In the research, GSE113486, GSE112264 and GSE113740 selected from the GEO database were analyzed with the bioinformatics tools to get the key blood miRNAs in gastric carcinoma. RRA analysis were performed to get the ranking of the corresponding miRNAs. Then the 20 most differentially expressed blood miRNAs (10 up-regulated and the other 10 down-regulated) became the aims to predict the target genes and the relative biological activities. There were 691 up-regulated differentially expressed genes and 1074 down-regulated genes which might be directly silenced by the 20 blood miRNAs. GO and KEGG enrichment analysis were proceeded on the differentially expressed genes by DAVID. The results suggested the biological processes and the signaling pathways in which the differentially expressed genes were mainly involved in this system. On this basis, analysis of the target genes – encoded proteins with PPI network suggested five crucial blood miRNAs which were hsa-miR-4276, hsa-miR-575, hsa-miR-125a-3p, hsa-miR-124-3p and hsa-miR-29b-3p. The results in human blood detection and survival curve analysis confirmed the important roles of the key blood miRNAs in gastric carcinoma. ROC analysis suggested hsa-miR-124-3p, hsa-miR-125a-3p and hsa-miR-29b-3p could be useful in gastric cancer diagnosis, especially in combination.
MiRNAs have been identified as a key regulator in complex biological processes, including the pathogenesis of cancer . Human cancers express the characteristics such as sustained proliferation signals, activation of invasion and metastasis, angiogenesis, escape from immune destruction and so on . MiRNAs can be involved in the every feature and affect multiple signal pathways . Blood miRNAs are becoming one of the most useful biomarkers in the diagnosis and treatment of tumors. MiRNAs have the specific expression profile in different cancer cells and tissues which can enter the circulation . Circulating free miRNA can be detected in blood, plasma and other body fluids . Mature miRNAs are very stable in body fluids  and have high specificity in different cancer states , which determines miRNAs a potential non-invasive tumor marker. There have been some research on the blood miRNAs in gastric carcinoma, such as miR-210, miR-1, miR-20a, miR-34a, miR-423-5p, and so on [17, 18]. In this research, with the bioinformatics tools, five new blood miRNAs, which were hsa-miR-125a-3p, hsa-miR-124-3p, hsa-miR-29b-3p, hsa-miR-4276 and hsa-miR-575, especially hsa-miR-124-3p and hsa-miR-125a-3p, might be the potential targets in gastric carcinoma diagnosis and treatment. The predicted key miRNAs and the relative target genes were also involved in the biological activities which are closely correlant to tumorigenesis. For example, platelet-derived growth factor receptor signaling pathway and transcription RNA polymerase II both play important roles in gastric carcinoma and development [19, 20]. Focal adhesion and PI3K-Akt signaling pathway are closely related to the carcinogenesis [21, 22]. These results detected the importance of the selected blood miRNAs in gastric carcinoma.
It has been explored that hsa-miR-125a-3p could influence breast cancer stem cells by targeting leukemia inhibitory factor receptor and regulating the Hippo signal pathway . In addition, hsa-miR-125a-3p is related to the tumorigenesis of prostate cancer, colon cancer, and so on . However, the research of hsa-miR-125a-3p on gastric carcinoma is still few. In this research, the abnormal expression of hsa-miR-125a-3p was identified in the blood of gastric cancer patients, and the correlation between its expression and disease might be related to age and gender. At the same time, the downstream target of hsa-miR-125a-3p was predicted as PR domain zinc finger protein 1 (PRDM1) gene. PRDM1 is the protein involved in regulating the differentiation of B cells and T cells, which plays an important role in immunosuppression . Previous reports have shown that PRDM1 is associated with many kinds of cancers, such as adrenocortical cancer, colon cancer, acute myeloid leukemia, brain cancer and lung cancer . The expression of PRDM1in non-germinal center B cell-like (non-GCB) patients was also associated with a worse prognosis . In this research, PRDM1also showed its correlation with the survival possibility of gastric cancer patients. Therefore, it may have high probability that hsa-miR-125a-3p is participating in the gastric carcinogenesis by inhibiting the expression of PRDM1, which need in-depth research later.
