| Background and Objective:Chemoradiotherapy is the main clinical treatment modalities of patients with lung squamous carcinoma now, but it turns up "bottleneck" phenomenon. So looking for a new, efficient, small toxicity anticancer drugs becomes basic and clinical research focus. TRAIL is a new biological agents, which can induce apoptosis of tumor cells, without significant damage to normal tissues. But most cancer cells are resistant to TRAIL, which have many mechanisms. Studies have been reported that the resistance to TRAIL may be related to of death receptor 4 (DR4) gene promoter methylation. Reversal of gene methylation may increase TRAIL-induced apoptosis of tumor cells. Therefore, this paper mainly studies lung squamous cell carcinoma and explores the relationship between DR4 gene promoter methylation and DR4 expression of the lung squamous carcinoma, in order to further study whether DR4 gene promoter methylation affects the TRAIL-induced apoptosis of lung squamouscarcinoma cell. Which can provide new therapeutic strategies for lung squamous cell carcinoma..Method:1.Elivision two-step method are employed to detect the expression of clinical tissue DR4;2.Methylation-specific PCR (MSP) are employed to detect methylation status of clinical tissue DR4;3.MSP is applied to detect the methylation status of DR4 gene promoter before and after lung squamous carcinoma cell lines (H226, SK-MES-1, H520) are processed by 5-Aza-CdR;4.RT-PCR and Western blot are applied to detect the expression of DR4 gene promoter mRNA and protein before and after lung squamous carcinoma cell lines (H226, SK-MES-1, H520) are processed by 5-Aza-CdR;5.MTT is used to detect the cell proliferation inhibition rate in lung squamous carcinoma cell;6.The flow cytometry is adopted to detect the apoptosis rate in lung squamous carcinoma cell.Result:1.The results of the clinical research are as follows,Methylation-specific PCR (MSP) result shows that 80.6% of 36 lung squamous carcinoma patients are in the methylation status of DR4 gene promoter that has no correlationship with clinicopathological features of lung squamous carcinoma patients in the lung age, gender, smoking, histological differentiation, TNM stage, lymph node metastasis (P>0.05); Immunohistochemistry indicates that the probability of DR4 low expression of lung squamous carcinoma patients is about 58.3%, there are four cases unexpressed, seventeen cases low expression, ten cases moderate and high expression, five cases high expression.which may be related to their gene promoter methylation (P＜0.05)2.The results of the in-vitro experiments are as follows, the MSP result suggests that DR4 genes of H226 and SK-MES-1 cells are in the methylation status and their mRNA and proteins all have low expression, while DR4 genes of H520 are in the unmethylation status and their mRNA and proteins all have high expression; after intervention with 5-Aza-CdR, DR4 genes of H226 and SK-MES-1 cells are in the unmethylation status and their mRNA and protein expression are significantly upregulated compared with the pre-interference ones, but such differences are of statistical significance (P＜0.05), while DR4 genes of H520 are still in the unmethylation status and their mRNA and protein expression have no obvious change compared with the pre-interference ones. In addition, the study confirms that TRAIL has varying degrees of proliferation inhibition effect and apoptosis effect on H226 and SK-MES-1 cells, but not sensitive, while after being interfered by 5-Aza-CdR, H226 and SK-MES-1 cells become significantly sensitive to TRAIL (P＜0.05).Conclusion:Lung squamous carcinoma has DR4 methylation modification universally, resulting in DR4 silence or low expression, while 5-Aza-CdR can reverse DR4 gene promoter methylation status to upregulate its expression, thereby increasing the TRAIL-induced apoptosis in lung squamous carcinoma cells. Therefore, combining 5- Aza-CdR and TRAIL is a new strategy for treating lung squamous carcinoma. |
PDF Full Text Request