Studies have shown that microRNA-148a (miR-148a) was proved to be silenced while DNA methyltransferase 1 (DNMT1) was over-expressed in gastric cancer. But the mechanism of aberrant expression of miR-148a and DNMT1 and their relationships in gastric cancer are still unknown. The aims of this study were to investigate the expression profile of miR-148a and DNMT1 and reveal whether they have any relationships. We used reverse-transcriptase quantitative real-time PCR, methylation-specific PCR and Western blot to measure the level of miR-148a expression, DNA methylation level and DNMT1 expression, respectively. Gastric cancer cells were transfected with plasmid or siRNA or treated with 5-aza-2'-deoxycytidine. Cell proliferation and apoptosis were detected by cell counting and flow cytometric analysis. In this study, we demonstrated that gastric cancer tissues and cell lines displayed a consistent down-regulation of miR-148a and hypermethylation of promoter region. DNMT1 was over-expressed in primary tumors and cell lines, while knockdown of DNMT1 using siRNA could decrease methylation level of miR-148a promoter and restore its expression. Furthermore, ectopic over-expression of miR-148a in cancer cell lines caused reduction in DNMT1 expression and inhibited cell proliferation, but no obvious change was found in apoptosis rate. These results suggest that miR-148a is inactivated by DNA hypermethylation of promoter region in gastric cancer, which is mediated through DNMT1 over-expression. Additionally, the silence of miR-148a reduces its suppression to DNMT1 in gastric cancer, and this may in turn result in over-expression of DNMT1 and promote DNA hypermethylation.
Underexpression of the gene runt-related transcription factor 3 (RUNX3), an important tumor suppressor, is known to contribute to gastric cancer progression. However, the mechanism underlying aberrant RUNX3 expression has not been fully elucidated. We investigated the role of microRNA-148a (miR-148a) and DNA methyltransferases (DNMTs) in RUNX3 promoter methylation and gene expression. RUNX3 mRNA, RUNX3 protein, and methylation levels were assayed in human gastric cancer tissues and matched normal tissues, and AGS and BGC-823 cells by real-time reverse transcription PCR, Western blot, and methylation-specific PCR, respectively. A correlation between RUNX3 mRNA levels and that of miR-148a was also investigated in gastric cancer tissues. We found that RUNX3 mRNA levels were significantly downregulated in gastric cancer tissues compared with their matched normal tissues, and were closely associated with miR-148a expression. After treatment of human gastric cancer AGS and BGC-823 cells with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, a significant increase in RUNX3 mRNA, RUNX3 protein, and the non-methylated form of the RUNX3 promoter were observed relative to untreated cells. Enforced expression of miR-148a, which can modulate DNMT1 and DNMT3B, also increased the expression of RUNX3 in gastric cancer cells. Knockdown of DNMT1 was associated with increased levels of RUNX3 mRNA and RUNX3 protein, while knockdown of DNMT3B did not have any effect on these in BGC-823 cells. Our results show that miR-148a may regulate RUNX3 expression through modulation of DNMT1-dependent DNA methylation in gastric cancer and highlight a miRNA-epigenetics regulation mechanism of gene expression.
IntroductionThe nutritional status of patients with gastric cancer (GC) after total gastrectomy continues to deteriorate and lasts a long time after discharge, which is an independent risk factor for mortality. Recent guidelines have recommended appropriate nutritional support after discharge for cancer surgery patients with malnutrition or nutritional risk. The evidence on the efficacy of oral immunonutritional supplement (INS) and its effect on long-term disease-free survival (DFS) in patients with GC is limited. This study was designed to test the hypothesis that oral INS compared to diet alone may improve 3-year DFS of GC patients with pathological stage III after total gastrectomy (Nutrition Risk Screening 2002 score ≥3 at discharge).Methods and analysisThis is a pragmatic, open-label, multicentre, randomised controlled study. 696 eligible GC patients with pathological stage III after total gastrectomy will be randomised in a 1:1 ratio to oral INS group or normal diet group for 6 months. The primary endpoint is 3-year DFS after discharge. The following secondary endpoints will be evaluated: 3-year overall survival; unplanned readmission rate at 3 and 6 months after discharge; quality of life, body mass index and haematological index at 3, 6 and 12 months after discharge; incidence of sarcopenia at 6 and 12 months after discharge; and the tolerance to chemotherapy. The adverse events of oral INS will also be evaluated during the intervention.Ethics and disseminationThis study was approved by the ethics committee of Jinling Hospital, Nanjing University (number 2021NZKY-069-01). The present study may validate the effectiveness of oral immunonutritional therapy in improving 3-year DFS for GC patients with pathological stage III after total gastrectomy for the first time. The results of this trial will be disseminated in peer-reviewed journals and at scientific conferences.Trial registration numberNCT05253716.
Background: STAD ranked 5th most common in the incidence of malignant tumors and 3rd most common in the death rate of cancer worldwide. CXC chemokines affect the biological progress of various tumors, resulting in therapeutic failure. The role of CXCL2 in STAD was still a mystery. Methods: The expression, prognostic value, and clinical function of CXCL2 were analyzed using several online bioinformatics tools and clinical tissues. Results: CXCL2 level was significantly upregulated in STAD tissues. Strong correlation was obtained between CXCL2 level and immune cells as well as immune biomarkers. High CXCL2 expression in STAD was correlated with a favorable prognosis. Further analysis revealed that CXCL2, pTNM stage and age were independent factors affecting the prognosis of STAD patients. A predictive nomogram indicated that the calibration plots for the 1-year, 3-year and 5-year OS rates were predicted relatively well compared with an ideal model in the entire cohort. Validation analysis revealed that CXCL2 expression was upregulated in STAD and high CXCL2 level had a better overall survival. CXCL2 was associated with resistance to numerous drugs or small molecules in STAD. Conclusions: We identified CXCL2 as a novel therapeutic target and associated with immune infiltration in STAD.
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