Expression Of EZH2 In Transitional Cell Carcinoma Of The Bladder Cell Lines And Tissues And Its Significance

Bladder cancer is the most common cancer in urological system in China. Transitional cell carcinoma (TCC), which refers to cancers arising from the transitional epithelium of the bladder, accounts for >90% of bladder cancers. According to its invasive depth, it can be divided into two groups: superficial tumors and invasive tumors. Although superficial tumors have a low risk of progression to muscle invasion, they have a high risk of recurrence. The prognoses of invasive tumors, which have high malignancy, are poor. The reports that the survival rates of invasive tumors are more than 50% are rare. And its basic treatment is total cystectomy. Qualities of life of patients who deserve that operations drop seriously. Therefore, it is critical to explore an efficient treatment for the bladder carcinoma.EZH2 (enhancer of zeste homolog 2) is the human homolog of enhancer of zeste gene in Drosophila melanogaster, and it is an important member of Polycomb group of genes. PcG proteins control the transcription remember system, repress transcription and maintain the inactive status of homeotic genes. PcG proteins may also be involved in the control of cellular proliferation, as several PcG complexes have been shown to act either as proto-oncogenes or as tumor suppressors in vertebrates. EZH2, which is an important member of PcG, plays a critical role in maintaining the inhibition state of Hox genes. It is also involved in X-inactivation, germline development, stem cell pluripotency and cell proliferation. It has been reported that the overexpressions of EZH2 gene are found in many human malignancy, but little or even non-expression in the corresponding normal tissues.The study detects the expression information of transitional cell carcinoma of the bladder cell lines, carcinoma tissues and normal bladder tissues. It may be contribute to clarify the roles of EZH2 in bladder carcinoma.1. Expression of EZH2 gene in TCC cell lines.1.1 Semiquanitative RT-PCR analysis of EZH2 mRNA expression in cell lines1.1.1 Establishing the semiquanitative RT-PCR methodThe RT-PCR was done by the way that EZH2 gene and GAPDH gene were amplified in the same tube. We analyze the IOD of RT-PCR products when we used different cycles and different ratios of EZH2 primer and GAPDH primer. We found that when the cycle was 28 and the ratio was 3/1, the efficiency of amplification was similar, and we used this condition for our future studies.1.1.2 EZH2 mRNA expression in TCC cell linesWe used semiquanitative RT-PCR with the condition that we described previously to detect the EZH2 mRNA in four TCC cell lines T24,EJ,MGH-U1,BIU-87 and prostate cancer cell line PC-3M. We found that all bladder carcinoma cell lines and prostate cancer cell line had the EZH2 mRNA expression. 1.2 Western blot analysis of EZH2 protein expression in TCC cell lines.We extract proteins from cultured TCC cell lines T24,EJ,MGH-U1,BIU-87 and prostate cancer cell line PC-3M. We detected expressions of EZH2 proteins in these cell lines. We found that all cell lines have EZH2 protein expression, which was in agreement with RT-PCR results.1.3 Immunocytochemistry analysis of EZH2 protein expression in TCC cell linesWe used immunocytochemistry to stain the sections of four cultured TCC cell lines T24,EJ,MGH-U1,BIU-87 and prostate cancer cell line PC-3M. We found that EZH2 proteins are positive in all five cell lines. The positive reaction represents yellow, and the EZH2 protein was located in cellular nucleus.2. Expression of EZH2 gene in TCC and normal bladder tissues.2.1 Semiquanitative RT-PCR analysis of EZH2 mRNA expression in TCC and normal bladder tissuesOne of 12 normal bladder tissues was detected EZH2 mRNA expression and the positive rate was 8.3%. 37 of 45 TCC tissues were detected EZH2 mRNA expression and the positive rate was 82.2%. The positive rate of superficial tumors (Tis, Ta, T1) was 74.2%(23/31),while the positive rate of invasive tumors (T2,T3,T4)was 100%(14/14);The positive rate of TCC G1 was 61.5%(8/13),G2 was 85.7%(18/21),G3 was 100%(11/11)。2.2 Western blot analysis of EZH2 mRNA expression in TCC and normal bladder tissuesWe used Western blot to detect the expression of EZH2 protein from the same tissues, and we found that the expression results in agreement with RT-PCR results2.3 The relationship between EZH2 gene expression in normal bladder tissues and TCC tissues and the tumor grades and stages.Through statistics analysis, the EZH2 expression rate difference between normal bladder tissues and TCC was significance (P<0.01); there is no difference in EZH2 expression rate between superficial tumors and invasive tumors (P=0.094). the EZH2 expression rate difference between TCC G1 and G3(P=0.041); And there is no difference between G2 and G3 (P=0.534), G1 and G2 (P=0.211).2.4 Immunohistochemistry analysis of EZH2 protein expression in TCC tissuesWe used immunohistochemistry to stain 40 TCC paraffin section and count the positive cell rate. Through statistics analysis, there is no difference in EZH2 protein nuclei expression rate (P=0.388); there is also no difference between G1 and G2 (P=0.626), G2 and G3 (P=0.698), G1 and G3 (P=0.348).From our studies, we conclude that the deregulation of EZH2 gene correlate with the cancerogenesis of transitional cell carcinoma and it can be used as a new oncogene and tumor bio-marker. Its deregulation also correlates with the malignancy of TCC and may correlates with the invasion of TCC. So EZH2 could be used as a potential gene therapy target. EZH2 gene is detected in four TCC cell lines T24,EJ,MGH-U1,BIU-87 and prostate cancer cell line PC-3M, and these cell lines would be used for in vitro experiment study in which EZH2 may be the gene therapy target.