| Part IThe expression of Methyl-CpG Binding Protein 2 in laryngealsquamous cell carcinomaObjective: To investigate the expression of Methyl-CpG Binding Protein 2(MBD2) in laryngeal squamous cell carcinoma and evalue the role of MBD2 in the development of laryngeal squamous cell carcinoma.Methods: MBD2 expression were detemined in 40 laryngeal squamous cell carcinoma cases, 40 samples from tissues adjacent tumor and 10 normal laryngeal tissues were detemined by RT-PCR.and Western Blot.Results: RT-PCR showed that the presense of MBD2 mRNA was observed in 30 specimens of 40 laryngeal squamous cell carcinoma and all the specimens from tissues adjacent tumor and normal tissues. There was a higher expression of MBD2 mRNA in specimens of laryngeal squamous cell carcinoma than in tissues adjacent tumor and normal tissues(P<0.01). There is no significant difference between tissues adjacent tumor and normal tissues((P>0.05). There is no significant difference of MBD2 mRNA expression among the samples of different histological grade or those of different T stages(P>0.05). Western Blot showed that the presense of MBD2 protein was observed in 29 specimens of 40 laryngeal squamous cell carcinoma all the specimens from tumor, tissues adjacent tumor and normal tissues. There was a higher expression of MBD2 protein in specimens of laryngeal squamous cell carcinoma than in tissues adjacent tumor and normal tissues(P＜0.01). There is no significant difference between tissues adjacent tumor and normal tissues((P>0.05). There is no significant difference of MBD2 mRNA expression among the samples of different histological grade or those of different T stages(P>0.05).Conclusion: The over expression of MBD2 may play a important role in laryngeal squamous cell carcinoma, MBD2 is possible concerned with occurrence of laryngeal squamous cell carcinoma.Part II Construction and identification of the eukaryotic expression plasmidfor MBD2 specific shRNAAbjective: To construct and identify the eukaryotic expression short hairpin RNA(shRNA) of MBD2.Methods: According to the Homo sapiens methyl-CpG binding domain 2 protein (MBD2) mRNA sequence, four strands of oligonucletide were designed and chemically syntheized, then annealed into two double strands in vitro. The two DNA strands were ligated with plasmid vector respectively, named pGenesill-MBD2 shRNA -1 and pGenesill-MBD2 shRNA -2.These plasmaids were transformed into compenent cells(DH5α). After selective culture, recombined plasmids were extracted from E.coli, sequence was used to make sure that the two strands were inserted into plasmaids in correct sequence and direction respectively.Results: The synthesized strands of oligonucletide contained correct and complete sequence of the shRNA. the MBD2 specific shRNA has been inserted into eukaryotic expression.Conclusion: The eukaryotie expression plasmid pGenesil-MBD2 shRNA has been constructed sueeessfully. It make a good prepration for the next step to RNA interference of MBD2 expression in Hep-2. Part IIIEffect of Knockdown of MBD2 Expression by shRNA on the Characteristics of laryngeal cancer Cell Line in vivo and vitro andmethylation of P16 geneAbjective: To study the effect of knockdown of MBD2 expression on the characteristics of human laryngeal carcinoma cell line Hep-2 and the methylation of P16 gene.Methods: The pGenesil-1-MBD2shRNA and pGenesil-1-C were transfected into Hep-2 by jetPEI-RGD and the three group of Hep-2 were named pGenesil-l-MBD2shRNA1-Hep-2, pGenesil-l-MBD2shRNA2-Hep-2 andpGenesil-1-C -Hep-2. pGenesil-l-MBD2shRNA1-Hep-2 was chosen for next tests. Then the cells were devided into four groups. Group A was Hep-2 , group B was Hep-2 treated with 5-Aza-2 -deoxycytidine, group C was Hep-2 transfected with the pGenesil-MBD2shRNAl, group D pGenesil-1-MBD2shRNA1-Hep-2. Then observe the characteristics of the five groups by MTT. The activity of caspase-3 was detected.The expression of MBD2 was detected by RT-PCR and Western blot and the methylation of p16 gene was detected by MSP. The expression of pl6 mRNA was detected by RT-PCR .Then created the models of xnograft were created in nude mice using all this five group cells. Observe growth of those xnografts in nude mice and draw growth curve of xnografted. After 8 weeks, the xnografts' expression of MBD2 was detected by RT-PCR and Western blot and the methylation of P16 gene was detected by MSP. The expression of p16 was detected by RT-PCR .Results: Group B,D can surpress the growth of Hep-2. The assay of caspase 3 activity indicates that the rate of apoptosis group B, D were higher than group A and C. The result of RT-PCR and Western blot show that the expression of MBD2 mRNA and protein was down regulated in group D. P16 was unmethylated in group B cell and methylated in group A, C and D. The express of p16 mRNA was positive in group B ,D and was negative in group A and C. Volum of xnografts in group B and D nude mice were smaller than group A and C nude mice, weight of xnografts in group B and D nude mice were lighter than group A and C nude mice . Significant diffence of MBD2 mRNA and protein was found between group B, D and group A,C xnografts. P16 was unmethylated in group B xnografts and methylated in group A, C and D xnografts. The express of pl6 mRNA was positive in group B ,D xnografts and was negative in group A ,C xnografts.Conclusions: The results indicated that down-regulation of MBD2 could surpress the proliferation and accelerate the apoptosis of Hep-2. The down-regulation of MBD2 can't transform the hypermethylated p16 into unmethylated p16 , but can suppress the proliferation of Hep-2 more stronglier than 5-Aza-2' -deoxycytidine and reactive the p16. We presume that the down-regulation of MBD2 may remove its founction of surpression to transcription. The exact role of MBD2 in DNA methylation and occurrence of laryngeal squamous cell carcinoma need more experiments.
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