Studies On Expression Of Proto-oncogene C-met In Esophageal Squamous Carcinoma And Its Correlation With COX-2 Expression, COX-2 Expression And Its Correlation With Cell Proliferation And Apoptosis

ObjectiveEsophageal carcinoma is one of common malignant carcinomas of digestive tract, involving activation of multiple proto-oncogenes in the pathogenesis. C-met is a protein product coded by C-met proto-oncogene, it is the receptor of hepatocyte growth factor, with activities of tyrosine kinase, correlated with many oncogene proteins and adjusting proteins. It participates in transduction of cell signal, adjustment of cytoskeleton rearrangement, and it is the important factor for cell proliferation, differentiation and migration. It is believed at present that, C-met is closely correlated with genesis and metastasis of various carcinomas. It is indicated in many studies that excessive C-met expression and genetic amplification occurs in genesis and metastasis of carcinomas.Currently, C-met expression is rarely reported in literatures on esophageal carcinoma. Therefore, we have adopted reverse transcription-polymerase chain reaction (RT-PCR) to detect expression of C-met mRNA in esophageal carcinoma of 57 patients and normal esophageal tissues of 20 patients, and analyze the expression of C-met protein; and also immunohistochemical method to detect C-met protein and COX-2 protein in esophageal carcinoma of 57 patients and normal esophageal tissues of 20 patients, thus to investigate the correlation between C-met protein and COX-2 protein. We still have applied flow cytometry to detect and analyze proliferation activity and apoptosis level of esophageal carcinoma cells, with the hope to confirm that COX-2 expression is correlated with proliferation and apoptosis of carcinoma cells.Materials and methodsⅠ. Experimental materialsEsophageal squamous carcinoma of 57 patients and normal adjacent esophageal tissues of 20 patients were obtained from Department of Chest Surgery, Provincial Hospital of Shandong University. There were 46 males and 11 females aged 34-72 years old, with a median age of 52.9 years. Of carcinoma tissues of 57 patients, carcinoma tissues were low differentiated in 13 patients, moderately differentiated in 28 patients, and highly differentiated in 16 patients, and 26 patients had lymphatic metastasis. Of these patients,28 patients had carcinoma located in middle segment of esophagus, and 29 patients had carcinoma in inferior segment; 32 patients had carcinoma≤5cm, and 25 patients had carcinoma>5cm. According to the clinical staging standard modified in 1997 by International Union against Cancer (UICC), there were 31 patients of stageⅡa, 26 patients of stageⅡb+Ⅲ, patients with carcinoma in superior segment of esophagus, of stage T4 and M1 (stageⅣ) were not enrolled in the operation. No radiotherapy and chemotherapy were conducted in all patients.Ⅱ. Experimental methods1. C-met expression detected by reverse transcription PCRTake 100mg tissues to be tested and add 1ml Trizol solution to completely homogenate. Further extract total RNA according to the instructions of Trizol kit, and determine the RNA concentration and purity by DNA/RNA analyzer. Add 10×RNA PCR Buffer 1μl, dNTP 1μl, MgCl2 2μl, Random 9mers 0.5μl, RNA 1μl, RNase inhibitor 0.25μl, Reverse Transcriptase 0.5μl and ddH2O 3.75μl. Reaction conditions:30℃10min,42℃30min,99℃5min and 5℃5min.Primer sequence of C-met:5'-TTCACCGCGGAAACACCCATC-3'(upper stream) and 5'-GTCTTCCAGCCAGGCCCAGT-3'(downstream). In 12.5μl reaction system, it contained sterilized ddH2O 7.16μl,20pmol/L upper and down stream 0.15μl, cDNA2.5μl, Taq Hs 0.04μl,5×Buffer 2.5μl. Reaction conditions:95℃2min,95℃1min,58℃1min,72℃1min,30 cycles, elongate at 72℃for 7 min. The internal referenceβ-actin was used to detected reverse transcription rate. PCR product was treated by 8% polyacrylamide gel electrophoresis at 100 V for 1.5 h. After electrophoresis, automatic gel imaging system was used to take images and analyze. Electrophoresis straps were determined with 100 bp gradient Makers (TaKaRa) as the molecule control.2. Expression of C-met and COX-2 proteins detected by using immunohistochemical methodStreptavidin-peroxidase (S-P) immunohistochemical method was adopted, using first antibody C-met and COX-2 rabbit anti-human polyclonal antibody (BIOCHEMICALS, USA), working concentration 1:100; S-P kit was the product of zhongshan company. Experimental procedures were conducted according to the kit instructions. The confirmed positive gastric cancer sections were taken as positive controls, PBS was used as the negative control to replace first antibody. Cytoplasm stained yellowish to brown yellow was the marker of positive cell. Three grades were classified according to the quantity and color intensity of positive cells:weak positive (+), positive (++) and strong positive (+++).3. Proliferation activity and apoptosis level of esophageal carcinoma cell detected by flow cytometryCut tissues blocks preserved under profound hypothermia, and prepare single cell suspension by using mesh screening method, and adjust cell number to 1×106/ml by using PBS. In a portion of suspension, add 1ml PI staining solution (PI 5mg, Rnase 2mg, TritonX-1001ml, normal saline 65ml, natrium citricum 100mg, and add distilled water to 100ml), place in a 4℃refrigerator, stain