Study Of Carcinogenesis Associated With Expression Of BNIP3 Gene And Its Promoter Hypermethylation In Laryngeal Squamous Cell Carcinoma

Laryngeal carcinoma, one of the frequent malignant tumol/Lors in the department of Otolaryngology, has a tendency of increasing recently, especially in northeast where the morbidity is high than that of others. It was reported that the development and metastasis of Laryngeal carcinoma was in relationship with activation of some oncogenes and transcription silencing of anti-oncogenes, such as nm23/NDPK, Myc gene and CD44molecule included. Furthermore, P53 genetic mutation was closely associated with metastasis of Laryngeal carcinoma. Discovering the relative bionomics on tumol/Lor has recently come to a hot spot on early diagnosis and treatment of Laryngeal carcinoma in Otolaryngology research.DNA methylation could control genetic expression on transcriptional level (major in the beginning of transcription). Gene promoter and methylation of cytosine in CpG island around it closes unnecessary genes of the tissue and cell, while unmethylation instructs activation of tissue specificity or phase specificity genes which allow the gene expression of space-time. In normal tissue, generally expressed gene promoter region CpG island was under unmethylated condition while in tumol/Lor cells, the CpG island was usually under condition of methylation with its relative genetic expression closed too. So, besides the DNA mutation, abnormal methylation of anti-genetic promoter is also an important factor of tumol/Lor development. BNIP3 structure and function: BNIP3 coding output contain apoptosis effect structural domain (Bcl-2 homologue 3, BH3) and transmembrane domain (trans-membrane, TM), who were members of BH3-only promoting apoptosis family, could interact with anti-apoptosis protein such as Bcl-2, Bcl-x and EIB19k etc. and promote apoptosis. Inhibition of apoptosis signal access was intimately correlated with the occurrence development of malignant tumol/Lor. Tumol/Lor cell with non-expression or poor-expression of BNIP3 and BNIP3L could tolerant hypoxia and keep on growing while with high-expression of BNIP3 and BNIP3L the tumol/Lor cell became apoptosis and then death. Accordingly, down regulation of BNIP3L expression escaped the malignant cell from hypoxia induced apoptosis which maybe one of important events during the development of tumol/Lor.This study is to explore that the relationship of BNIP3 and carcinogenesis in laryngeal squamous cell carcinoma and influence on proliferation of laryngeal squamous cell and the mechanism, according to analysis the expression of BNIP3 gene and HIF and promoter hypermethylation in laryngeal squamous cell carcinoma .Immunohistochemical assay method was administered for the expression and location of HIF and BNIP3 in Laryngeal carcinoma tissue. It was shown that most of the HIF, whose express intensity was increased accompany with rising of pathology grade, was expressed in the cytoplasm. BNIP3 was both expressed in nucleus and cytoplasm, as to the Laryngeal carcinoma, BNIP3 was mainly expressed in cytoplasm and some of the nucleus. The masculine cell chiefly occurred around the tumor or necrosis and the edge of cancer soakage. The strongest BNIP3 expression in cytoplasm lied in Laryngeal carcinoma grade I and gradually decreased, while the strongest BNIP3 expression in nucleus was Laryngeal carcinoma grade III, grade II second and none grade I of BNIP3 expression in nucleus was found. Comparing with the regularity of HIF and BNIP3 expression , we found that they were closely related together in Laryngeal carcinoma. Studies on precancerous lesions of Vocal polyp and Laryngeal carcinoma presented that no expression of BNIP3 protein existed in normal laryngeal mucosa, but in Vocal leukoplakia and advanced atypical hyperplasia BNIP3 protein were all highly expressed and in leukoplakia the most.MTT method was administered to examine the effect of different concentration of DNA methyltransferase inhibitor 5-aza-deoxycytidine (5aza-dCyd) on Hep-2 cells proliferation, so that we could determine the transcription and expression mechanism in Laryngeal carcinoma tissue. And we found that continuous application of 5aza-dCyd