With the development of technology, the diagnosis and treatment of GC have made great progress. But the recurrence and metastasis of tumor are important factors affecting prognosis. Then, Lymph node metastasis is the most common form of metastasis in GC. To investigate the mechanism of lymph node metastasis and to estimate the status of its are urgent. Intestinal-type GC is preceded by premalignant lesions, including chronic atrophic gastritis and intestinal metaplasia. Diffuse type GC is more common in young patients, in whom there is a female preponderance and behaves more aggressively than the intestinal type17,18. So, we thought that investigating GC as the whole could lose the specific subtype related discoverable points. Previous studies showed that ceRNA network19-25 and immune cells fraction 26 27 28 existing in or influencing the many carcinomas’ tumorigenesis, progress and therapy. We supposed that these may also have an effect on lymph node metastasis. In present study, we screened differentially expressed mRNAs and lncRNAs and constructed ceRNA network for GC’s lymph node metastasis to reveal its potential functions and mechanisms. The immune cell fractions were evaluated via CIBERSORTx. The lncRNAs, mRNAs and immune cell fractions selected by means of SVM-RFE were used to establish the SVM model for predicting the status of lymph node metastasis in the whole, intestinal and diffuse cohort, respectively. The results showed that the predicting effect of model in the intestinal and diffuse cohort were superior to that in the whole cohort, which proved their excellent clinical application.
In this study, we systematically integrated gene expression profiles and identified lncRNAs aand mRNAs in GC. And we didn’t identify differentially expressed miRNAs, the reason is that we thought miRNA work as intermediate in the ceRNA network and an extreme example proving our ideas is that when the miRNA’s expression is constant, the ceRNA network still works. So, using differentially expressed miRNAs for constructing ceRNA network could lead to the neglect of important lncRNA-miRNA-mRNA triples. We investigated the DElncRNAs, miRNAs and DEGs in the ceRNA network and took the intersection to RNAs of three cohort, respectively. There are not only common RNAs, but also specific subtype’s RNAs, which proved our hypothesis that the behavior of lymph node metastasis is different in Lauren subtype. Of course, the detailed mechanism needs us to further explore.
The performance of prediction model of three cohort using significant signatures after SVM-RFE perfectly illustrated different Lauren subtypes had different mechanism of lymph node metastasis. Probably because of fewer mRNAs, the few KEGG enrichment pathways were significant (p < 0.05). The KEGG enrichment pathways and GO terms of the diffuse cohort didn’t need to be paid more attention, which the reason was that the number of selected mRNAs of the diffuse cohort was small. We found the intrinsic component of membrane of GO terms were common in the whole and intestinal cohort, which meant the intrinsic component of membrane maybe related with lymph node metastasis. In addition, the cell-cell signaling by wnt was specific for the whole cohort and vesicle lumen, catalytic activity, positive regulation of nitrogen compound metabolic process, cellular anatomical entity and so on were specific for the intestinal cohort.
We observed the differences in the components of immune cells between lymph node metastasis and non-metastasis and found significant NK cells activated, Macrophages M0, Macrophages M2, Mast cells resting and Neutrophils for the whole cohort, NK cells resting, dendritic cells resting and mast cells resting for the intestinal cohort and B cells memory, Plasma cells, T cells CD8 and Macrophages M0 for the diffuse cohort. The tumor microenvironment contains innate and adaptive immune cells, which display pro or anti-tumor functions 29. Evidence accumulated from many cancer models suggested that macrophages 30 31 32, Dendritic cells 33 34, Mast cells 35 36 and NK cells 37 contributed to the lymph node metastasis of tumors. Significantly different immune cells just supported their association with lymph node metastasis.
In the co-expression of the intestinal cohort, our study suggested that dendritic cells were significantly associated with CD70. CD70 is not only implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27 38. Moreover, CD70 was reported that it was related with dendritic cells and NK cells 39 40. Thus,
There were several limitations of our study that should be acknowledged. The public data we use are all data on the population of Western countries, and this conclusion should be applied cautiously to Asian countries. Because we're analyzing Lauren subgroup population data, the number of cases in the subgroup of diffuse is relatively small, which may lead to less reliable results. Moreover, many inferences need to be proved by further experiments.
Although there are some inadequacies in our research, it still has many bright spots. First of all, our research established the ceRNA network and combined immune cells using CIBERSORTx, and these are based on two Lauren subgroups, which can reduce the effect of tumor heterogeneity. Then, the prediction results of SVM model also demonstrated that it was correct for us to classify and analyze patients with GC. The good performance of SVM model meant it could be used in clinical diagnosis.