| Purpose: Gastric cancer is the one of the most common malignancy in China.Although developments in diagnostic modalities have increased the diagnosis of early gastric cancer,most patients present with advanced diseases when they are diagnosed with gastric cancer.The Borrmann classification system is now widely used to classify advanced gastric cancer.Borrmann type 4 gastric cancer is characterized by a thickened and indurated gastric wall without a clear margin of tumor;this shows a special biological behavior compared to other types of gastric cancer.Several studies have reported that patients with Borrmann type 4 gastric cancer have a significantly poorer prognosis compared to those with other types of gastric cancer.However,all of these studies included both patients who underwent radical surgery and those who did not undergo radical surgery.It has been confirmed that surgical curability is one of the independent prognostic factors for the survival of Borrmann type 4 gastric cancer patients,and the prognosis of patients with Borrmann type 4 gastric cancer could be markedly increased by R0 resection.Thus,whether Borrmann type 4 gastric cancer still has prognostic significance in gastric cancer patients when only patients who underwent radical surgery were selected is highly uncertain.At present,the current tumor-node-metastasis(TNM)staging system,which consists of penetration depth of the primary tumor(pT),the number of metastatic lymph nodes(pN),and the presence of distant metastasis(pM),are considered to be the most important prognostic factors in patients with gastric cancer.Recently,several papers reported that addition of macroscopic pathological features of primary tumor,such as macroscopic serosal classification and width of serosal invasion,might improve pT classification prediction of prognosis in patients with advanced gastric cancer after radical surgery.Unfortunately,the impact of Borrmann classification on pT classification has not been reported so far.In the first part of the study,we aimed to investigate the prognostic value of Borrmann type 4 gastric cancer in advanced gastric cancer patients who underwent radical surgery with special attention paid to the feasibility of Borrmann type 4 gastric cancer incorporated into the pT classification.Clinically,Borrmann type 4 gastric cancer often involves the whole layer of the gastric wall and spreads to the whole stomach,accompanied by extensive lymph node metastasis and peritoneal metastasis.Even after comprehensive treatment such as surgery and chemotherapy,recurrence and metastasis will occur in a short period of time.With the improvement of diagnosis and treatment of gastric cancer,the overall efficacy and long-term survival of advanced gastric cancer have been improved in recent years,but the therapeutic effect of Borrmann type 4 gastric cancer is still poor.The special malignant biological behavior of Borrmann type 4 gastric cancer is the main reason for its poor therapeutic effect.Therefore,it is very important to study the molecular mechanism of Borrmann type 4 gastric cancer’s malignant biological behavior.High-throughput screening is an effective way to identify tumor regulators.Through high-throughput screening technology,researchers can quickly and efficiently detect differentially expressed genes,and combine various gene analysis software and databases to screen out potential tumor regulators for further research and verification.Currently,two high-throughput screening tools are commonly used in tumor research: gene chip and high-throughput sequencing.Compared with the traditional chip hybridization platform,high throughput sequencing has the advantages of more accurate quantification,higher detection flux and wider detection range.In the second part and the third part of this study,we detected differentially expressed genes between OCUM-1 and AGS by high-throughput RNA-seq.With genetic analysis software and network database analysis,we identified LIPC(lipase C,hepatic type)might be a regulative gene of Borrmann type 4 gastric cancer.Next,we detected the expression of LIPC in Borrmann type 4 gastric cancer tissues and analyzed its relationship with clinicopathological features and prognosis.Then we examined the regulation effect of LIPC on the malignant biological behavior of Borrmann type 4 gastric cancer and explored the molecular mechanism.Methods: The first part of the study: 1622 patients with gastric cancer who underwent surgery at the Department of Surgical Oncology,First Affiliated Hospital of China Medical University,from January 1980 to December 2005 were collected.All statistical