Liver Cancer Susceptibility Gene Pfk-2/fbpase-2 Sequence Analysis And Gnmt Polymorphism Study

Objective:Molecular cloning and sequence analysis of PFK-2/FBPase-2 in HepG2 cell,this work made it possible to analysis the glycol-metabolism for PHC.Methods:The total RNA of HepG2 cell was extracted with the guanidine isothiocyanate.The reverse transcription reaction was performed using the Ferments RevertAid^TMFirst-Strand cDNA Synthesis kit.PFK-2/FBPase-2 cDNA was amplified by PCR,and then cloned into pMD1g-T Vector and sequenced.Results:PFK-2/FBPase-2 cDNA was amplified by RT-PCR.DNA sequencing revealed that the cDNA was composed of 1150bp with an open reading frame encoding 377anino acids of protein.The PFK-2/FBPase-2 sequence has 90%,88%,80%,76%,63%and 50%DNA homology and 91%,90%,83%,81%,69%and 60%amino acid sequence homology to the PFK-2/FBPase-2 gene sequence of human(AY714243,BC096078),rat(Y00702,X58865),mouse (AY756065,AY756067),respectively.Conclusion:PFK-2/FBPase-2 was cloned in HepG2 cell and sequence alignment results displayed that it has higher homology with PFK-2/FBPase-2 of human,rat and mouse,respectively. Objective:To investigate the characters of GNMT sequence,and its polymorphisms and genetic susceptibility to PHC in Guangxi Zhuang and Han people.Methods:Polymorphisms of GNMT gene were analyzed in 80 patients suffering PHC and 122 health controls.The partial of GNMT gene was scanned by molecular and genetic techniques,including PCR,PCR-SSCP,direct sequencing of PCR products.Results:(1)For length polymorphism of the region of promoter of GNMT gene diplo-nucleotide repeat sequence,five alleles were detected and the most alleles were 10,16,20 in two groups.And the frequency showed no difference between cases and controls(P＞0.05).(2)The insertion of GAGT in the region of promoter of GNMT gene,and the mutation frequency did not show apparent difference between patients and controls(x²=1.002,P=0.317).(3)The nucleotide substitution of C→T were found in 5-UTR of GNMT gene,and the frequency was significantly higher in patients than in control groups(x²=9.402,P=0.002).(4)No mutation was found in the intron lof GNMT gene either in cases or in controls.(5) If both the insertion of GAGT in he region of promoter and the mutation of C→T in 5-UTR were found in GNMT gene,it is obvious difference in the two groups (P=0.0001).(6)The mutation in the region of promoter and in 5-UTR of GNMT did not show significant difference between the Zhuang people and the Han people in Guangxi(P=0.518,P=0.606).Conclusion:(1)No association with the risk of PHC was observed for the GNMT gene diplo-nucleotide repeat sequence.(2)No association with the risk of PHC was found for the insertion of GAGT in the region of promoter of GNMT gene.(3)The mutation in 5-UTR of GNMT gene wound be a risk factor suffering PHC.(4)The region of intron 1 of GNMT gene was comparatively constant.(5) combining the mutation of the insertion of GAGT in the region of promoter and the mutation of C→T in 5-UTR wound increase the fatalness of suffering PHC.(6) The mutation in the region of promoter and in 5-UTR of GNMT did not show significant difference between the Zhuang people and the Han people in Guangxi.