| Renal cell carcinoma (RCC), which accounts for2-3%of all adult malignancies, is known as the second most common urologic tumors in China and its incidence has increased over the past few years. Approximately,80%of all RCC is clear cell renal cell carcinoma (CCRCC), which originates from renal proximal tubule cells. RCC is often deficient of clinical symptoms in early period. Despite the increasing number of cases diagnosed at small size due to extensive use of various image checks including ultrasonography, computer tomography, and magnetic resonance imaging, RCC presents with metastases in up to25%-30%of cases at the time of diagnosis. So far, nephrectomy is still the most effective treatment of clinically localized tumor and nearly25-50percent of the patients undergoing operation will eventually develop recurrence. Renal cell carcinoma is resistant to conventional chemotherapy and radiotherapy, with only small subsets of patients responding to immunotherapy. Although treatment has significantly improved with the introduction of targeted therapy, metastatic renal cell carcinoma has a poor prognosis with a progression-free survival (PFS) rate of about8～10months after the patients receive first-line targeted therapy. Up to now, the pathogenesis of renal cell carcinoma remains unclear. Current researches indicate that the incidence and development of renal cell carcinoma is a mutli-stage process marked by multi-factor and multi-gene abnormality. Actively exploring new cancer-related genes of renal cell carcinoma can help to reveal the molecular mechanisms of the disease and provide new clues and strategies for treatment of this tumor.In recent years, the homeobox genes and their roles in oncogenesis have been a hot topic of gene regulation in cancer research. The engrailed2(EN2) gene belongs to the non-class I homeobox gene family members. Human EN2gene is located on chromosome7q36, encoding333amino acids. The gene structure is composed of two exons and one intron. EN2protein contains five sub-regions with functional conservation, which are termed EH1-EH5from the N-ternimus to the C-ternimus. EN1and EH5regions can mediate transcriptional repression, with the former recruiting the co-repressor groucho. Meanwhile, EH2and EH3bind another homeodomain-containing transcription factor PBX that modifies significantly the DNA binding affinity and specificity of EN2, which enables EN2gene to play an activation role or inhibition role in process of regulating transcription. EH4is a highly conserved homeodomain, consisting of61amino acids. The structure of EH4is same with homology domain of other homeobox proteins. As a transcriptional factor, to combine the specific DNA is one of the important biological functions of EN2protein, which plays a regulatory role at transcriptional level. EN2protein can be secreted from cells and internalized by others. Its activities depend upon a conserved nuclear export sequence located in the EH4region, which is a tightly regulated process. As a substrate of serine/threonine protein kinase CK2, phosphorylation of a serine-rich domain within EN2by the protein kinase CK2specifically inhibits secretion and intemalization of EN2in cells. The down-expression of CK2enhances the activities of secretion and intemalization of EN2protein, and vice versa.EN2gene plays a key role in early embryonic development, particularly in nervous system development process. EN2gene may be involved in establishment and maintenance of space integrity in the specific region of developing tissues and organs. During embryonic development, gene mutations can cause malformations in organogeny, which result in tumorigenesis in adults. This study found that EN2was ectopically expressed in human breast cancer tissues. When the nontumorigenic mammary cell lines were engineered to ectopically express EN2, the cell lines had a significant reduction in their cycling time, losing cell contact inhibition, and becoming sensitive to anti-cancer treatment. Suppression of EN2mediated by siRNA can inhibit proliferation of human breast cancer cells. It is suggested that EN2is a candidate oncogene in human breast cancer. Another research indicated EN2was over-expressed in human prostate cancer. Down-regulation of EN2expression by siRNA resulted in a decrease of PAX2expression and a dramatic decrease in prostate cancer cell proliferation. The urinary EN2protein increased in prostate cancer and bladder cancer, which could be a sensitive and specific protein biomarker for prostate cancer and non-muscle-invasive