Expression And CpG Island Methylation Of FBXO32Gene In Esophageal Squamous Cell Carcinoma

Objective: The gastrointestinal malignancy is a serious threat to humanhealth. China is a country with high incidence regions of esophageal cancer,especially in Cixian and Shexian of Hebei province. Esophageal squamouscell carcinoma (ESCC) is the predominant type of esophageal cancer inNorthern China. Due to the occult symptoms, it is difficult to early detectionof ESCC. Extensive studies have been conducted to elucidate the etiology ofesophageal cancer in recent years, and as a main part of epigenetics, DNAmethylation has been proved to play an important role in the occurrence anddevelopment of ESCC.DNA methylation is the most common modification process in theeukaryotic cells, meanwhile, it has been participated in transcription regulationof mammals and negatively correlated with gene expression. A lot of studieshave shown that aberrant DNA methylation may be a frequent event in tumorsand may be used as promising molecular markers for early diagnosis andprognosis judgement of tumor. In the present study, mRNA and proteinexpression, and methylation status of muscle atrophy F-box protein (FBXO32,atrogin-1, MAFbx) were detected in esophageal cancer cell lines andesophageal squamous cell carcinoma to explore its roles in ESCC.Method:1Reverse transcription polymerase chain reaction (RT-PCR) methodwas used to detect the mRNA level of FBXO32gene in esophageal cancer celllines (TE1, TE13, T.TN, Yes-2) and the cell lines treated with5-aza-dC, ESCCtumor tissues and corresponding normal tissues.2Immunocytochemistry/Immunohistochemistry methods were used toexamine the protein expression of FBXO32in esophageal cancer cell lines andthe cell lines treated with5-aza-dC, ESCC tumor tissues and corresponding normal tissues.3Methylation specific PCR (MSP) method was used to examine themethylation status of FBXO32gene in esophageal cancer cell lines and thecell lines treated with5-aza-dC,51ESCC tumor tissues and correspondingnormal tissues.4SPSS13.0was applied to analyze the results of experiments.Result:1The mRNA and protein expression, and methylation status of FBXO32inesophageal cancer cell lines1.1The mRNA expression of FBXO32in esophageal cancer cell lines treatedand untreated with5-aza-dCmRNA expression of FBXO32gene was only detected in TE1cell line ofthe four cell lines. When the cell lines were treated with5-aza-dC, mRNAexpression of FBXO32can be detected in the treated four cell lines.1.2The protein expression of FBXO32in esophageal cancer cell lines treatedand untreated with5-aza-dCThe positive protein expression of FBXO32was only detected in TE1cell line of the four cell lines. When the cell lines were treated with5-aza-dC,protein expression of FBXO32can be detected in the treated four cell lines,indicating the restoration expression of FBXO32by treatment of5-aza-dC.1.3The methylation status of FBXO32in esophageal cancer cell lines treatedand untreated with5-aza-dCFBXO32gene was fully methylated in TE13, T.T N, and Yes-2cell lines,while semi-methylation of FBXO32was dected in TE1cells. After treatmentwith5-aza-dC, FBXO32gene was detected unmethylation status in the fourtreated cell lines2The relationship between mRNA and protein expression, and methylationstatus of FBXO32and ESCC clinical pathology2.1The mRNA expression of FBXO32in ESCCFBXO32mRNA expression in tumor tissues (0.24±0.15) wassignificantly lower than that in corresponding normal tissues (0.49±0.21) (P<0.05). The mRNA expression of FBXO32was correlated with status oflymphatic metastasis, differentiation and TNM stage of ESCC patients (P<0.05).2.2The protein expression of FBXO32in ESCCThe protein expression of FBXO32in tumor specimens (23.5%,12/51)was significantly lower than that in corresponding normal tissues (70.5%,36/51)(P<0.01). The protein expression of FBXO32was significantlyassociated with lymphatic metastasis of ESCC (P<0.05）, however, it was notassociated with differentiation and TNM stage (P>0.05).2.3The methylation status of FBXO32in ESCCThe promoter methylation frequency of FBXO32in tumor specimens(52.9%,27/51) was significantly higher than that in corresponding nomaltissues (27.5%,14/51)(P<0.05). Methylation frequencies of FBXO32gene inpoor differentiation group (84.0%,21/25) was much higher than that inmoderate and high differentiation group (23.1%,6/26)(P<0.05). Methylationfrequencies of FBXO32in lymph node metastasis group (80.8%,21/26) wassignificantly higher than that in non lymph node metastasis group (24.0%,6/25)(P<0.05). Methylation status of FBXO32gene was not associated withTNM stage, age, and gender (P>0.05).2.4Relationship between methylation status of FBXO32and its expressionThe mRNA expression of FBXO32in tumor tissues with positive proteinexpression of the gene (0.33±0.19) was significantly higher than that in tumortissues with negative protein expression of the gene (0.21±0.13)(P<0.05). ThemRNA expression of FBXO32in tumor tissues with promoter hymethylationof the gene (0.19±0.12) was significantly lower than that in tumor tissues withunmethylation of the gene (0.30±0.17)(P<0.05). The positive proteinexpression of FBXO32in tumor tissues with promoter hymethylation of thegene (11.1%,3/27) was significantly lower than that in tumor tissues withunmethylation of the gene (37.5%,9/24)(P<0.05).Conclusions:1FBXO32gene promoter methylation may be an important mechanism for loss of expression of the gene in esophageal cancer cells and FBXO32maybe used as a demethylation therapy target for its restoration expression by5-aza-dC.2The methylation status of FBXO32promoter was not correlated withage, gender and TNM stage of patients. However, it was significantly relatedto differentiation and lymph node metastasis, indicating that promotermethylation of FBXO32may be associated with tumor prognosis.3There seemed to be a correlation between the low expression ofFBXO32and tumor prognosis, and promoter hypermethylation of FBXO32may be one of the reasons that lead to down-expression of FBXO32in ESCC.