Ow-up period for patients was 23 months (range, 0?6 months).Four patients in the unmethylated group and 17 patients in the methylated group were lost to follow-up. Kaplan eier plots indicated that methylation of HOXD10 was associated with poor 3-year overall survival (OS) (P = 0.048, Fig. 2b). While, according to Cox proportional hazards model analysis, HOXD10 methylation was not an independent prognostic factor for 3-year OS after adjusting for tumor differentiation, vessel cancerous embolus, and TNM stage (P = 0.127, Table 2). The expression of HOXD10 was evaluated by immunohistochemistry (IHC) in 40 cases of available matched primary HCC and adjacent tissue samples. Staining of HOXD10 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 was mainly localized in the nucleus, and itsFig. 2 The expression and methylation status of HOXD10 in primary HCC. a Representative MSP results of HOXD10 in NSC309132 msds normal liver tissue samples and primary HCC samples. N: normal liver tissue samples; HCC: primary HCC samples. b The 3-year overall survival curves for patients in the methylated and unmethylated groups (P < 0.05). c Representative IHC results show HOXD10 expression in HCC tissue and adjacent tissue samples (top: ?00; bottom: ?00). d The expression of HOXD10 and DNA methylation status is shown as a bar diagram. Reduced expression of HOXD10 was significantly associated with promoter region hypermethylation. *P < 0.05, r = 0.33. e HOXD10 expression scores are shown as box plots; horizontal lines represent the median score; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; vertical bars represent the range of data. Expression of HOXD10 was significantly different between adjacent tissue and HCC tissue in 40-matched HCC samples. ***P < 0.Guo et al. Clinical Epigenetics (2017) 9:Page 6 ofTable 1 Clinical factors and HOXD10 methylation in 117 cases of HCC samplesClinical factor Age (year) < 60 60 Gender Male Female HBV infection Yes No Liver cirrhosis Yes No Tumor size (cm) 5 >5 Number of lesions 1 1 Differentiation Well Moderate Poor TNM stage Stage I + stage II Stage III + stage IV Lymph node metastasis Negative Positive Vessel cancerous embolus Negative Positive 87 30 24 3 63 27 112 5 26 1 86 4 0.049* 52 65 14 13 38 52 0.867 8 74 35 4 19 4 4 55 31 0.377 92 25 22 5 70 20 0.044* 37 80 6 21 31 59 0.681 88 29 17 10 71 19 0.231 85 32 19 8 66 24 0.093 101 16 21 6 80 10 0.762 80 37 19 8 61 29 0.248 No. HOXD10 methylation status Unmethylated n = 27 (23.1 ) Methylated n = 90 (76.9 ) 0.799 *P value*P values are obtained from chi-square test, significant difference, *P < 0.expression was significantly reduced in primary HCC compared to adjacent tissue samples (P < 0.001, Fig. 2c, e). In 40 cases of available primary HCC, loss or reduced expression of HOXD10 was found in 28 cases. Of these 28 case samples, 26 cases were methylated and 2 cases were unmethylated. Loss or reduced expression of HOXD10 was significantly associated with promoter region hypermethylation (P < 0.05, r = 0.33, Fig. 2d). These results indicate that HOXD10 expression is regulated by promoter region methylation in primary HCC.HOXD10 suppresses proliferation of HCC cellsTo evaluate the effects of HOXD10 on HCC cell proliferation, cell viability was determined by the MTT assay. The OD value was 0.63 ?0.05 vs. 0.50 ?0.05 (P < 0.01) and 0.68 ?0.01 vs. 0.53 ?0.02 (P < 0.05) before and after restoration of HOXD10 expression in HOXD10 unexpressed SMMC7721 and Huh7 cells, respectively. The OD value was reduced sig.
