A Preliminary Study On The Expression And Clinical Significance Of HOXB13 In Gastric Adenocarcinoma

Objective:Gastric cancer(GC)is the third most common cause of cancer death globally.Compared with stomach adenocarcinoma(STAD),gastric signet ring cell carcinoma(GSRCC)is highly malignant and the prognosis is poor.To investigate driver genes in GC and find the biomarkers for the early diagnosis and prediction of efficacy of immunotherapy in GC,we identified HOXB13,the co-upregulated genes in both high-grade and low-grade malignant tissues,and further explored the expression of it in GC.We also investigated the effects of HOXB13 on proliferation,migration,and invasion of gastric adenocarcinoma cells and the value of it in predicting the response of immunotherapy.Methods:Samples of GSRCC and STAD were compared to those of paracancerous mucosa using Agilent microarray technique,and HOXB13 was identified as the most significant differentially expressed gene(DEG)in GSRCC and STAD tissues.HOXB13 expression in GC and non-GC tissues of GC patients were analyzed using Gene Expression Profiling Interactive Analysis(GEPIA)and Oncomine Database Analysis.The Kaplan-Meier plotter was used to analyze the prognostic value of HOXB13 in GC.The expression of HOXB13 in GC and its relationship with various clinicopathological parameters were analyzed by The Cancer Genome Atlas(TCGA)database.Correlation and enrichment analyses of HOXB13 were performed.We collected 60 pairs of surgical specimens of gastric adenocarcinoma and corresponding paracancerous gastric tissues.q PCR and Western blot were used to detect the HOXB13 m RNA and protein levels in samples.Gastric adenocarcinoma cell lines(AGS and MKN45 cell lines)with HOXB13 knockdown were established.The cell proliferation was detected by CCK8 assay and cell migration and invasion abilities were examined by Transwell assays.The Tumor Immune System Interactions Database(TISIDB)was used to analyze the relationships between expression of HOXB13 and immune subtypes and molecular subtypes in GC.Correlation between HOXB13 and immune cell infiltration,immunostimulator,immunoinhibitor,MHC,chemokine and its receptors and targets in cancer immunotherapy was analyzed by TISIDB.The Tumor Immune Dysfunction and Exclusion(TIDE)algorithm was employed to predict the relationship between HOXB13 expression level and immunotherapy response in GC.Results:1.High expression of 9 genes was observed in both GSRCC and STAD tissues compared to their paracancerous tissues respectively and HOXB13 was chosen as the most significant DEGs(P < 0.05).2.Both GEPIA and Oncomine Database showed that the expression of HOXB13 was significantly increased in GC tissues compared with paracancerous tissues.We discovered that HOXB13 expression was significantly correlated with age and histologic grade in STAD by TCGA database(P < 0.05).Enrichment analysis revealed that HOXB13 was implicated in several biological process(BP)including pattern specification process and cell fate specification.3.The results of q PCR showed that the expression of HOXB13 was higher in GC samples compared with normal samples(P < 0.01).Western blot revealed that HOXB13 protein expression was higher in GC samples compared with normal samples(P < 0.05).4.The m RNA and protein expression level of HOXB13 were significantly increased in gastric csncer cell lines(AGS and MKN45)compared with normal gastric mucosa cell lines(GES-1).5.Cellular proliferation,migration and invasion abilities in the HOXB13 knockdown group(sh-HOXB13 AGS、sh-HOXB13 MKN45)were significantly lower than those in the control group,the differences were statisticlly significant(P < 0.05).6.A negative correlation between HOXB13 expression and immunoinhibitory immune cell infiltration was identified by TISIDB.HOXB13 expression was also correlated with immunostimulator,immunoinhibitor,MHC,chemokine and its receptors and targets in immunotherapy.7.High level of HOXB13 expression was associated with lower TIDE score(P <0.05),the difference was stastically significant(P < 0.05).8.KM-plotter showed that patients with high HOXB13 expression had a significantly better OS and DFS than those with low HOXB13 expression(P < 0.05).Conclusions:We identified HOXB13,the co-upregulated gene expressed in both high-grade malignancy and low-grade ones,as a potential driver gene that might involve in the beginning event of tumor development.The expression of HOXB13 was significantly increased in GC tissues compared with paracancerous tissues.Inhibition of HOXB13 attenuated GC cell proliferation,migration and invasion in vitro.These data were not consistent with our clinical results.Besides tumor-cell intrinsic alterations,HOXB13 also displayed reprogramming of tumor microenvironment.We found that the high expression of HOXB13 suggested decreased immune suppression and a better response to immunotherapy.