with the concentration over 1.0μmol/L for 7 days could effectively inhibit the proliferation of hep-2 cell, and the more the concentration the stronger the inhibition. Observe the expression of BNIP3 protein by fixing hep-2 cell whichwasprocessedby 5aza-dCyd for 5 days and immunofluorescence staining. BNIP3 antibody was marked with Alexa Fluor555 antibodies and through confocal microscopy, positive BNIP3 protein presented to be red fluorescent labelling, which was mainly located in cytoplasm. Relatively quantitate to BNIP3 expression shown that normally cultivated hep-2 presented few BNIP3 positive cells, and after 5aza-dCyd, BNIP3 expression were increased. The result told us that removal of some genes'methylation could inhibit proliferation of Hep-2 cells and reinforce BNIP3 protein expression. Further MSP method (Methylation-specific PCR) enlarging on BNIP3 promoter methylation shown that extraction of Hep-2 cells DNA after processed with different concentration of 5aza-dCyd (0.1, 0.5, 1.0, 5.0 and 10.0μmol/ L) for 5 days, amplifying BNIP3 promoter with methylation or unmethylation primer, primers of methylation and unmethylation in the Hep-2 cells with no drug action group could both amplify specific strap. While after 5aza-dCyd action with concentration of 1.0μmol/L-10μmol/L, only the primer of unmethylation could amplify specific strap. So we proved that 5aza-dCyd could eliminate methylation state on BNIP3 promoter. The level of BNIP3 mRNA in the cell was examined with immediate RT-PCR method. After the effect of 1.0μmol/L and 5.0μmol/L concentration of 5aza-dCyd on the cell, the transcription level of BNIP3 mRNA increased which was three times more than that of the control group and the group with the concentration of 0.1μmol/L, and it showed significantly different (P<0.05). Combined with MSP, we found that the unmethylation of 5aza-dCyd with 1.0μmol/L and 5.0μmol/L concentration was in accordance with the reinforcement of BNIP3 genetic transcription.MSP method was administered to examine BNIP3 genic methylation in 26 Laryngeal carcinoma tissues and immediate RT-PCR method was administered to examine the level of BNIP3 genetic expression. The incidence of BNIP3 methylation in the 26 Laryngeal carcinoma tissues was 42% (11/26) while in the 20 normal laryngeal mucosa incidence was 10% (2/20). The incidence of BNIP3 methylation in normal laryngeal mucosa was lower than that of the malignant tissue (X2=5.82, P<0.05). BNIP3 genic methylation in Laryngeal carcinoma tissue has no association with clinical pathology factor such as tumol/Lor soakage, but association with neoplastic differentiation degree and lymph node metastasis. The incidence of BNIP3 methylation in Laryngeal carcinoma tissues was consistent with the expression of BNIP3.Conclusion:1. Laryngeal carcinoma tissue expresses BNIP3 protein, its location and intensity is closely related with the neoplastic differentiation. degree.The expression of BNIP3 protein in cytoplasm decreased with degrade of differentiation, while as in the nucleus it increases.2. It can be supposed that BNIP3 would take part in cancerogenesis of Laryngeal carcinoma and is early event in development of Laryngeal carcinoma, because of its overexpression protein in advanced atypical hyperplasia of Vocal leukoplakia .3. Remover of DNA methylation (5aza-dCyd) could inhibit proliferation of Hep-2 cells and reinforce BNIP3 expression intensity.4. 5aza-dCyd could eliminate methylation state on the BNIP3 promoter and reinforce BNIP3 genetic transcription.5. The incidence of BNIP3 methylation in normal laryngeal mucosa was lower than that of the malignant tissue. The methylation of BNIP3 exists in Laryngeal carcinoma tissue and correlates with neoplastic differentiation degree and lymph node metastasis. The methylation of BNIP3 in laryngeal carcinoma tissue followed the down regulation of transcriptional level and decrease of cytoplasm expression protein. Combining the expression of HIF, we infer that the methylation interrupt the effective site about HIF with BNIP3 and interfere with transcriptional level of BHIP3.6. The expression in the nucleus and the methylation of BNIP3 may be two independent events in cancerogenesis of laryngeal carcinoma.