Expression Of P73 MRNA And Protein In Laryngeal Carcinoma

Object: Tumorigenesis progression of malignant tumor is a complex and multi-step process involving poly gene regulation. p53 gene is putative and the most frequently mutated gene in human cacer. It is reported that it acts as important role in tumorigenesis progression and metastasis of laryngeal carcinoma. But its structural homologs of the p53 genes, p73, have recently been identified. The p73 gene is located on human chromosome Ip36.3, a region found to be frequently deleted in neuroblastoma and other tumors. It is monoallelically expressed in neuroblastoma, in addition to its resemblant structure and function, taken together, so it is deemed a tumor suppressor gene. But many scholar found its high expression, few mutation, biallelically expression, so it is not deemed a tumor suppressor gene. To this day, it is not reported about expression of p73 gene in laryngeal carcinoma. This reseach analyzed the expression of p73 mRNA and protein in larygeal carcinoma by RT-PCR and Flow Cytometry (FCM). Our aim is to investigate mechanism of tumorigenesis progression in laryngeal carcinoma.Methods: 42 laryngeal carcinoma fresh samples were analyzed and normal mucosa that was furthest from the tumor specimen. All subjects were the admitted patients for operative treatment form 2000 to 2001 in our department (including male in 38, female in 4, aged from 42 to 74, average 57.8 years old; Smoking exceed 30 years in 17, under 30 years in 25, tumor size more than 3cm in 25, less than 3cm in 17; have metastasis of lymph node in 7,have not metastasis of lymph node in 35;superglortic cancer in 24,glortic cancer in 16,subglotric cancer in 2;stage I II in 25, stage III IV in 17; grade I in 5, grade II in 35, grade III in 2). All specimen was mensurated by RT-PCR,38 of laryngeal carcinoma and 12 of normal mucosa was mensurated by Flow Cytometry (FCM). RT-PCR method: firstly isolate RNA by Trizol Reagent, then appraise its pureness and completeness, at last RT-PCR. P73 primes is 5' -AAC GCT GCC CCA ACC ACG AG-3' and 5'-GCC GGT TCA TGC CCC CTA CA-3' ,under the following conditions: 95 1 minute, 60 1 minute, 72 1.5 minute for 33 cycles. PCR product is 231bp. Result judgement : After 2% gelose gel electrophoresis of 8 u 1 PCR product, then analysize photon intensity of DNA ladder by gel imaging system, specific value of p73 and 0 -actin act as standard of quantitative expression of p73. FCM method: the first labeling antibody is rabbit-derived polycolonal antibody to p73. the second antibody for p73 isFLIT-IgG, a sheep-derived polycolonal antibody to rabbit. The measure was performed with a flow cytometer(FACC420BD USA). Result judgement: Fluorescence Index was defined as the quantitative expression index of p73 gene pyotein. The FI value more than xn + 2s of normal laryngeal mucosa should be consided positive expression(p73 FI>1.06), whereas negative expression.Results: 1.Expression of p73 mRNA in laryngeal carcinoma and normal mucosa: 32 of 42 (76.2%)laryngeal carcinoma are p73 positive expression, 9 of 42 (21.4%)laryngeal carcinoma are p73 negative expression, p73 mRNA positive expression rates in laryngeal carcinoma is higher than those in normal mucosa, it has very remarkable difference in statistics(p=0.001). specific value of between p73 and B-actin is 0.67 + 0.25 in laryngeal carcinoma, specific value of between p73 and B-actin is 0.15 + 0.06 in normal mucosa, expression quantity of p73 in laryngeal carcinoma is obvious higher than those in normal mucosa, it has very remarkable difference in statistics(p=0.0001). 2. relation between p73 mRNA and characteristic of clinical pathology in laryngeal carcinoma: specific value of between p73 and 3 -actin in stageIII IV (0.94 + 0.31) were remarkable higher than those in stage I II(0.42 + 0.19), it has very remarkable difference in statistics(t=3.59, p=0.001). In pathology specific value ofbetween p73 and B-actin in grade III(2.26+0.83) was also higher than those in grade I II(0.76+0.22,0.60 + 0.17), it has very remarkable difference in statistics(p=0.0002). The ex...