Analysis Exon Sequence,mRMA Level,Protein Expression Of Puma In Skin Squamous Cell Carcinoma

Object To explore whether there is change of puma exons sequence, puma mRNA and protein expression in skin squamous cell carcinoma.Methods Optimization and ultimately established the amplification conditions of three exons of puma with high GC content. using t RT-PCR detected the level of puma mRNA.Developed Western blot analysis of PUMA protein expression.1. By exploring the experiment conditions of PCR, the ultimate reaction conditions of three exons of puma were eatablished.(1) The reaction system of Exon2was:2×Mastermix30pμ1, primers(10μmol/L)1.5μ1, template DNA3.0μ1, ddHaO24μ1, the total volume was60μ1. The amplification conditions were:After5min at95℃, reactions were cycled through45s denaturation at95℃,45s annealing at68℃, and45s extension at72℃for33cycles,then followed for5min at72℃.(2) The reaction system of Exon3was the same as Exon2.The amplificat conditions were:45s annealing at63℃, other amplification condition were the same as Exon2.(3) The reaction system of Exon4was the same as Exon2.The amplificat conditions were:45s annealing at55℃, other amplification conditions were the same as Exon2.2. By exploring the conditions, the RT-PCR was developed for the study of puma gene expression level in human SSCC tissues. The reaction mixtures contained2×SYBR premix10μ1; the forward and reverse primers0.4μ1each; ROX0.4μ0; cDNA1.0μ1; ddH2O7.8μ1; the total reaction volume was20μ1. The amplification conditions were as follow:95℃for5minutes; followed by5cycles of95℃for10sonds,60℃for5sonds and72℃for10sonds; then followed by40cycles of95℃for10sonds,60℃for5sonds,72℃for10sonds and89℃for27sonds(fluorescence measurement); and65℃for5minutes. The reaction system of GAPDH in RT-PCR was the same as puma,and the detecting was performed under the following conditions:95℃for5minutes; followed by5cycles of95℃for15sonds,64℃for5sonds and72℃for10sonds; then followed by40cycles of95℃for15sonds,64℃for5sonds,72℃for10sonds,78℃for3sonds and72℃for30sonds(fluorescence Measurement); and65℃for5minutes.(pMD18-T(+)/puma and pMD18-T(+)/GAPDH plasmids for the standard curve were successfully constructed by TA-colony in the preliminary experiments.)3. The expression of PUMA protein was detected by Western Blot,and?-actin was used as a reference gene.Results1. By using the optimized reaction conditions, the Exon2to4of puma gene in32cases of skin squamous cell carcinoma tissues were amplificationed.The amplification productions of puma exon2to4were detected by sequencing technique,and then compared to DNA bank sequence by Blast2.none of mutation was found.2. By using RT-PCR,puma mRNA was expressed in skin squamous cell carcinoma tissues and normal skin tissues,but the expression levels between them were no statistically significant (P>0.05).3. The expression of PUMA protein was detected by Western Blot,and?-actin was used as a reference gene.?-actin was expressed in all32cases of skin squamous cell carcinoma tissues and2cases of normal skin control tissues,but only8cases of skin squamous cell carcinoma tissues and1cases of normal skin control tissues expressed PUMA, and the expression levels were relatively low.Conclusions1. The three Exons coded sequences of puma gene are conservative and stable.2. The relative expression levels between skin squamous cell carcinoma tissues and normal skin tissues were no statistically significant (P>0.05).3. May be the expression of PUMA protein in the skin tissues was relatively low.