bladder cancer.However, the expression of EN2and its significance in CCRCC has not yet been elucidated. In this study, we used immunohistochemistry (IHC), Western blot analysis and real-time quantitative polymerase chain reaction (RT-qPCR) to investigate the expression of EN2in CCRCC tissues and cells. Moreover, in order to explore the effect of EN2in cell proliferation, apoptosis and invasive ability of CCRCC, human CCRCC cell line786-O was engineered to over-express EN2by plasmid construction and gene transfection.1. Expression of EN2in clear cell renal cell carcinoma tissuesObject:EN2gene, a member of HOX genes family of homeodomain-containing transcription factors, is found to be over-expressed and a potential oncogene in a number of cancers. Nevertheless, the expression status of EN2in CCRCC remains unclear. The purpose of the study is to investigate the expression and significance of EN2in clear cell renal cell carcinoma.Methods:The three sample groups included in this study were:1) tissue from CCRCC tumors (n=35, experimental group);2) tissue from normal kidney adjacent to the tumors collected in group one (n=3S, control group1); and3) tissue from normal renal kidney from patients that underwent nephrectomy for renal injury and multiple angiomyolipoma of the kidney (n=12, control group2). The expression of EN2were detected in these tissues by immunohistochemistry (IHC), Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot respectively. Results:Cells were stained for the presence of EN2using IHC. The CCRCC tissue, adjacent tissue to the tumor and normal renal parenchyma tissue showed stained cells with percentages of22.8%,100%and100%respectively, the difference was significant shown by Fisher’s exact test (χ2=59.39, P=0.000). No difference was identified between the two control groups. A significant negative correlation was found between EN2expression and histological grade (Spearman correlation coefficient was-0.382, P=0.003). There was no statistical association between EN2expression level and the patient’s sex (Spearman correlation coefficient was0.074, P=0.674), age (Spearman correlation coefficient was0.126, P=0.0.473), tumor size (Spearman correlation coefficient was0.193, P=0.266), or tumor stage (Spearman correlation coefficient was0.077, P=0.660). EN2protein was present mainly in cytoplasm and nucleus of the normal renal tubular cells and renal tubule, but invisible in the interstitial tissue between renal tubules and renal corpuscles. EN2protein was distributed only in the cytoplasm in few tumor tissues. EN2mRNA expression was3.9-fold higher in adjacent tissues compared with tumor tissues (t=4.863, P=0.001) by RT-qPCR. As expected, the expression of EN2protein in adjacent tissues increased compared with the CCRCC tissues by Western blot, the difference was significant showed by Paired-sample t test (t=5.362, P=0.000).Conclusion:Our study provides vigorous evidence that EN2is expressed at high levels in renal tubule epithelial cell in adulthood and can be secreted into renal tubule. EN2is down-regulated and ectopically expressed in CCRCC and absence of EN2expression is associated with poor histological grade.2. Expression of EN2in clear cell renal cell carcinoma cell lineObject:EN2is found to be down-regulated and ectopically expressed in CCRCC tissues. The purpose of this study is to investigate the expression of EN2in Human CCRCC cell lines786-0and human normal kidney cell line HK-2in vitro.Methods:786-O and HK2were cultured in vitro. The expression of EN2mRNA and protein were detected in786-0and HK-2by RT-qPCR, Western blot, and Immunofluorescent respectively.Results:The result of QRT-PCR indicated the EN2mRNA expression was1.2-fold higher in HK2cells than in786-0cells, however, the difference was not significant shown by Independent-sample t test (t=2.85, P=0.065). EN2protein was high-expressed in HK-2cells compared with786-O cells by Western blot. EN2protein was present mainly in the cytoplasm and nucleus of HK-2cells in Immunofluorescent analysis.Conclusion:EN2was expressed at high levels in HK-2cells and down-regulated in786-0cells, Which is coincident with CCRCC tissue and normal renal tissue adjacent to tumor.3. Construction and identification of the eukaryotic expression vector of EN2and the stably transfected cell lineObject:We constructed eukaryotic expression vector PcDNA3.1+EN2and transfected it stably into the human clear cell renal cell cancer786-0cell in order to research the effect of high-expression of EN2on the biological function of CCRCC.Methods:The