away from light for 30 min and inject into the analyzer. Add 5μl FITC-AnnexinV and PI into another portion, and stand under room temperature away from light from 15 min, inject into the machine and analyze.The applied flow cytometry was FACSCalibur (B.D, USA). The light source was a 488 nm argon ion laser, green florescence was irradiated after FITC was stimulated, PI radiated red florescence. From each portion of specimen,10 000 cells were counted, and then relevant software was utilized on Macintosh 9.5 computer for data analysis. Before determination, human lymphocytes were used as standard products to adjust the CT value of the machine within 3.0%. MetaMorph (CellQuest 3.0) cell cycle analysis software was applied to calculate the percentages of different phases in DNA histogram, and proliferation index (PI) was used to represent proliferation activity. In 2D lattice diagram, cells with high FITC staining and low PI staining (FITC+PI-) were early apoptotic cells, and its percentage was calculated.Ⅲ. Statistical analysisThe numeration data was tested by X2 test, measurement data by t test. All tests were conducted by using SPSS13.0 software.Results1. Expression of C-met gene in esophageal carcinomaThe positive rate of C-met gene in 57 patients with esophageal carcinoma was 52.6% (30/57), but it was lowly expressed in normal adjacent tissues of 20 patients, the positive rate was only 5.0%(1/20), with significant difference between them (P<0.01).2. Correlation of C-met gene with clinical pathological properties of esophageal carcinoma tissuesExpression of C-met gene was not correlated with patient age, gender, lesion length and differentiation degree (P>0.05), but relevant with infiltrating depth of carcinoma tissues (P<0.05), lymphatic metastasis and TNM stages (P<0.01).3. Correlation of C-met protein expression in esophageal carcinoma tissues with expression of COX-2 proteinThe positive rate of C-met protein in 57 patients with esophageal carcinoma was 52.6%(30/57), but it was lowly expressed in normal adjacent tissues of 20 patients, the positive rate was only 5.0%(1/20), with significant difference between them (P<0.01). In esophageal carcinoma tissues, expression of C-met protein was correlated with infiltrating depth of carcinoma tissues, lymphatic metastasis and TNM stages, but irrelevant with patient age, gender, lesion length and carcinoma differentiation degree (P>0.05).The positive rate of COX-2 protein in 40 of 57 patients with esophageal carcinoma (positive rate was 70.2%), but only 1 case had positive expression of 20 patients with normal adjacent tissues (positive rate was 5.0%), with significant difference between them (P<0.01). In esophageal carcinoma tissues, expression of COX-2 protein was correlated with lymphatic metastasis and TNM stages (P<0.05), but irrelevant with patient age, gender, lesion length, carcinoma infiltrating depth and carcinoma differentiation degree (P>0.05).In esophageal carcinoma tissues of 30 patients with positive C-met protein expression, 26 patients had positive COX-2 protein expression; in esophageal carcinoma tissues of 27 patients with negative C-met protein expression,14 patients had positive COX-2 protein expression. By statistical detection, C-met protein expression was positively correlated with COX-2 protein expression (r=0.436,χ2=8.203, P=0.004).4. Correlation COX-2 gene expression with carcinoma cell proliferation in esophageal carcinoma tissuesIn esophageal carcinoma of 57 patients detected by using immunohistochemical method, the average proliferation index (PI) was (30.14±4.99)%. Forty patients had positive COX-2 protein expression,17 patients had negative COX-2 protein expression, and the proliferation indexes (PI) of esophageal carcinoma cell were respectively (31.41±5.14)% and (27.15±4.66)%, with significant difference (t=6.933, P<0.01).5. Correlation of COX-2 gene expression with early apoptosis of carcinoma cells in esophageal carcinoma tissuesIn esophageal carcinoma tissues of 57 patients detected by immunohistochemical method, the average apoptosis rate was (2.18±0.78)%. Forty patients had positive COX-2 protein expression,17 patients had negative COX-2 protein expression, and the early apoptosis rates were respectively (1.97±0.71)% and (2.66±0.94)%, with significant difference (t=4.831, P<0.01).Conclusion1. C-met gene is highly expressed in esophageal squamous carcinoma, and lowly expressed in normal adjacent esophageal carcinoma tissues.2. Expression of C-met gene is irrelevant with the patient age, gender, lesion length and tissue differentiation degree, but correlated with carcinoma infiltrating depth, lymphatic metastasis and TNM stages.3. COX-2 gene is highly expressed in esophageal squamous carcinoma, but lowly expressed in normal adjacent esophageal tissues.4. Expression of COX-2 gene is irrelevant with patient age, gender, lesion length, carcinoma infiltration depth and differentiation degree, but correlated with lymphatic metastasis and TNM stages.5. In esophageal squamous carcinoma tissues, C-met protein expression is closely correlated with COX-2 protein expression.6. COX-2 gene expression in esophageal squamous carcinoma is correlated with proliferation of carcinoma cells.7. COX-2 gene expression in esophageal carcinoma tissue is correlated with early apoptosis of carcinoma cells.