By tracing the upstream miRNAs of the target genes, the results of Cytoscape which screened out key node genes from the PPI network showed the appearance frequency of hsa-miR-124-3p was the highest in the key miRNAs. However, there was no clear conclusion about the relationship between hsa-miR-124-3p and tumorigenesis in the miRbase database. Both the examination in human blood species and the analysis of the survival curve suggested the correlation between hsa-miR-124-3p and the gastric carcinogenesis. The further ROC analysis also suggested the importance of hsa-miR-124-3p in gastric cancer diagnosis. In the predicted target genes of hsa-miR-124-3p, a number of genes had a higher correlation with the prognosis of gastric cancer, such as ANXA5 and CAV1. ANXA5 is a member of the Annexin family and is involved in the tumorigenesis and development of a variety of cancers . It plays the role on gastric cancer by regulating the ERK signal pathway . Then low expression of CAV1 means decreased expression of E-cadherin, cell morphology changes and an increase in the migration ability of gastric cancer cells . Other miRNAs, such as miR-6792-3p, can also be involved in gastric carcinogenesis by their inhibitory effect on the target CAV1 . So, it can be speculated that hsa-miR-124-3p may facilitate gastric carcinoma by negatively regulating multiple downstream target genes in one network.
Hsa-miR-29b-3p plays a key role in the tumorigenesis of glioblastoma  and metastatic colorectal cancer . Hsa-miR29b-3p can also regulate the expression of VEGFA in pancreatic ductal adenocarcinoma . In addition, as one of the important target genes of hsa-miR-29b-3p, activation of PER1 can inhibit the progression of pancreatic cancer . Although the blood check results didn’t display the correlation between hsa-miR-29b-3p and gastric cancer, the analysis of the patient prognosis and diagnostic value analysis detected the importance of hsa-miR-29b-3p in gastric cancer diagnosis. Blood miR-125-3p, hsa-miR-124-3p and hsa-miR-29b-3p together showed the highest AUC value in ROC curve, and the second one is for the combination of miR-125-3p and hsa-miR-124-3p, while the third is among the single miRNAs. The joint detection of the key blood miRNAs could be the biomarkers for the diagnosis and treatment monitor in gastric cancer. The role of the miRNAs on the negatively regulation of target genes can also be studied deeply.
MSRB3 has been identified to be the key protein that can regulate the proliferation and migration of gastric cancer cells which might be an effective marker to predict gastric cancer peritoneal metastasis and poor prognosis [36, 37]. MSRB3 was predicted the target gene of hsa-miR-575 in this research. It was also detected in blood check that hsa-miR-575 was differentially expressed in gastric cancer, and the correlation between its expression and disease might be related to age and gender. Hsa-miR-575 might has the trend correlated with the prognosis of gastric cancer patients, but without significant difference. More research will be needed to determine the role of hsa-miR-575 which can negatively regulateMSRB3 in gastric carcinogenesis.
Hsa-miR-4276 is involved in the apoptosis process of pulmonary endothelial cells during acute lung injury . There is few relevant research to prove that hsa-miR-4276 is related to tumorigenesis, also with its target genes RNF217 and IP6K1. On the other hand, among the key nodes obtained by Cytoscape, the number of downstream genes of hsa-miR-4276 was 24 times. This suggested that hsa-miR-4276 and its target genes may have a strong correlation with gastric cancer. Unfortunately, none of the results from patient blood check, prognosis analysis, and ROC analysis suggested this possibility. However, it might need more in-depth exploration.
In summary, all the research implied the accuracy and feasibility with the abnormal expression level of the key miRNAs in blood for gastric cancer prevention, diagnosis and treatment result testing. Through bioinformatics analysis and check in human blood species, five key blood miRNAs could be involved in gastric carcinoma which were hsa-miR-125a-3p, hsa-miR-124-3p, hsa-miR-29b-3p, hsa-miR-575 and hsa-miR-4276, especially the combination of hsa-miR-125a-3p, hsa-miR-124-3p and hsa-miR-29b-3p. More research is needed in the future to further confirm the mechanism and biological activities of the miRNAs in gastric carcinoma. The blood miRNAs may become new biomarkers for the early diagnosis of gastric cancer.