Expression Of EGFR,TGF-α,Signal Transducers And Activators Of Transcription 3 And Related Factors In Laryngeal Carcinoma

IntroductionLaryngeal carcinoma is a common head and neck malignancy and the 11th common malignancy in the world.Now surgery and radiation are the main therapies in laryngeal carcinoma;the contribution of chemotherapy to local control and prognosis still needs further research.During the last decades,total laryngectomy is decreaing along with increasing measures of larynx preservation but the survival rate did not increase significantly.Further studies about molecular mechanism of laryngeal carcinogenesis are very valuable to the prevention,diagnosis,and prognosis and drug selection of laryngeal carcinoma.EGFR(epidermal growth factor receptor) possess the acetivity as RTK(receptor tyrosine kinase),that's encoded by the gene of C-erbB1.EGFR expresses generally in epidermal cell and matrix cell.Clinical research indicates that overexpression of EGFR is common in lots of epitheliogenic malignant tumors.EGFR signal pathway is always closed in normal tissue,and will be activated in tumor tissue.The downstream signal transduction pathway chiefly contains Ras-ERK/MAPK,PI3K/AKT and JAK/STAT,in which the the JAK/STAT pathway correlates closely with the cell survival,growth, angiogenesis,invasion and immune escaping mainly through regulation of its target genes.Stat3 is a major member of STATs.Stat3 is latent in cytoplasm and activated through tyrosing phosphorylation.Phosphorylated stat3 forms homo- or hetero-dimer and translocates to nucleus to promote transcription.In normal cell stat3 activation is a short process lasting from several minutes to several hours.Stat3 activation has been found in various human malignancies and cell lines and was confirmed as an oncogene. Now carcinogenesis of head and neck is not clearly understood.Expressions of EGFR and relationship with its downstream signal transduction pathway stat3 in laryngeal carcinoma have not been reported around the world. ObjectiveTo investigate the expression and activation status of TGF-α,EGFR,Stat3,the relationship between activated Stat3 and TGF-α,EGFR.Materials and MethodsMaterialsPatients(n=62) with laryngeal carcinomas,who were treated at Shengjing Hospital of China Medical University,from March 2007 to Oct 2008 were included in this study.Mucosas,which were obtained from 13 patients with total laryngectomy and over 1.0cm away from tumor margin,were used as control.The mucosas were proved to contain no carcinoma cells pathologically.Reverse transcription-polymerase chain reactionTotal RNA was extracted by Trizol one-step method.After cDNA synthesis,PCR amplication was undertaken.The sequences of the human primers used were as follows: EGFR(187 base pair[bp]):sense,5'-GCCAAGGCACGAGTAACAAGC-3';antisense, 5'-AGGGCAATGAGGACATAACCAG-3';TGF-α(253 bp):sense,5'-CAGGGTGCGG AGATGGAAC-3';antisense,5'-CGAGGGCTCACGAGGAAGT-3';Stat3(149 bp): sense,5'-CACCAAGCGAGGACTGAGCAT-3';antisense,5'-GCCAGACCCAGAAG GAGAAGC -3';β-actin(473bp):sense,5'-GTGGGGCGCCCCAGGCACCA -3'; antisense,5'-CTCCTTAATGTCACGCACGATTTC -3'.Western blot analysisSamples were treated with lysis solution to get whole cell protein exracts and then electrophoresed through 10%sodium dodecyl sulfate(SDS)-polyacrylamide gel and were transferred onto a nitrocellulose membrane.Membranes were blocked and incubated with primary antibody.After incubation with alkali phosphatase(AP) conjugated secondary antibody.After developing,membranes were observed under UVP-8000.ImmunochemistrySP method was used and the procedures were followed by the instruction. Statistical analysisThe data were analyzed by the SPSS software v11.5.Measurement data was represented with average±standard deviation and t-rest was used.Chi-squared test was used in enumeration data.Pearson correlation analysis was applied for the bivariable relationship.Results1.In immunochemistry,EGFR protein was detected in cell membrane and cytoplasm.The positive rate of EGFR in 62 tumor was 77.4%with + 38.7%,++ 21.0%,+++ 17.7%.All 13 control mucosas were EGFR negative.The difference was statistically significant(x~2=18.61,P=0.000).2.EGFR protein was detected in 62 tumor and 13 control mucosa specimens in western blot.The EGFR protein value of tumor was 110.67±21.63 and that of control mucosa was 54.80±6.59.The difference was statistically significant(t=9.178,P=0.001).3.EGFR mRNA was detected in 62 tumor and 13 control mucosa specimens.The EGFR mRNA value of tumors was 70.49±17.09 and that of control mucosas was 31.99±2.99.The difference was statistically significant(t=8.055,P=0.000).4.In immunochemistry,TGF-αprotein was detected in cytoplasm.The positive rate of TGF-αin 62 tumor was 46.8%with + 22.6%,++ 19.4%,+++ 4.8%.All 13 control mucosas were TGF-αnegative.The difference was statistically significant (x~2=6.839,P=0.009).5.TGF-αprotein was detected in 62 tumor and 13 control mucosa specimens in western blot.The TGF-αprotein value of tumor was 84.14+15.04 and that of control mucosa was 38.19±3.96.The difference was statistically significant(t=10.879, P=0.000).6.TGF-αmRNA was detected in 62 tumor and 13 control mucosa specimens.The TGF-αmRNA value of tumors was 49.69±10.09 and that of control mucosas was 25.99±2.15.The difference was statistically significant(t=8.383,P=0.000).7.EGFR protein was positively correlated with TGF-αprotein.Pearson correlation coefficient was 0.896(P=0.000).8.In immunochemistry,Stat3 protein was detected in both cytoplasm and nucleus. The positive rate of Stat3 in 62 tumor was 61.3%with + 25.8%,++ 21.0%,+++ 14.5%. 100%in control mucosa was Stat3 negative.The difference was statistically significant (x~2=16.151,P=0.020). 9.In immunochemistry,p-Stat3 protein was detected in nucleus.The positive rate ofp-Stat3 in 62 tumor was 43.5%with + 16.1%,++ 19.4%,+++ 8.1%.100%in control mucosa was p-Stat3 negative.The difference was statistically significant(x~2=8.846, P=0.003).10.Stat3,p-Stat3 protein was detected in 62 tumor and 13 control mucosa specimens in western blot.The Stat3 protein value of tumor was 114.91±25.45 and that of control mucosa was 57.64±17.67.The difference was statistically significant (t=6.152,P=0.000).The p-Stat3 protein value of tumor was 70.65±9.53 and that of control mucosa was 13.77±2.33.The difference was statistically significant(t=8.890, P=0.000).11.Star3 mRNA was detected in 62 tumor and 13 control mucosa specimens.The value of Stat3 mRNA of tumor was 68.34±10.59 and that of control mucosa were 33.49±2.61.The difference was statistically significant(t=11.721,P=0.000).12.p-Stat3 protein was positively correlated with EGFR protein.Pearson correlation coefficient was 0.698(P=0.000).Conclusions1.There is high expression of EGFR mRNA and protein in laryngeal carcinoma.2.There is high expression of TGF-αmRNA and protein in laryngeal carcinoma.3.The aotucrine system consisted of TGF-αand EGFR might play an important role in the occurance and development of laryngeal carcinoma.4.There are high expressions of p-Star3,Stat3 mRNA and protein in laryngeal carcinoma.5.EGFR might have a relationship with Stat3 pathway activation in laryngeal carcinomas.