analyses were performed with the SPSS 19.0 statistical package(SPSS Inc.,Chicago,IL,USA).The categorical variables were compared using a 2-tailed chi-square test.The continuous data was presented as a mean±standard deviation(SD),and 2-tailed t test was used for the statistical comparisons.All the clinicopathologic features that were significantly associated with Borrmann type IV gastric cancer were included in the logistic regression analysis(stepwise forwards method)to identify the independent predictors of the presence of Borrmann type IV gastric cancer.Overall survival rates were determined using the Kaplan–Meier estimator,and the log-rank test was used to identify differences between the survival curves of different patient groups.Cox’s proportional hazard model was used as multivariate analysis to identify independent factors predicting patients’ prognosis.For all analyses,P values were 2-tailed,and only P values<0.05 were considered significant.The second and third part of the study: Illumina HiSeq 4000 system was used for high-throughput detection of differentially expressed genes between Borrmann type 4 gastric cancer cell line OCUM-1 and low-differentiation gastric cancer cell line AGS.GO analysis software and Pathway analysis software were used to analyze the gene functions of differentially expressed genes.The prognostic significance of LIPC in gastric cancer was analyzed with network database KM plotter.Immunohistochemistry was used to detect the expression of LIPC in gastric cancer and adjacent normal tissues.A shRNA lentiviral plasmid targeting LIPC was constructed to infect Borrmann type 4 gastric cancer cell line OCUM-1,and a stable OCUM-1 cell line with low LIPC expression was constructed.We used Transwell invasion assay,Transwell migration assay,CCK-8 assay and flow cytometry to detect the effect of knockdown of LIPC expression on invasion,migration,proliferation,cell cycle and apoptosis of OCUM-1.In addition,Western Blot was used to detect the expression changes of EMT-related factors and invasion-related factors of OCUM-1.Results: 1.Borrmann type IV gastric cancer was one of the independent prognostic factors for patients with advanced gastric cancer who underwent radical surgery.Further analysis demonstrated that the prognosis of Borrmann type IV gastric cancers was homogeneous with that of pT4 b cancers.2.The results of gene RNA-seq showed that,compared with AGS,a total of 2,544 genes were up-regulated and 2,775 genes were down-regulated in OCUM-1.Both GO analysis and Pathway analysis of differentially expressed genes indicated that some differentially expressed genes were closely related to metabolic functions,and LIPC,a lipid metabolism-related gene,was 44 times more expressed in OCUM-1 than in AGS.Network database KM plotter analysis showed that the survival of gastric cancer patients with positive LIPC expression was significantly worse than that of patients with negative LIPC expression.3.Immunohistochemical results showed that LIPC was almost not expressed in adjacent normal tissues,while the positive expression rate was significantly increased in gastric cancer tissues,and the positive expression rate of LIPC in Borrmann type 4 gastric cancer tissues was significantly higher than that in other Borrmann type gastric cancer tissues.Further analysis revealed that positive expression of LIPC in Borrmann type 4 gastric cancer was correlated to lymph node metastasis,TNM stage and worse prognosis.Multivariate analysis showed that positive expression of LIPC was an independent prognostic factor for Borrmann type 4 gastric cancer.4.Transwell invasion assay,Transwell migration assay,CCK 8 assay,and flow cytometry showed that LIPC regulated migration and invasion ability of OCUM-1,but had no effect on proliferation,cell cycle and apoptosis.5.Western Blot showed that knockdown of LIPC expression of OCUM-1 up-regulated E-cadherin expression and down-regulated Snail and MMP2 expression.Conclusions:1.Borrmann type IV gastric cancer is one of the independent prognostic factors for patients with advanced gastric cancer who underwent radical surgery.Borrmann type 4 gastric cancer should be classified as pT4 b disease.2.Positive expression of LIPC in Borrmann type 4 gastric cancer is correlated to lymph node metastasis,TNM stage and worse prognosis.Positive expression of LIPC is an independent prognostic factor for Borrmann type 4 gastric cancer.3.LIPC regulates invasion and migration of Borrmann type 4 gastric cancer cells.The regulation may be through Snail/ E-cadherin pathway and Snail/ MMP-2 pathway. |
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