total sequence of coding region of EN2gene was amplified by PCR, and the eukaryotic expression vector PcDNA3.1+EN2was constructed by DNA recombination technique. The eukaryotic expression vector pcDNA3+EN2was identified by PCR, restriction enzyme analysis and sequencing. Two plasmids including a recombined pcDNA3.1+EN2and empty PcDNA3.1were transfected into786-0cells cultured in vitro by liposome-mediated gene transferred method. After being screened with786-0, the stable transfectants were obtained. RT-PCR and Western blot were used to access the expression of EN2mRNA and encode protein in786-0cells before and after transfection.Results:Restriction enzyme analysis and sequencing results showed that the sequence of this gene fragment was identical with EN2gene sequence reported in GenBank. The results of RT-qPCR and Westen blot showed that the expression of EN2mRNA (F=571.48, P=0.000) and protein (F=6.861, P=0.028) were higher in cells transfected with PcDNA3.1+EN2compared with the un-transfected cells group and empty vector-transfected cells group, and there was no significant difference between the two control groups.Conclusions:The eukaryotic expression vector PcDNA3.1+EN2was successfully constructed and was transfected to786-0cells, which markedly enhanced the expression of the EN2mRNA and encode protein in the786-0cells.4. Effects of EN2on biological characteristics of human CCRCC cellPurpose:In former studies we found that EN2gene was down-regulated and ectopically expressed in CCRCC as well as constructed eukaryotic expression vector pcDNA3.1+EN2and transfected successfully to786-0cells. To further research into the functions of EN2, MTS test, flow cytometry, and transwell invasion test in vitro experiment were conducted to detect biological characteristics of CCRCC cell.Methods:The eukaryotic expression vector pcDNA3+EN2was constructed and transfected successfully to786-0cells. The786-0cell line transfected eukaryotic expression vector pcDNA3.1+EN2was experimental group, with the cell line transfected empty PcDNA3and786-0cell un-transfected being control groups. MTS colorimetric assay was conducted to detect its activity of proliferation. Apoptosis of cancer cell line was analyzed by flow cytometry and invasion efficacy was accessed by transwell invasion test in vitro experiment.Results:MTS colorimetric assay showed that in the first day after transfection, the OD values of PcDNA3.1+EN2, PcDNA3.1and un-transfected786-0group were0.54±0.03,0.64±0.02, and0.72±0.01respectively. The second day the values were0.84±0.02,1.07±0.04, and1.1±0.08respectively and the third day the values were1.13±0.05,1.59±0.08, and1.58±0.06respectively. Urivariate analysis of variance showed that the experimental group showed a significant slow proliferation compared with two control groups (F=11.73. P=0.000). The results indicated that the over-expression of EN2could significantly slow down proliferative activity of786-0cells.On the one hand, the percentage of cell number of experimental group in S period increased in the first and second day after transfection while the percentage in G0/G1period decreased in the second and third day after transfection by flow cytometry. On the other hand, the percentage of apoptosis cell of the experimental group increased and the number of active cells reduced significantly compared with two control groups. The percentages of apoptosis cell of PcDNA3.1+EN2, PcDNA3.1 and786-O group were2.93%±0.92,1.01%±0.33and0.51%±0.17respectively. Urivariate analysis of variance showed that the percentage of apoptosis cell was different among three groups (F=72.72, P=0.000).The results suggested that high-expression of EN2gene could enhance apoptosis in786-0cells.Transwell invasion test showed that after transfection for48h, the cell numbers through matrigel film of pcDNA3.1+EN2, PcDNA3.1and un-transfected786-0group were1.38±2.2、59.00±26.73, and31.50±15.47respectively, One-way ANOVA test showed that the cell number through matrigel film was different among three groups (F=20.80, P=0.000). The experimental group was fewer compared with two control groups. The results suggested that high-expression of EN2gene could weaken the invasion of786-0cells.Conclusion:After being transfected pcDNA3.1+EN2plasmid, proliferative activity of786-0cells could slown down, the percentage of cell apoptosis was enhanced and invasive ability was weakened, which indicated that EN2might play an anti-oncogene role in oncogenesis and development of CCRCC. |
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