Empirical Study Of The Expression Of NDRG2 In Renal Carcinoma And The Mechanism Of Inhibiting The Proliferation Of Human Renal Carcinoma Cell Line A-498

Aim: NDRG2 is a new gene cloned from the healthy human brain cDNA library in 1999 [GenBank accession number: AF159092], and the chromosomal localization is 14q12.1 which consists of 15 introns and 16 extrons. The total length of the cDNA of NDRG2 is 2024 bp which encodes the protein composed of 357 amino acid residues and the molecular weight is about 41 kD. The studies indicated that the expressions of NDRG2 in many tumorous tissues or cell lines, such as colon carcinoma, neurogliocytoma, leukemia, leucoma and carcinoma of parotid gland, are negative or very low, on the contrary, this gene in those corresponding normal tissues of the above mentioned tumors is of high level expression. Moreover, it is highly expressed in the cerebral cortex, substantia alba and nerve corpuscle of the central nervous system as well as in the endothelial cells of salivary gland and skeletal muscle cells. Meanwhile, the gene transfection researches indicated that NDRG2 can inhibit the transition of neurogliocytoma cell line BT325 from G1 phase to S phase. Thus, it is postulated that NDRG2 may be a new anti-oncogene. The purpose of this study is to discuss the role of NDRG2 in the pathogenesy of renal carcinoma and to ascertain the function of NDRG2 in order to provide the theoretical bases for the prevention and treatment of renal carcinoma.Methods: the RT-PCT, Western blot and immunohistochemistry are applied to determine the expressions of NDRG2 in the renal carcinoma cell line, the renal cell line; normal tissue and tissue of renal carcinoma; construct the recombinant adenovirus expression vector of NDRG2 and perform the packaging of the recombinant adenovirus and titer determination to obtain the recombinant adenovirus vector of NDRG2 with high titer for the subsequent study of cell function; transfect the recombinant adenovirus vector of NDRG2 into the suprarenal epithelioma cell line A-498 which has been confirmed to be with low expression of NDRG2 to increase the expression level of NDRG2 in this cell line, and analyze the cell cycle, detect the apoptosis by flow cytometry and use the Western blot to detect the changes of cell cycle-related protein; observe the effect of p53 gene to the expression of NDRG2 through the transfection of the recombinant adenovirus vector of p53 into the renal carcinoma cell line A-498. Results: 1. The NDRG2 protein and expression of the mRNA in human tissue of renal carcinoma and the renal carcinoma cell lines were detected and the results indicated that the renal cell lines HKC and HK-2 are of high expression levels of NDRG2 mRNA, however, the expression levels of NDRG2 mRNA in the renal cell carcinoma cell lines 786-O and A-498 were comparatively lower; the expression levels of NDRG2 mRNA in the tissue of carcinoma were lower than those in the tissue beside the carcinoma in 18 cases of patients with renal carcinoma and the expression level was directly correlated with the pathological grade; in this study, there were only 6 cases of patients with renal carcinoma in which the expression levels of NDRG2 mRNA in the tissue of carcinoma were higher than those in the corresponding tissue beside carcinoma; otherwise, there were 14 cases in which there was no difference between the expression levels of NDRG2 mRNA between the tissues. 2. The recombinant shuttle vector containing NDRG2 was firstly constructed by the mature adenovirus expression system pAdTrack-CMV from American Novagen company, the linearized recombinant shuttle vector and the vector containing the gene of skelemin were transfected into the cell line HEK293 through the liposomal transfection to obtain the prime viral strains, the single plaque bacteriophages were selected to further infect HEK293 cells to acquire the viral lysate of single plaque bacteriophage. Based on these operations, we established the prime seed lot, major seed lot and working seed lot and finally accomplished the production, purification and titer determination of the recombinant adenovirus pAd-GFP-NDRG2, and the final virus titer of the recombinant adenovirus was 1.1×1011 pfu/ml. 3. The results of Western blot confirmed that the transfection of the recombinant adenovirus pAd-GFP-NDRG2 increased the expression level of the endogenous NDRG2 in the cell line A-498. The subsequent results of MTT detection indicated that the OD values of MTT at different detected time points within 48 h in the A-498 group transfected by the recombinant adenovirus were all significantly lower than those corresponding values in the groups without transfection and transfected by the blank plasmid, which suggested that the expression of NDRG2 inhibited the growth of the cell line A-498. The further analysis of cell cycle showed that the plasmid transfection induced the G1 phase arrest in A-498, which implied that NDRG2 might inhibit the proliferation of A-498 through the cell cycle arrest. The results of the apoptosis analysis indicated that the proportion of the apoptosis cells in the A-498 cells infected by the recombinant adenovirus containing NDRG2, which was 12.% at 48 h after infection, was significantly higher that those in the A-498 cells without transfection (1.0%) and transfected by the recombinant adenovirus without NDRG2 (2.0%). Finally, the results of the Western blot indicated that, in the A-498 cells tranfected by human NDRG2, the expression levels of cyclin D1 and cyclin E decreased, but there was no significant change on the expression levels of cyclin D2, cyclin D3 and cdk2. Thus, it is postulated that NDRG2 may inhibit the cell proliferation through the effect on cyclin D1 and cyclin E. 4. It was discovered that A-498 cells express inactive p53 through the Western blot and RT-PCR, however, the expression of NDRG2 was almost undetectable. The increased expression level of NDRG2 was observed when the A-498 cells were transfected by the p53 gene in activated form, which was dose-dependent. This result suggested that NDRG2 is regulated by p53 gene, thus, NDRG2 is also a downstream gene of p53.Conclusions: NDRG2 is of high expression level in normal renal tissue but of low expression level or without expression in the tissue of renal carcinoma. NDRG2 can inhibit the proliferation of the renal carcinoma cell line A-498 cells, induce arrest at G1 phase and inhibit the expressions of cyclin D1 and cyclin E. The expression of p53 can up-regulate the expression of NDRG2. Thus, the result in this study further confirmed that NDRG2 is a novel anti-oncogene and the strategy of gene therapy taking this gene as the target might provide a new way to the gene therapy of renal carcinoma.