Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells
Introduction
Ki-67 is an established proliferation maker which is used extensively to estimate the proliferation fraction of tumors. Ki-67 labeling index is an independent prognostication for assessing the clinical outcome of individual patients with cancer including renal cell carcinoma (Tannapfel et al., 1996). The Ki-67 antigen is a large nuclear protein (Schlüter et al., 1993). The sequence of entire gene locus has been cloned (Duchrow et al., 1996). The Ki-67 antigen is known to accumulate from G1-phase to mitosis, where it is found at its highest content. The amount of the antigen decreases to a minimal level immediately after mitosis (Du et al., 1991). Detailed cell cycle analysis reveals that the antigen is present in nuclei of proliferating (G1-, S- and G2-phases and mitosis) cells but not in nuclei of quiescent cells (G0-phase) (Gerdes et al., 1984).
Recently it was demonstrated that the Ki-67 protein belongs to the family of MPM-2 antigens and further that phosphorylation of the Ki-67 protein during mitosis is associated with condensation of the chromosomes and separation of sister chromatids (Endl and Gerdes, 2000, MacCallum and Hall, 1999). Furthermore, a C-terminal domain of the Ki-67 protein (Kon21) is able to bind to all 3 members of the mammalian heterochromatin protein1 (HP1) family in vitro and in vivo, suggesting a role for the Ki-67 protein in the control of higher order chromatin structure (Scholzen et al., 2002).
Since transformation of malignant cells is frequently associated with uncontrolled cell proliferation and since proliferation is tightly associated with the Ki-67 protein labeling index, this antigen may represent a potential target for cancer therapy. Schlüter et al. reported that Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of human multiple myeloma cell line IM-9 cells (Schlüter et al., 1993). Kausch et al. reported antisense-mediated inhibition of Ki-67 expression led to significant inhibition of proliferation and tumor growth in vitro and in vivo (Kausch et al., 2003, Kausch et al., 2005). We recently found antisense oligoxydeonucleotides (ASOs) and peptide nucleic acids (PNAs) targeted against Ki-67 mRNA can inhibit the proliferation and induce apoptosis by blocking Ki-67 expression of human renal carcinoma 786-0 cells (Zheng et al., 2005).
RNA interference (RNAi) provides a new approach for suppression of gene expression. RNAi is a sequence-specific, post-transcriptional gene silencing mechanism induced in animals and plants by the introduction of double stranded RNA (dsRNA) homologous in sequence to the silenced gene (Bass, 2000, Cogoni and Macino, 2000, Hammond et al., 2001, Tuschl, 2001). dsRNA, a hybrid consisting of a sense and antisense strand of an endogenous mRNA, can initiate a cell response results in the sequence-specific degradation of homologous single-stranded RNA (Sharp, 2001). It is evident that two ways of inducing RNAi are available, first using long dsRNA and second using small interfering RNA (siRNA). In mammalian cells, long dsRNA (> 30 bp) led to activation of a global, sequence unspecific response resulting in blockage of initiation of protein synthesis and mRNA degradation. SiRNA, 21-ntRNA, with 2-nt 3′overhang can mediate strong and specific suppression of gene expression (Billy et al., 2001, Harborth et al., 2001, Yang et al., 2001). Many examples (Filleur et al., 2003, Martinez et al., 2002) demonstrate the usefulness of siRNA as an effective method for inhibiting the expression of oncogenes. RNAi with siRNA opens new avenues for cancer gene therapy.
In the present study, we evaluated the inhibitory effect of siRNA directed against Ki-67 on Ki-67 expression in human renal carcinoma cells (cell line 786-0). Incubation of human renal carcinoma 786-0 cells with appropriate siRNA resulted in Ki-67 mRNA and protein reduction and apoptotic cell death. SiRNA-mediated inhibition of Ki-67 expression led to significant inhibition of proliferation in vitro.
Section snippets
SiRNA preparation
The siRNAs duplexes were synthesized, purified, and annealed by Ambion Company (USA). Ki-67 siRNA targeted the region containing the 364∼382 bases of Ki-67 complementary DNA (cDNA). The sequence of Ki-67 siRNA: sense sequence 5-′GGAGGCAAUAUUACAUAAUtt-3′; antisense sequence 5-′AUUAUGUAAUAUUGCCUCCtt-3′. The selected sequence was submitted to BLAST searche to assure the only the selected gene was targeted. A scrambled siRNA was purchased from Ambition (silencer™ Control siRNA#3) and was used as
Effect of siRNA treatment on Ki-67 mRNA expression
Ki-67 mRNA expression was examined by the methods of RT-PCR and in situ hybridization. As shown in Fig. 1A, cells treated with 100 nM Ki-67 siRNA exhibited significantly decreased Ki-67 mRNA content compared with 786-0 cells treated with control siRNA in the RT-PCR assay (Fig. 1A,B). Cultures examined by in situ hybridization also demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells (Fig. 1C).
Effect of siRNA treatment on Ki-67 protein expression
The effect of Ki-67 siRNA on Ki-67 protein expression was evaluated by
Discussion
Renal cell carcinoma (RCC) is the most common malignant disease of the adult kidney. Approximately 30–40% of all patients afflicted with metastasis disease at the time of diagnosis have a poor prognosis (Sokoloff et al., 1996). The tumor is considered to be relatively resistant to radiotherapy and chemotherapy and immunotherapy (Mulders et al., 1997, Vogelzang et al., 1993). These clinical facts suggest new therapeutic regimens must be explored in the quest to develop an effective therapy for
Acknowledgements
We thank ZHENG GY for very useful comments and ZONG YY for technical assistance. This project is supported by grants from the National Natural Science Foundation of China (No.30370331) and Health Departmental Science Research Funds of Jiangsu Province, China (No.H200153).
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