The Expression Of Enhancer Of Zeste Homolog2in Renal Cell Carcinoma And Its Clinical Significance And The Experimental Study On RNA Interference Targeting EZH2in Renal Cancer Cell Lines ACHN

Background:Renal cell carcinoma (RCC) represents2-3%of all adult cancers, with the highest incidence occurring in Western countries. In Europe, until recently, there was a general annual increase of2%in the incidence. The use of imaging techniques such as ultrasound (US) and computerised tomography (CT) has increased the detection of asympomatic RCC. However, incidence rates of RCC have now stabilized or declined in some north European countries (Sweden and Denmark), while other European countries are still showing an upward trend in the incidence of RCC. In China, RCC is considered to be the second most common genitourinary tract malignancy, and only next to bladder cancer. The peak incidence of RCC occurs between60and70years of age, with a1.5:1ratio of men to woman. Aetiological factor include lifestyle factors, such as smoking, obesity and hypertension. Because of the masked location of the kidney in our body and the changeable clinical symptoms of RCC, the early diagnosis is not easy. More than50%of RCCs are diagnosed incidentally. The classic triad of flank pain, gross haematuria and palpable abdominal mass is seldom found (6-10%). When the final diagnosis is reached, lymph node metastasis occurs in about35%of patients. Once lymph node metastasis occurs, even if the radical nephrectomy with retroperitoneal (lymph node) bloke dissection for RCC is given, the patient survival time is less than5years. The prognosis is significantly bad when the liver, lung and/or bone metastases occur. Because of the unsatisfactory results for RCC, radiotherapy and chemotherapy are not used as the adjunctive conventional therapy for RCC. The patients with RCC may benefit from application of gene therapy targeted for the expression of specific biomarker. But there is no satisfactory and reliable tumor cellular biomarker as the gene therapeutic target. Thus, it is a great need to search for the molecular biomarker of the tumor at the time of diagnosis, and which will help to guide the clinical therapy strategies and supervise the RCC prognosis.Enhancer of zeste homologue2(EZH2), a homolog of the Drosophila Enhancer of zeste (E(z)), is a conserved Polycomb group (PcG) protein. PcG and Trithorax group (TrxG), which play an antagonistic role in maintaining the homeotic genes expression or repression, are evolutionarily conserved modifier genes in the chromatin. The TrxG proteins promote gene expression, while the PcG proteins maintain the state of gene inactivation. In1996, EZH2gene was first found by Chen et al. when they researched the adjacent genes to virulence gene of Down’s syndrome. Accumulating evidences indicate that EZH2was of overexpression in many kinds of human cancers and maybe promoted the proliferation and diffusion of these tumor cells. EZH2was also shown to be a tumor-suppressor gene inhibitor. Hence, the characterization and identification of EZH2during the development of RCC may offer a satisfactory and reliable target for RCC treatment strategies.RNA interference (RNAi), as a gene silencing technique, was first invented in1998by Fire et al. There are many advantages of RNAi, such as high degree of specificity and efficiency, diffusive interfering effect and relatively simple experimental operation. RNAi, which was applied for some gene therapy, could precisely suppress the proto-oncogene expression. The main mechanism of RNAi is post transcription inhibition, and the basic process includes the initiation and effector phases. RNAi mediated plasmid, virus, chemosynthesis or other vector could effectively depress endogenous RNA expression. Through RNAi, we can silence EZH2gene expression and investigate its effect during the product and progress of RCC. What’s the relationship between EZH2and the WNT/β-catenin signaling pathways? And what’s the possible mechanism? To the best of our knowledge, there are no studies in the literatures, in vitro or in vivo, which answer these series of questions. Considering the questions and background above, our study includes the following four parts:1. The