Research Of Association Of Single Nucleotide Polymorphisms Of PTEN Gene With China Laryngeal Cancer

As one of the common upper respiratory tract malignantcarcinomas, Laryngeal carcinoma has aroused more and moreattentions among the research groups all over the world.Laryngeal squamous cell carcinoma is recognized as the mostfamiliar Laryngeal carcinoma, and the squamous cell in thelaryngeal is genesis. Most studies shows that smoking has asignificant influence on the genesis of Laryngeal carcinoma, forpeople who smoke and drink frequently, the risk of Laryngealcarcinoma is obviously high and for those group of people whodo not smoke or drink, the risk is extremely low. Thenosogenesis of Laryngeal carcinoma is a serious of complicateprocess of gene changed, genetic factors, environmental factorsand immune factors have all been tested playing relevant roles inthis process. To definitude the nosogenesis of Laryngealcarcinoma is benefit for the prevention, early diagnosis and thetreatment of this disease, it is also useful for evaluating theprognosis and the activity of the treatment. From this point ofview, many researchers world round performed large numbers ofstudies in search of the relevant genes of the laryngealcarcinoma, they have acquired some advancement, especially inthe aids of the rapid progress of genetic technology.Objective:To study the association of single nucleotide polymorphisms(SNP) of the phosphatase and tensin homologue (PTEN) geneamong the cases of North-China Han population with laryngealcarcinoma.Materials and methods:1. To produce the template DNAAdd 900ul cell lysis buffer into Eppendorf tube, and then add300ul premelt blood into it and mix carefully, after turning 3minutes in the centrifugal machine, pour the upper liquid, andadd the nuclear lysis buffer, put the Eppendorf into 37℃ waterfor 30-40 minutes. Add 1.5ul Rnase into the Eppendorf and mixagain, then put it into 37 ℃ water 15 minutes, and add 100 ulProtein Precipitation Solution , after mix carefully, exposure tothe room temperature for 5 minutes, and turn 3 minutes again,absorb the upper liquid to a tube with 300ul lsopropylalcohol,react in the room temperature for 15 minutes. After turning 3minutes, pour the upper liquid gentlely, add 300ul 70% alcohol,wash once carefully and turn 3 minutes, pour the upper liquidand turn 3 minutes again, dry in the room temperature for about20 minutes, and add 100ul DNA Lysis Buffer, put the tube into 65℃ water 2 minutes, and conserve it in the -20℃ in the end.2. Polymerase Chain ReactionSelect a 25 ul reaction tube, add water, PCR buffer, dNTPs,Sense, Anti-sense, template DNA, Tap DNA Polymerase into itrespectively, put it into a PCR machine, and set the reactiontemperature and time, after the action finished, reserve the tubeinto 4℃ refrigeratory.3. Agarose Gel ElectrophoresisPrepare gel tray by sealing ends with tape. Place comb ingel tray about 1 inch from one end of the tray and position thecomb vertically such that the teeth are about 1-2 mm above thesurface of the tray. Scale the agarose and microwave it withelectrophoresis buffer until agarose is dissolved. After cool to 50℃, add ethidium bromide into it and mix throughfully and pourthe gel solution carefully. Put the gel in the room temperature for30-40 minutes. Add the electrophoresis butter, make the surfaceis higher than the gel surface about 1-2mm. Prepare samples forelectrophoresis and put the sample into the gel well.Electrophorese at 120 volts until dye markers have migrated anappropriate distance. The result is observed under short waveUV light.4. Restricted enzyme incising in the amplification productionFirstly ,add quantitive PCR production, enzymolysisbalanced solution , incision enzyme , water distilled twice into a1.5ml centrifuge tube in order;Secondly , centrifuge tube isagitated on the whirlpool agitate;Thirdly centrifuge tube isincubated in the thermostatic waterbath for 4 hours beforechecked.5. Statistics methodAll measurement data is denoted by M 士 SD inexperimental data. T test is applied in group comparision;X2 testis applied in the contrast between genotypic frequency and allelefrequency in the case group and control group. All statisticsanalysis is analyzed by SPSS software. P<0.05 is forsignificance standard.Result:By detecting 3 SNPs of the PTEN gene, we find that thegenotype distribution G/G: G/C: C/C was 13 (G/G): 21(G/C):18(C/C) and 29(G/G): 57(G/C): 18(C/C) in laryngocarcinomapatients and normal control groups respectively. The genotypicfrequencies of this SNP were significantly different betweenlaryngocarcinoma and the control group (P=0.049).Conclusion:1. Our data showed the suggestive association for thers2735343 polymorphism in PTEN gene with carcinoma of larynxin North China Han population, the result indicates thatrs2735343 maybe relevant to the nosogenesis of Laryngealcarcinoma.2. There is no statistically significance between SNP(rs701848,rs1903858)polymorphism in PTEN gene in the contrast groupwith carcinoma of larynx group, these 2 SNPs maybe aren'tinvolved in the genesis of laryngeal carcinoma.