue. HGC-27 and AGS with low expression miR-32 were transfected with mimic, while MGC8-03 and MKN-45 that displayed high expression miR-32 were transfected with inhibitor. The xCELLigence RTCA-MP system was used to monitor cell proliferation ability and transwell assays were used to detect cell migration and invasion abilities. TargetScan7.0 predicted and luciferase reporter assays results showed that miR-32 directly bound to the 3’UTR of KLF4. The effect of miR-32 on GC cell proliferation, migration and invasion ability was observed. Detecting the expression of KLF4 mRNA in GC by qPCR and analyzing the correlation between miR-32 and KLF4 mRNA expression.(4) MicroRNA arrays were applied to screen differentiated expression between GSRCC(MKN-45 and KATO-III) and non-GSRCC(MGC8-03, SGC-7901 and AGS), and it was found that miR-935 was lower expressed in GSRCC and the function of miR-935 in tumor was unclear. Normal gastric tissue, human gastric epithelial cell line GES-1, seven non-GSRCC cell lines and two GSRCC cell lines were used to detect the expression of miR-935. MKN-45 and KATO-Ⅲ with low expression miR-935 were transfected with mimic, while MGC8-03 and HGC-27 that displayed high expression miR-935 were transfected with inhibitor. The xCELLigence RTCA-MP system was used to monitor cell proliferation ability and transwell assays were used to detect cell migration and invasion ahilities. TargetScan7.0 predicted and luciferase reporter assays results showed that miR-935 directly bound to the 3’UTR of Notch1. Detecting the expression of Notch1 mRNA in GC tissue and paired adjacent normal tissue by qPCR and analyzing the correlation between miR-935 and Notch1 mRNA expression.Results:(1) Male accounted for 66%(97/147) and female accounted for 34%(50/147) in the 147 patients with advanced gastric cancer. The lesion site before treatment:the upper part of the stomach accounted for 23%(34/147), the middle part of the stomach 31%(46/147), gastric antrum accounted for 46%(67/147). R0 resection in 135 cases accounted for 91.8%. The patients received neoadjuvant chemotherapy with an average of 3.7 cycles. During the operation, the lymph node dissection number range from 15 to 77, with the average of 28, and the lymph node positive rate was 68.7%(101/147). Among the 147 patients, the evaluation of neoadjuvant chemotherapy was CR (complete remission) in 2 cases, PR (partial remission) in 74 cases, SD (stable) in 55 cases, and PD (progression) in 16 patients. Chemotherapy efficiency (CR+PR) was 51.7%(76/147), and the disease control rate was 89.1%(131/147).45.5%(67/147) of the patients had decreased N staging; 55.6%(5/9) of T2 patients had decreased T staging; 42.6%(29/68) of T3 patients had decreased T staging; 58.6%(41/70) of T4 in patients had decreased T staging. The TNM staging was significantly associated with prognosis (P< 0.01).(2) miR-301a is upregulated in gastric carcinoma tissue and plasma. Comparing with CA199, CEA and CA724, plasma miR-301a level is more significantly associated with the treatment response in patients receiving neoadjuvant chemotherapy. Upregulation of miR-301a attenuates the gastric carcinoma cells apoptosis induced by neoadjuvant chemotherapy in vitro. GLUL is the target gene of miR-301a, and there is a significant inverse correlation between miR-301a and GLUL in GC.(3) miR-32 is upregulated in GC tissue, plasma and cell line. Enhanced expression of miR-32 could promote the proliferation, migration and invasion of GC cells. KLF4 is the target gene of miR-32, and there is a significant inverse correlation between miR-32 and KLF4 in GC.(4) miR-935 is lower expression in GSRCC cell line and tissue than non-GSRCC cell line and tissue. Increased expression of miR-935 could inhibit the proliferation, migration and invasion of GSRCC cells. Notch1 is the target gene of miR-935, and there is a significant inverse correlation between miR-935 and Notch1 in gastric carcinoma.Conclusions:(1) Neoadjuvant chemotherapy has a good chemotherapy effectiveness and disease control rate for patients with advanced gastric cancer. The prognosis of patients received neoadjuvant chemotherapy is associated with tumor staging, and the prognosis is better in patients with earlier stage after neoadjuvant chemotherapy. Nutritional support can improve the nutritional status of patients, maintain body weight and reduce the incidence of adverse drug reactions.(2) For plasma miR-301a level is more sensitive than traditional biomarker in reflecting the treatment response in patients receiving neoadjuvant chemotherapy, it could be a potential biomarker for GC patient diagnosis and neoadjuvant chemotherapy effect. miR-301a attenuates the gastric carcinoma cells apoptosis by targeting GLUL 3’UTR.(3) miR-32 is upregulated in GC tissue, plasma and cell line. miR-32 promotes GC cell proliferation, migration and invasion by targeting KLF4.(4) miR-935 is downregulated in GSRCC tissue and cell line. miR-935 inhibits GSRCC cell proliferation, migration and invasion by targeting Notch 1, and acts as a tumor suppressor gene in GC.