Expression Of Histone Deacetylation 6 (HDAC6) In Esophageal Squamous Cell Carcinoma And Its Significance

Oesophageal squamous cell carcinoma (ESCC) is a common digestive tract malignancy, it is the sixth most common fatal cancer in the world, and is one of most hazard malignancy. China is a high incidence of esophageal carcinoma, especially in Lin Zhou city, Henan province. ESCC was dianogosed in an advanced stage with poor prognosis and rapid progression. With the improvement of medical treatment, there has greatly improved in therapy by surgical operation combined radiotherapy and chemotherapy, however, the prognosis of ESCC remains very poor. To date, there is no ideal prognosis and long term survival markers to detect the patients with ESCC, thereby there is no obvious progression in therapy. The poor prognosis of ESCC is not only related to the resistance of radiotherapy and chemotherapy, but also associated with diffusion of tumor cells via lymph and vein, which results in the lymph node and distant metastasis. Therefore, it is very necessary to identify invasion and metastasis related molecules of ESCC, which will lay a foundation for clinical therapy, prevention and the improvement of patient's survival rate and quality of life.HDACs are grouped into three classes based on their primary homology to three Saccharomyces cerevisiae HDACs. The class IHDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are most related to the yeast transcriptional regulator yRPD3. ClassⅡHDACs, which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, share domains similar to yHDA1. HDAC11 is most closely related to classⅠHDACs; however, no classification has been given since its sequence similarity is too low. ClassⅢHDACs are similar to the NAD+-dependent ySIR2. In recent years, more and more studies have demonstrated that HDAC6 as an important member of HDACs play a critical role in the occurrence and development of ESCC and inhibition of HDACs activity and expression may be as an important molecular target for malignancy. At the present time, HDAC6 displays at high level at multiple different tumor including glioma, breast carcinoma, lung cancer, prostate cancer, multiple myeloma, oral squamous cell carcinoma, and so on, suggesting high expression of HDAC6 is closely associated with tumors.However, up to now, there was no report about the role and expression of HDAC6 in ESCC in the world. To elucidate the possible role of HDAC6 in ESCC, in this study, expressions of HDAC6 mRNA and proteins were investigated by in situ hybridization, immunohistochemistry, semi-quantitative RT-PCR and Western blotting methods and correlations between expressions of HDAC6 and clinicopathological features were analyzed. The study above will provide the theoretical basis for HDAC6 mediated gene target therapy of ESCC.Methods:(1) Expression of HDAC6 mRNA was detected by in situ hybrid method in 75 cases of ESCC tissues,43 cases of mucosa adjacent to cancer, and 75 cases of normal esophageal mucosa, and correlations between expression of HDAC6 mRNA and clinicopathological features was analyzed.(2) Expression of HDAC6 protein was detected by immunohistochemistry method in 75 cases of ESCC tissues,43 cases of mucosa adjacent to cancer, and 75 cases of normal esophageal mucosa, and correlations between expression of HDAC6 protein and clinicopathological features was analyzed.(3) Correlation between HDAC6 mRNA and protein was analyzed by SPSS 13.0 software.(4) Randomly 3 cases of ESCC and paired normal epithelium tissues were selected to investigate the relative level of HDAC6 mRNA by semi-quantitative RT-PCR method.(5) Randomly 3 cases of ESCC and paired normal epithelium tissues were selected to explore the relative level of HDAC6 protein by Western blotting method.(6) Statistical analysis:The data were carried out with chi square test, t test, analysis of variance and correlation using SPSS version 13.0. In all statistical analyses, a P-value <0.05 was considered statistically significant.Results:(1) The results of in situ hybrid revealed that HDAC6 mRNA was mainly localized in cytoplasm, appearing ianthinus granules. There was high expression of HDAC6 mRNA in ESCC, and positive ratio was 92.00%, significantly higher than those of mucosa adjacent to cancer (20.93%) and normal esophageal mucosa epithelial tissue (13.33%), there were significant differences among three groupsχ2=107.122,P=0.000).(2) HDAC6 protein was mainly localized in cytoplasm, showing pale yellow to brown granules. There was high expression of HDAC6 protein in ESCC, and positive ratio was 88.00% (66/75), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 23.26%(10/43) and 12.00%(9/75), respectively, there were significant differences among three groups (χ2=97.587, P=0.000).(3) Expression of HDAC6 mRNA is closely related TNM staging and lymph node metastasis (χ2=6.020 and 4.112, respectively; P value=0.014 and 0.043, respectively, both<0.05), but not related to histologic grade (χ2=0.956, P=0.620) and invasion depth (χ2=0.379, P=0.538).(4) Expression of HDAC6 protein is closely related TNM staging and lymph node metastasis (χ2=5.576 and 6.448, respectively; P value=0.018 and 0.011, respectively, both<0.05), but not related to histologic grade (χ2=0.699, P=0.705) and invasion depth (χ2=0.594, P=0.441).(5) The results of correlation analysis of HDAC6 demonstrated that 69 cases of HDAC6 mRNA positive expression included 64 cases of HDAC6 protein positive expression in the 75 cases of ESCC tissues, while 6 cases of HDAC6 mRNA negative expression included 4 cases of HDAC6 protein negative expression. HDAC6 mRNA and protein expression displayed positive correlation (y=0.496, P=0.000).(6) Relative expression levels of HDAC6 mRNA in randomly selected samples 1, 2 and 3 were 1.912±0.044,0.347±0.013 and 0.872±0.021, respectively. While only sample 3 appeared HDAC6 mRNA expression and the relative expression level was 0.208±0.015, but there was no expression of HDAC6 mRNA in the two other samples. There was significant difference in HDAC6 mRNA expression between ESCC tissues and normal esophageal epithelium tissues (P<0.05).(7) Relative expression levels of HDAC6 protein in randomly selected samples 1, 2 and 3 were 1.635±0.033,0.781±0.027 and 1.109±0.041, respectively. While only sample 3 appeared HDAC6 protein expression, and the relative expression level was 0.129±0.015, and HDAC6 protein expression in ESCC tissues was evidently higher than that in normal esophageal epithelium tissues, and.there was significant difference (P<0.05).Conclusion:(1) Expressions of HDAC6 mRNA and protein in ESCC tissues are significantly higher than those in mucosa adjacent to cancer and normal esophageal epithelium tissues, and HDAC6 at high level plays an important role in occurrence and development of ESCC.(2) Expressions of HDAC6 mRNA and protein are both associated with TNM staging and lymph node metastasis, but not related to histologic grade and invasion depth, suggesting HDAC6 may be an important molecular marker for diagnosis of malignant degree of ESCC.(3) HDAC6 mRNA and protein expression displays positive correlation.(4) The results of semi-quantitative RT-PCR and Western blotting demonstrate that HDAC6 is expressed at high level, suggesting HDAC6 may be tightly associated with the occurrence and development of ESCC.