| Background and AimsThe homeobox(Hox)superfamily genes,including A,B,C and D clusters,encode transcription factors and usually regulate the expression of target genes at transcription level.Dysregulation of Hox genes will cause the abnormality in individual development and tissue formation,and may also lead to malignant transformation.So far,the study of HoxC10 in cancer is relatively limited.Recently,it was found that the expression of HoxC10 was elevated in primary breast cancer,and even more significantly in distant metastasis arising after failed chemotherapy.High HoxC10 expression was related to the poor survival in patients with estrogen receptor-negative breast cancer undergoing chemotherapy.Another study demonstrated that HoxC10 was overexpressed in cervical cancer tissues and linked with the invasiveness of cervical cancer cells.To date,the function and molecular mechanisms of HoxC10 in GC remains poorly understood.Gastric cancer(GC)is one of the most common malignancies worldwide.The development of GC is considered to be a process with multiple stages.A multitude of factors contribute to the progression of GC,including activation of oncogenes and inactivation of tumor suppressors.Finding key molecules and their mutual relations in gastric carcinogenesis is of great significance for the diagnosis and treatment of GC.Our group have previously screened by GC tissue microarray and found that the expression of HoxC10 was remarkably upregulated in GC.To clarify the function and molecular mechanism of HoxC10 in GC,we carried out a further study.Material and Methods1.The expression of HoxC1O in GC tissues and its clinical significance:By using two GC tissue cohorts from Sir Run Run Shaw Hospital and the First Affiliated Hospital of Zhejiang University,we analyzed HoxC10 expression in GC and the corresponding adjacint non-tumor tissues,as well as its correlation with clinical pathological parameters.In addition,we also verified the results by utilizing The Cancer Genome Atlas(TCGA)database and KM-plotter survival analysis database.2.The effects of HoxC10 on GC cell biology behaviors:By altering the expression of HoxC10(overexpressing and knocking down)and combination of in vitro cell function tests and in vivo nude mice models,we studied the effects of HoxC10 on GC cell biology behaviors(cell proliferation,cell cycle,cell migration and invasion).3.The screen and verification of potential downstream targets of HoxC10:We screened HoxC10 potential downstream targets by using gene expression profile microarray and verified the results by real-time quantitative PCR(RT-qPCR);we chose p21 for further study due to the changes of p21 expression after HoxC10 alteration and the transcriptional binding sites in p21 promoter by bioinformatics prediction.4.The molecular mechanism of HoxC10 in the regulation of p21 transcription:By altering HoxC10 expression,RT-qPCR and western blotting,we studied the effects of HoxC10 on p21 expression and its downstream cell cycle-related proteins and validated the correlation of HoxC10 and p21 expression in cell and tissue levels;by application of dual-luciferase reporter assay and chromatin immunoprecipitation(CHIP),we explored the molecular mechanism of HoxC10 in regulating p21 transcription repression in GC.Results1.The expression of HoxC10 in GC tissues and its clinical significance? Using RT-qPCR,we found HoxC10 mRNA expression were significantly higher in fresh frozen GC tissues than in matching adjacent non-tumor tissues(the proportion of upregulation 91.43%,64/70,P<0.01).Western blotting showed that the protein level of HoxC10 expression was also upregulated in GC tissues.HoxC10 expression level was related to related to tumor size,invasive depth,lymph node metastasis and tumor stage(P<0.01).? Using Tissue microarray made by GC paraffin specimens,immunohistochemical staining showed that HoxC10 protein expression was widely upregulated in GC tissues(91.3%,137/150,P<0.01)and was closely correlated with gender,invasive depth and tumor stage(n=195,P<0.01).Kaplan-Meier survival analysis showed that high HoxC10 expression was related to the poor prognosis of patients(hazard ratio,HR=2.223;95%CI 1.361-4.186).Multiariable COX regression analysis showed that high HoxC10 expression was an independent predictor of poor prognosis.? In TCGA database,HoxC10 expression in GC tissues was upregulated by 122 times than in matching adjacent non-tumor tissues(n=33,P<0.01),In KM-plotter survival analysis database,high HoxC10 expression was found related to poor prognosis of GC patients(HR=1.8;95%CI 1.5-2.16).2.The effects of HoxC10 on GC cell biology behaviors? CCK8 and clony formation assays showed that HoxC10 overexpression promoted GC cell proliferation,while HoxC10 knocking-down repressed GC cell proliferation.? Flow cytometry cell cycle detection showed that HoxC10 overexpression could promote G1-S phase transformation significantly and HoxC10 knocking-down induced cell cycle arrest in G1 phase.Application of the cell cycle synchronization drug Nocodazole further verified that knocking down of HoxC10 induced GC cell cycle arrest in G1 phase.? Flow cytometry cell apoptosis assay showed that HoxC10 overexpression could inhibit GC cell apoptosis,while HoxC10 knocking-down facilitated cell apoptosis.? Transwell assays showed that overexpression of HoxC10 could promote cell migration and invasion,whereas knocking-down of HoxC10 expression could significantly inhibit the ability of GC cell migration and invasion.? In nude mice model of GC subcutaneous transplantation,overexpression of HoxC10 significantly accelerated the growth of tumors,whereas knocking-down of HoxC10 expression inhibited the growth of tumors.3.The screen and verification of potential downstream targets of HoxC10? We knocked down the expression of HoxC 10 in BGC823 cells and conducted expression profile microarray analysis.By a difference ratio>1.3 with gene functionanalysis,a series of differential expression genes were selected,such as ELF5,PRDM2,AKT3,CDKN1A(p21),SLC39A10,ZEB1,NRP2 and so on.? By using RT-qPCR weverified the alteration of downstream targets in GC cells.Therein,genes such as PRDM2,AKT3 and p21 were found upregulated significantly in HoxC10 knocking-down GC cells,and genes such as ZEB1 and NRP2 were found downregulated(P<0.05).? With Genomatix and JASPAR online transcription factor binding sites prediction databases,multiple potential binding sites of HoxC10 were found in p21 promoter.4.The molecular mechanism of HoxC10 in the regulation of p21 transcription? After altering HoxC10 expression in GC cells,we found that overexpressing HoxC10downregulated the expression level of p21,while knocking-down HoxC10 achieved an opposite effect;in nude mice model of GC subcutaneous transplantation,immunohistochemical staining showed that the expression of HoxC10 and p21 was also negatively correlated;in the stable HoxC10 knocking down cells,the expression of p21 and its downstream cell cycle-related proteins CDK2,CDK4,c-Myc,CyclinD1 and pRb was changed.? Dual-luciferase reporter assay showed that overexpressing HoxC10 inhibited p21 transcription,whereas knocking-down HoxC10 increased the transcriptional activitiy of p21.? We verified that HoxC10 could bind to the p21 promoter by CHIP assay with primers specific to the promoter region of p21.Conclusions1.The expression of HoxC10 was widely upregulated in GC tissues and related to the depth of tumor invasion,tumor stage and the prognosis of patiens.High HoxC10 expression was an independent predictor of poor prognosis.2.HoxC10 could regulate G1 cell cycle and inhibit GC cell apoptosis,promote GC cell proliferation and nude mice transplantation tumor formation,promote GC cell migration and invasion,so as to play the role of cancer-promoting gene.3.HoxC10 was able to bind to the promoter of p21 and induce the decreased activity of p21 transcription,negatively regulate the expression of p21 and influence downstream cell cycle-related signaling pathways. |
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