expression of EZH2in the tissue of RCC and human RCC cell line and the clinical significances;2. The construction of RNA interferencing EZH2sequences;3. The effect of gene EZH2silencing via siRNA transfection on proliferation, apoptosis and invasion of the RCC cell line ACHN;4. The mechanism of inhibition and the effect on WNT/β-catenin signaling pathways of EZH2in ACHN cell line.Objective:1. To study the expressive level of EZH2in the renal cell cancer tissue and the "normal" kidney tissue.2. To compare the mRNA and protein expressive level of gene EZH2between the renal cancer cell line ACHN and the normal human proximal tubule epithelial cell line HK-2.Methods:1. Immunohistochemistry analysis was used to detect the expression of EZH2in66cases of renal cell cancer and8cases of "normal" kidney tissues. Meanwhile, the clinical information and pathological data of all66patients were analyzed.2. RT-PCR was used to detect the mRNA level of gene EZH2of the cell line ACHN and HK-2, while western blot is used to detect the EZH2protein level of the both cell lines.Results:1. The result of EZH2expressive level in66cases of renal cancer tissues:positive48cases, negative18cases, and the positive ratio was72.7%. The expression result of EZH2in8cases of "normal" kidney tissues:positive1case, negative7cases, and the positive ratio was12.5%. The EZH2positive ratio of RCC was higher than that of "normal" kidney tissue obviously (P=0.002).2. The expressive level of EZH2was correlated with the RCC clinical Robson grade, the size of tumor, lymph node metastasis, and pathologic type of renal cancer (P<0.05), but it was not correlated with the sex and age of patients, Fuhrman grade (P>0.05).3. RT-PCR was used to detect the level of the EZH2mRNA and IODEZH2/IODGAPDH indicated the expressive level. Of the renal cancer cell line ACHN, the EZH2mRNA was0.648±0.003; of the normal human proximal tubule epithelial cell line HK-2was0.333±0.025. The ACHN had obvious higher level of EZH2mRNA than HK-2(P<0.01).4. Western blot was used to detect the level of the EZH2protein and IODEZH2/IODGAPDH indicated the expressive level. The EZH2protein level of cell line ACHN was0.290±0.009; HK-2was0.136±0.012. The ACHN also had obvious higher level of EZH2protein than HK-2(P<0.01).Conclusion:1. The EZH2protein expression were elevated in the human renal cancer. Because of EZH2involved in the development and progression of renal cancer, it maybe a reliable biomarker for the diagnosis of RCC and may be used as a target of RCC gene therapy.2. Because of the cell line ACHN had higher level of EZH2mRNA and protein than HK-2, the cell line ACHN can be used in the follow-up experiments. Objective:To investigate the construction and identification of RNA interference EZH2sequences, and to confirm whether the EZH2gene expression can be suppressed by the EZH2-RNA interference.Methods:According to the literature report, the region from461to481bases of EZH2complementation DNA was selected as the target and constructed EZH2siRNA expression plasmid. We designed the DNA model-strand to product the small hairpin (shRNA) of EZH2. Then the model strand was annealed and cloned into pGPU6/GFP/Neo plasmid to build the recombinatant plasmid pGPU6/GFP/Neo-EZH2shRNA, which was confirmed by sequencing. We used lipofectamine to transfect the pGPU6/GFP/Neo-EZH2shRNA into ACHN cell. Western blot was used to detect the EZH2expression after the RNAi. Results:We established three parallel groups:Group A (no treatment group, in which no liposome was transfected into the ACHN cell), Group B (negative control group, in which we used the GPU6/GFP/Neo plasmid without EZH2siRNA to transfect the ACHN cell) and Group C (interference group, in which we used lipofectamine to transfect the pGPU6/GFP/Neo-EZH2siRNA into ACHN cell). Western blot was used to detect the EZH2protein level of these groups and IODEZH2/IODGAPDH indicated the level. The Group A and B had higher level of EZH2protein than Group C (P<0.01). But there was no significant difference of the EZH2protein level between Group A and B (P>0.05).Conclusion:1. The expressing vector of pGPU6/GFP/Neo-EZH2siRNA was successfully constructed.2. The cell that stable expressed the EZH2siRNA was successfully constructed.3. The