| Background and aimWilms tumor (WT) or nephroblastoma is the most common primary malignant tumor of kidney in children. It accounts for about8%of all pediatric malignant tumors. Although the long-term survial rate of WT has been enhanced by surgery, chemotherapy and radiotherapy in recent years, there still have some patients who were dead because of recurrence, metastasis and drug resistance. Therefore, it is very meaningful to explore the mechanism and find the biomarker of WT. Astrocyte elevated gene-1(AEG-1), also called metadherin (MTDH) and lysine-rich CEACAM1coisolated (LYRIC), was first identified and cloned by subtraction hybridization of genes expressed at elevated levels in primary human fetal astrocytes (PHFAs) infected by human immunodeficiency virus-1(HIV-1) in2002. Recent studies demonstrate that AEG-1is a pleiotropic protein that contributes to diverse signaling pathways such as PI3K/AKT, NF-κB, MEK/ERK, WNT/β-catenin. Also, AEG-1is correlated with tumor cell proliferation, invasion, metastasis, chemoresistance, and angiogenesis. Aberrant elevation of AEG-1expression frequently occurs in human cancers, including prostate cancer, breast cancer, lung cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, neuroblastoma, glioma, renal cancer, colorectal carcinoma, ovarian carcinoma, gastric cancer, osteosarcoma and salivary gland carcinoma. However, whether AEG-1deregulation also occurs in WT remains unclear. In this study, we will exploer the role of AEG-1in WT. Materials and methods1. The expression and prognostic value of AEG-1in WT:A total of42patients with a pathology confirmed diagnosis of WT were retrieved from the files of Department of Pediatric Surgery, Provincial Hospital Affiliated to Shandong University. The expression of AEG-1in WT was detected by immunochemistry. The relationship between the expression of AEG-1and the clinicopathologic factors was evaluated. Prognostic value of AEG-1was determined using Kaplan-Meier analysis.2. Silencing of AEG-1in cell line G401mediated by lentivirus vector:The expression of AEG-1in cell line CCC-HEK-1and G401were detected by real time quantitative polymerase chain reaction (real-time PCR) and western blot. Lentiviral vectors (LV-RNAi), containing U6promoter and green fluorescentprotein (GFP), were constructed to deliver small hairpin RNA (shRNA) targeting AEG-1into G401cell according to the manufacturers’instruction. The control group was the G401cell transfected with negative vector. The transfection efficiency was determined by detecting the GFP expression with the fluorescent microscopy. The expression level of AEG-1mRNA was examined by real-time PCR. The expression level of AEG-1protein was finally examined by western blot for identifing the inhibitory efficiency of RNAi.3. Effects and molecular mechanism of AEG-1gene silence on biological behavior of cell line G401:The cell proliferation was detected by MTT assay. Motility and migrating velocity of cells was tested by scratch wound healing assay. Invasion of cells were tested by transwell method. The expression of MMP2and MMP9related to invasion and metastasis of tumor was tested by western blot. The rates of apoptosis and cell cycle changes of the G401were studied by flow cyometry. The expression of Bcl-2, Bax, Caspase-3related to apoptosis and Cyclin D1, CDK2, p21Cip1, p27Kipl related to cell cycle were tested by western blot. Drug sensitivity was tested by MTT assay. The expression of MDR1related to drug resistance of tumor was tested by western blot.Results1. The expression and prognostic value of AEG-1in WT:High expression of AEG-1was detected in50%(21/42) of WT tissues, while no expression of AEG-1was detected in adjacent non-cancerous samples. AEG-1expression was significantly correlated with stage (P=0.019), histological type (P=0.048) and status of recurrence (P=0.015). However, no significant associations were found between AEG-1expression and other clinical features including age at diagnosis (P=0.354) and gender (P=0.751). Patients with high AEG-1expression had a shorter DFS and OS compared with those with low AEG-1expression (P=0.006and P=0.007).2. Silencing of AEG-1in cell line G401mediated by lentivirus vector:real-time PCR and western blot showed that AEG-1was high expressed in cell line G401compared with CCC-HEK-1. Two RNAi lentivirus expression vectors targeting to various sites of AEG-1gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by sequencing assay. The lentivirus was packaged in293T cells with high titer; shRNA targeting AEG-1was transfected into G401cells. The efficiency of AEG-1RNAi sequence against the expression of AEG-1was detected by real-time PCR and western blot. Real-time PCR and western blot showed that the relative mRNA levels and the protein levels were significantly reduced in both RNAi groups and the strongest inhibition of the expression of AEG-1was AEG-1RNAi1, which was choosed for the following experiments to investigate the effects and molecular mechanism of AEG-1gene silence on biological behavior of cell line G401.3. Effects and molecular mechanism of AEG-1gene silence on biological behavior of cell line G401:AMTT assay revealed that treatment with AEG-1RNAi did reduce the number of viable cells, compared with the control groups (P<0.0001). Knockdown of AEG-1significantly reduced the migration (P=0.0413) and invasion (P=0.0238) and the expression of MMP2and MMP9were down regulated (P=0.0395, P=0.0213). More silencing cells were at G0/G1phase and less were at S phase (P<0.001). The expression of p21Cip1and p27Kip1were up regulated (P<0.001). AEG-1silencing increased apoptosis level (P=0.0024). The expression of Bax and Caspase-3were up regulated (P<0.001), but the expression of Bcl-2were down regulated (P=0.024). The sensitivity of AEG-1silenced cells to doxorubicin or actinomycin D were increased (P<0.001) and the expression of MDR1were down regulated (P=0.0018).Conclusions1. AEG-1expression was significantly correlated with stage, histological type and status of recurrence. However, no significant associations were found between AEG-1expression and other clinical features including age at diagnosis and gender. Patients with high AEG-1expression had a shorter disease-free survival and overall survival compared with those with low AEG-1expression.2. AEG-1was high expressed in cell line G401compared with CCC-HEK-1. The construction and production of lentiviral vectors was successful. The relative mRNA levels and the protein levels were significantly reduced in both RNAi groups and the strongest inhibition of the expression of AEG-1was AEG-1RNAi1.3. AEG-1silencing affected the process of proliferation, invasion, metastasis, cell cycle, apoptosis, and chemoresistance. AEG-1overexpression may enhance characteristics of malignant aggressiveness by some critical molecules. |
PDF Full Text Request