ABSTRACT
Objective To observe the RASSF1A expression in laryngeal keratosis and laryngeal squamous cell carcinoma and investigate the relationship between the gene and the occurrence and development of this disease. Methods The specimens of keratosis of larynx(43 cases)and laryngeal squamous cell carcinoma(31 cases)in Oto-laryngology-Head and Neck surgery of Anhui provincial hospital from Dec 2009 to Dec 2012 were collected.Nor-mal vocal fold mucosa from the cadaveric head as control group(8 cases)were collected.Immunohistochemical meth-ods were used to detect the expression of RASSF1A protein in these tissues.The diffuse distribution of brown gran-ules in the cell cytoplasm yielded positive results and the nucleus was not coloring.The percentage of positive cells of every section,and the average standard deviation(￣x±s)were calculated.SPSS17.0 statistical analysis software was used to analyze the data,P0.05).RASSF1A expression in the clinical stage from I to IV of laryngeal squamous cell carcinoma gradually de-clined.During the clinical stage,the difference had statistics significance (P0.05).ConcIusion The reduction of RASSF1A expression correlated with laryn-geal squamous cell carcinoma,which has certain significance for the different ion of carcinoma and laryngeal precan-cerous lesion.
ABSTRACT
Background and purpose:Loss or altered expression of Ras association domain family 1A gene (RASSF1A) through DNA methylation has been associated with the pathogenesis of a variety of cancers, which suggests the tumor suppressor function of this gene. This study aimed to explore the effect of DNA methyltransferase inhibitor 5-Aza-2’deoxycytidine (5-Aza-dc) on demethylation and expression of RASSF1A in cervical cancer cell lines. Methods:HPV positive cervical cancer cell lines HeLa and Caski, HPV negative cell lines HT-3 and C-33A were treated with two different concentration of 5-Aza-dc (5 μmol/L, 10 μmol/L). MSP (methylation-specific PCR) and Bisulfite genomic sequencing PCR (BGS) combined with TA clone were used to investigate methylation status of RASSF1A gene promoter and exon 1 before and after treatment of 5-Aza-dc. RASSF1A gene mRNA expression was detected by RT-PCR. Results:Two HPV positive cell lines showed hypomethylated RASSF1A promoters and expressed RASSF1A mRNA, and after treatment with 5-Aza-dc, the mRNA expression of RASSF1A did not change significantly (FHeLa=3.003, P=0.125; FCaski=0.045, P=0.956). Two HPV negative cervical cancer cell lines showed hypermethylation status of RASSF1A promoter and silenced RASSF1A. After treatment with 5-Aza-dc, demethylation occurred in the promoter region of RASSF1A gene, which subsequently induced re-expression of this gene in HT-3 and C-33A. The F test (FHT-3=18.002, P=0.03;FC-33A=17.179, P=0.03) and LSD-t test (P<0.05) demonstrated that significant difference in the expression of RASSF1A was found upon two different concentrations drug treatment.Conclusion:The methylation status of promoter and exon 1 of RASSFIA gene in HPV positive and HPV negative cervical cancer cell lines are different. The promoter hypermethylation is correlated with RASSF1A gene expression in HPV negative cervical cancer cell line HT-3 and C-33A, and plays a key role in RASSF1A silencing. 5-Aza-dc may effectively reverse the methylation status of RASSFIA gene promoter in cervical cancer HT-3 and C-33A cells and reactivate gene expression silenced by aberrant hypermethylation in a dose-dependent manner within certain extent.