The Expression And Significance Of Stk15 In Lung Squamous Cell Carcinoma And Adenocarcinoma

INTRODUCTIONIn most tumor, the chromosome instability is a common phenomenon, it often shows the chromosome gained or loss. The mechanism of chromosome instability remained unknown, but the function of centrosome during mitosis in remaining the stability of chromosome is important and chromosome instability associates with centrosome.Stk 15 (serine threonine kinase 15) is a centrosome - associated gene , the amplification or high expression of stk15 cause centrosomal anomalies, anuploidy formation and cell transformation . The expression level of stk15 in most normal tissues remains low, but the overexpression of stk15 is a common event in tumor.Recently, the studies on stkl5 expression in pancreatic tumor,esophageal cancer, gatric cancer and some other tumors were more. But the study in lung neoplasm were little. In the present study we detected the stk15 expression in lung squamous cell carcinoma and adenocarcinoma by immunohitochemitry and explored the correlation between stk15 expression and clinicopathological param-ers. At the same time we investigated the difference of stk15 expression between the normal lung tissues and lung squamous cell carcinoma and adenocarcinoma by Western Blot, immunohistochemistry and RT-PCR.MATERIALS AND METHODS1. MaterialsAll the 80 lung squamous cell carcinoma and adenocarcinoma and 40 nor-mal samples (fixed with 10% formalin and embeded in paraffin blocks) were obtained from the surgery in the first affiliated hospital of China Medical University between 2002 and 2003.2. ReagentsThe rabbit - anti - human polyclonal antibody of stkl5 was bought from No-vus Biologicals Company. The Ultro -- sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company. The goat - anti - rabbit second antibody was bought from Huamei Biological Company. The primer of stkl5 and RT kit were bought from Takala Biological Company.3. Methods3.1 ImmunohistochemistryDetect the stkl5 expression by immunhistochemitry S - P method. Scoring of the results; 100 cancer cells were counted in 10 good fields. The extent of staining was scored as ( - ) indicating < 20% of tumor area stained; ( + ) 20 -50% stained; ( + + ) >50% stained.3.2 Western BlotRapidly homogenize tissues (1 - 2g) in 5 volumes of lysis buffer ( 50mM Triscl PH7.4,150mM Nacl.O. 1%SDS,1%Triton - 100,lmM EDTA PH8.0, Aprotinin 12ul/10ml,PMSF60ul/10ml) ,Centrifuge the homogenate(13000rpm, 4) for 30 minutes to pellet insoluble material. ?Electrophoresis (^transfer proteins from gel to membrane <3) blocking (J) incubation with primary antibody (D enzyme conjugate incubation ?substrate incubation ?scan the results to computer3.3 RT-PCRExtract RNA from tissues in routine mothod by trizol regents,RT the RNA into cDNA and per the cDNA, take the per product to agarose electrophoresis, analyse the result by FluorChemV2.0 Stand Alone.4. Statistical analysisSPSS for Windows 11.5 statistical software was used to solve the data and Log - rank test to analyze the differences. Paired - T test was used to solve the data of Western Blot and RT - PCR. The significance level chosen was P value less than 0.05.RESULT1. The results of immunohistochemistryStkl5 expression in normal lung tissues and lung squamous cell carcinoma and adenocarcinomaThe expressin of stkl5 was located in cytoplasm and nucleus in lung squamous cell carcinoma and adenocarcinoma and normal lung tissues, and yellow could be observed. No color was found in cell membrane.X2 analysis suggested that the difference of stkl5 expression between normal lung tissues and lung squamous cell carcinoma and adenocarcinoma was signifi-cant(P<0.05).1.2 The relation of the expression of stkl5 and clinicopathological parametersNo potentially confounding correlation was observed between stkl5 expression and histological type, lymphatic metastasis( P >0.05). \ analysis suggested that the difference of stkl5 expression between different tumor cell differentiation was significant ( P <0.05). 2. The result of Western BlotThe analysis for the results showed that significant difference of stkl5 expression was found between the normal lung tissues and lung squamous cell carcinoma and adenocarcinoma The majority of lung squamous cell carcinoma and adenocarcinoma showed the specific staining band (46 kD) and the normal tissues have no specific band.3. The result of RT - PCRThe analysis for the results showed that significant difference of stkl5 expression was found between the normal lung tissues and lung squamous cell carcinoma and adenocarcinoma The expression level of stkl5 in 29 tumor samples (total number was 40) were higher than that in paired normal tissues.CONCLUTIONS1. Stkl5 protein and mRNA expression in lung squamous cell carcinoma...