HOXA13 Promotes The Proliferation And Metastasis Of Gastric Cancer Partly Via Activating Erk1/2

Part Ⅰ Homeobox A13(HOXA13)expression in gastric cancer tissuesObjective: To study the expression of HOXA13 in gastric cancer(GC)tissues,and to analyze the relationship between HOXA13 and Ki67.Methods: We examined the expression of HOXA13 in normal mucosae and GC tissue specimens by q RT-PCR、Western blot and IHC.Searching the microarray data sets from the Oncomine database,we analyzed the expression of HOXA13 in GC tissues.We analyzed the correlation between HOXA13 and Ki67 using Pearson’s correlation.Results:(1)We found that the expression of HOXA13 was higher in GC tissues than in normal mucosae.(2)A statistically significant positive correlation between HOXA13 and Ki67 expression was verified(r = 0.539,P < 0.01).Conclusion:These findings suggested the overexpression of HOXA13 in GC tissues,and a positive correlation is detected between HOXA13 and Ki67 expression.Part Ⅱ The effect of HOXA13 on the proliferation and metastasis of GC cells in vitroObjective: Using cell functional assays to study the effect of HOXA13 on the proliferation and metastasis of GC cells,to study the role of HOXA13 in GC development.Methods: We overexpressed HOXA13 in AGS by infecting the cells with the lentiviral vector containing HOXA13 coding region,and knockdowned HOXA13 expression in SGC-7901 by infecting the cells with the lentiviral vector containing HOXA13 sh RNA.To observe the effect of HOXA13 expression on cell proliferation,we performed CCK-8 and plate colony formation assays.Flow cytometry-based cell cycle analysis was used to analyze the effect of HOXA13 on cell cycle.To evaluate whether HOXA13 affected GC cell migration and invasion,we performed wound healing and Transwell assays.We also examined Cyclin D1 and biomarkers related to EMT process by Western blot.Results:(1)The overexpression of HOXA13 increased cell proliferation in GC cells in vitro.(2)Flow cytometry-based cell cycle analysis showed that the overexpression of HOXA13 increased the percentage of cells in the S phase.(3)HOXA13 promoted GC cells migration and invasion in vitro.(4)The overexpression of HOXA13 led to elevated expression of Cyclin D1,and both N-cadherin and Vimentin were increased with HOXA13 overexpression,conversely,E-cadherin expression was decreased.Conclusion: HOXA13 promotes the proliferation and metastasis behavior of GC cells in vitro.Part Ⅲ The potential mechanisms of HOXA13-mediated proliferation and metastasis in GC cellsObjective: To further explore the potential mechanisms of HOXA13-mediated proliferation and metastasis in GC cells.Methods: RNA-Seq transcriptome analysis was used to compare the transcriptome in AGS-HOXA13 and AGS-Vector cells.Gene ontology(GO)analysis was performed to elucidate the biological implications of unique genes in the significant or representative profiles.All data were analyzed to characterize biological processes(BP),cellular components(CC)and molecular functions(MF).Pathway analysis was performed to identify the significant pathways of the differentially expressed genes according to the KEGG database.This result was confirmed by Western blot and q RTPCR.Results:(1)A total of 1042 genes were certified to be differentially expressed(HOXA13/Vector ratio >1.50-fold,P <0.05).(2)The interactive network among these enriched pathways revealed that the MAPK signaling pathway was located in the core position.Therefore,we inferred that the MAPK signaling pathway may be activated in HOXA13 overexpression cells,and its activation may mediate malignant biological properties resulting from HOXA13.(3)Western blot showed that HOXA13 overexpression increased Erk1/2 phosphorylation,whereas HOXA13 knockdown decreased Erk1/2 phosphorylation.Interestingly,we found that VEGFA,EGFR,FGF2 and FGFR2,which exhibited positive regulation of the Erk1/2 cascade according to GO analysis,were increased according to RNA-Seq transcriptome analysis,which was confirmed by q RT-PCR.Conclusion: HOXA13-mediated proliferation and metastasis might occur through the activation of Erk1/2 signaling.Part Ⅳ The effect of HOXA13 on the proliferation and metastasis of GC cells in vivoObjective: To explore the role of HOXA13 in the tumorigenicity and metastasis of GC cells in vivo.Methods: Four-week-old male BALB/C nude mice were used to establish GC xenografts and metastasis in vivo.The mice were then randomly divided into four groups(n = 5)to establish GC xenografts.Then,AGS-HOXA13,AGS-Vector,SGC-7901-Scramble or SGC-7901-sh-HOXA13 was injected into the subcutaneous tissues of nude mice.All mice were sacrificed three weeks after injection.Tumor volume was calculated.Paraffin sections of the xenograft tissues were prepared for IHC staining.For metastatic assays,SGC-7901-Scramble or SGC-7901-sh-HOXA13 were injected into the abdominal cavities of nude mice.Each group included three mice.After four weeks,all mice were sacrificed,and the peritoneal metastatic nodules were measured.Results:(1)HOXA13 overexpression promoted the tumorigenicity of GC cells in vivo.(2)HOXA13 knockdown inhibited GC cells metastasis in vivo.Conclusion: HOXA13 enhances tumor growth and metastasis of GC cells in vivo.