| 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,prognosis and drug selection of laryngeal carcinoma.Signal transducers and activators of transcription(STATs)were found through the study of transcription activation in response to interferon alpha(IFNα)and interferon gamma(IFNγ)in 1990s.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.Star3 takes,functions in tumor cell survival,growth, angiogenesis,invasion and immune escaping mainly through regulation of its target genes.Main target genes of Stat3 are Cyclin D1,Bcl-xL and c-Myc et al.Now carcinogenesis of head and neck is not clearly understood.Expression of Stat3 and relationship with its downstream target gene cylin D1 in laryngeal carcinoma have not been reported around the world. ObjectiveTo investigate the expression and activation status of Stat3,the relationship between activated Stat3 and its target gene Cyclin D1 and related factors of activated Stat3 in laryngeal carcinoma.Materials and MethodsMaterialsPatients(n=64)with laryngeal carcinomas,who were treated at the First Affiliated Hospital of China Medical University,from Jan.2005 to June 2005 were included in this study.Mucosas,which were obtained from 12 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 sequence of the human primers used were as follows: Stat-3(463 base pair[bp]):sense,5′- GGCATTCGGAAAGTATT G-3′;antisense,5′-TCACCCACATTCACTCATT-3′; CyclinD1(378 bp):sense,5′-CCCACTCCTACGA TACGC-3′;antisense,5′'-AGCCTCCCAAACACCC-3′;β-actin(473bp):sense,5′-AAA TCGTGCGTGACATTAA-3′;antisense,5′-CTCGTCATACTCCTGCTTG-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.Immunochemistry SP 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.Stat3 mRNA was detected in 64 tumor and 12 control mucosa specimens.The Stat3 mRNA value of tumors was 69.49±10.78 and that of control mucosas was 33.24±2.89.The difference was statistically significant(t=11.518,P=0.000).2.Stat3 protein was detected in 64 tumor and 12 control mucosa specimens in western blot.The Stat3 protein value of tumor was 118.83±25.86 and that of control mucosa was 72.46±6.58.The difference was statistically significant(t=6.142, P=0.000).3.In immunochemistry,Stat3 protein was detected in cytoplasm and nucleus.The positive rate of Stat3 in 64 tumor was 67.2%with + 21.9%,++ 20.3%,+++ 25.0%. All 12 control mucosas were Stat3 negative.The difference was statistically significant (x~2=18.568,P=0.000).4.p-Stat3 protein was detected in 64 tumor and 12 control mucosa specimens in western blot.The p-Stat3 protein value of tumor was 61.89±18.80 and that of control mucosa was 13.65±2.62.The difference was statistically significant(t=8.821, P=0.000).5.In immunochemistry,p-Stat3 protein was detected in nucleus.The positive rate of p-Stat3 in 64 tumor was 48.4%with + 23.4%,++ 17.2%,+++ 7.8%.All 12 control mucosas were p-Stat3 negative.The difference was statistically significant(x~2 =9.817,P=0.002).6.There were no statistical differences of the p-Stat3 protein level in different sex, age,cell differentiation and tumor site.The p-Stat3 protein level in stage T1,T2 was higher than that in stage T3,T4.7.CyclinD1 mRNA was detected in 64 tumor and 12 control mucosa specimens. The value of CyclinD1 mRNA of tumor was 73.67±17.16 and that of control mucosa were 31.43±3.31.The difference was statistically significant(t=8.454,P=0.000).8.Cyclin D1 protein was detected in 64 tumor and 12 control mucosa specimens in western blot.The Cyclin D1 protein value of tumor was 113.12±23.65 and that of control mucosa was 55.32±7.55.The difference was statistically significant(t=8.343, P=0.000).9.In immunochemistry,Cyclin D1 protein was detected in nucleus.The positive rate of Cyclin D1 in 64 tumor was 43.7%with + 25.0%,++ 15.6%,+++ 3.1%.91.7% in control mucosa was Cyclin D1 negative;8.3%was +.The difference was statistically significant(x~2=5.371,P=0.020).10.p-Star3 protein was positively correlated with CyclinD1 mRNA.Pearson correlation coefficient was 0.827(P=0.000).Conclusions1.There is high expression of Stat3 mRNA and protein in laryngeal carcinoma.2.Abnormal activation of Star3 exists in laryngeal carcinoma.3.The level of activated Stat3 is high in early stage of laryngeal carcinomas.4.Activated Stat3 may take function through transcription activation of its downstream target gene Cyclin D1.
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