EZH2expression was significantly suppressed by EZH2siRNA in ACHN cell line. Key words ACHN cell line; EZH2; siRNA; Western blotObjective:Using the special siRNA interference EZH2sequence for transfection to the renal cancer cell line ACHN to silence the gene EZH2, and then to investigate its effect on proliferation, apoptosis and invasion of ACHN cell.Methods:We established three parallel groups:Group A (no treatment group, in which no liposome was transfected into the ACHN cell), Group B (negative control group, in which we used the GPU6/GFP/Neo plasmid without EZH2siRNA to transfect the ACHN cell) and Group C (interference group, in which we used lipofectamine to transfect the pGPU6/GFP/Neo-EZH2siRNA into ACHN cell). We used lipofectamine to transfect the pGPU6/GFP/Neo-EZH2siRNA into ACHN cell of these groups. The proliferation, apoptosis and invasion of the transfected ACHN cell was respectively assessed with MTT, flow cytometry (Annexin V/PI double staining) and transwell invasion assay.Results:1. We used the flow cytometry to detect the apoptosis index (AI) of ACHN cell in these three groups. The AI of Group C was higher than Group A and B (P<0.05). But there was no significant difference of the AI between Group A and B (P>0.05).2. We used the MTT assay to detect the proliferation of ACHN cell in three groups at four different times after transfection. The optical density at570nm of Group C was conspicuously less than Group A and B at48hours,72hours and96hours after transfection (P<0.01). But at24hours after transfection, there was no significant difference of the optical density among three groups (P>0.05).3. We used the transwell invasion assay to detect the invasion of ACHN cell in three groups. The cells in the lower chamber of Group C were conspicuously less than Group A and B (P<0.01). And there was no significant difference of the cells in the lower chamber between Group A and B (P>0.05).Conclusion:1. EZH2siRNA markedly promoted renal cancer cell line ACHN apoptosis.2. EZH2siRNA markedly inhibited ACHN cell proliferation in a significant time-dependent manner.3. EZH2siRNA markedly inhibited ACHN cell invasive capability. Objective:To investigate the molecular biological mechanism of inhibition and the effect on WNT/β-catenin signaling pathway system of EZH2siRNA in human renal cancer cell line ACHN.Methods:We still established three parallel groups just like part III: Group A (no treatment group, in which no liposome was transfected into the ACHN cell), Group B (negative control group, in which we used the GPU6/GFP/Neo plasmid without EZH2siRNA to transfect the ACHN cell) and Group C (interference group, in which we used lipofectamine to transfect the pGPU6/GFP/Neo-EZH2siRNA into ACHN cell). RT-PCR was used to detect the mRNA level of gene WNT3a,(3-catenin and GSK-3β of the cell line ACHN in these three groups, while western blot was used to detect the corresponding protein levels of these genes.Results:1. RT-PCR was used to detect the level of the WNT3α, β-catenin and GSK-3β mRNA, and IODEZH2/IODGAPDH, IODβ.catenin/IODGAPDH and IODGSK-3p/IODGAPDH indicated the corresponding expressive levels of these three genes. According to the results, the GSK-3P mRNA level of ACHN cell in Group C was higher than Group A and B (P<0.01, P<0.05). But the WNT3α and β-catenin mRNA level of ACHN cell in Group C was less than Group A (P<0.01, P<0.01) and B (P<0.01, P<0.05). And there was no significant difference of these three genes mRNA levels of ACHN cell between Group A and B (P>0.05).2. Western blot was used to detect the level of the WNT3a, β-catenin and GSK-3β protein, and IODEZH2/IODGAPDH, IODβ-catenin/IODGAPDH and IODGSK-3p/IODGAPDH indicated the corresponding expressive levels. According to the results, the GSK-3β protein level of ACHN cell in Group C was conspicuously higher than Group A and B (P<0.01). But the WNT3a and β-catenin protein level of ACHN cell in Group C was conspicuously less than Group A and B (P<0.01).Conclusion:1. EZH2siRNA markedly inhibited the expression of gene WNT3α and β-catenin.2. EZH2siRNA markedly promoted the expression of gene GSK-3β.3. EZH2siRNA could inhibit the occuring and developing of renal cell cancer via WNT/β-catenin signaling pathway.