The Cytological Characteristics And Molecular Mechanism Of Lymphatic Metastasis In Gastric Cancer Were Studied By Single Cell RNA Sequencing

Objective To investigate the changes of cytological characteristics and molecular mechanism related to lymphatic metastasis in the development and progression of gastric adenocarcinoma.Methods The gastric cancer and adjacent tissues of patients with positive and negative lymph node metastasis who underwent radical gastrectomy in the gastrointestinal surgery of the Second Affiliated Hospital of Anhui Medical University were collected.The cytological characteristics and related marker genes in the samples were revealed by single-cell RNA sequencing.The expression levels of chief cell 3 marker gene XIST and pit mucous cell 3 marker gene CXCL5 were detected by real-time quantitative PCR.The expression levels of XIST and CXCL5 in gastric cancer tissues of patients with positive and negative lymph node metastasis were analyzed by independent sample t-test.The difference was statistically significant（P < 0.05）.Results（1）Single cell RNA sequencing was performed on gastric cancer and adjacent tissues of two patients.A total of 11 main cell types and corresponding marker genes were identified.Comparing the cell types of gastric cancer,the proportion of chief cells in lymph node metastasis positive gastric cancer tissues was significantly higher than that in lymph node metastasis negative gastric cancer tissues.（2）According to the automatic clustering of the transcription spectrum of the chief cells,the chief cells were divided into four subtypes.Through the analysis of the transcription trajectory,it was found that the transcription transition state from chief cell 2,chief cell 3 and chief cell 1to cancer cells.Chief cell 3 was significantly enriched in gastric cancer tissue samples from patients with positive lymphatic metastasis.（3）According to the automatic clustering of the transcriptional spectrum of pit mucous cells,pit mucous cells were divided into three subtypes.Among them,pit mucous cells 3 were mainly enriched in gastric cancer tissue samples of patients with positive lymphatic metastasis,and had a strong interaction with cancer cells.（4）The expression level of XIST was（0.297 ±0.261）in gastric cancer tissues with positive lymphatic metastasis and（0.025 ± 0.023）in gastric cancer tissues with negative lymphatic metastasis.The expression level of XIST in gastric cancer tissues with positive lymphatic metastasis was significantly higher than that in gastric cancer tissues with negative lymphatic metastasis.The expression level of CXCL5 was（0.036 ± 0.019）in gastric cancer tissues with positive lymphatic metastasis and（0.008 ± 0.007）in gastric cancer tissues with negative lymphatic metastasis.The expression level of CXCL5 in gastric cancer tissues with positive lymphatic metastasis was significantly higher than that in gastric cancer tissues with negative lymphatic metastasis.（5）The expression levels of XIST and CXCL5 were associated with overall survival and progression free survival,and XIST was associated with worse overall survival and progression free survival in patients with gastric cancer.Conclusions Chief cell 3,pit mucous cell 3 and their marker genes XIST and CXCL5 are expected to be molecular biomarkers for predicting lymphatic metastasis and treatment of gastric cancer.