Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Key Laboratory of Glycoconjugate Research, Ministry of Public Health, Shanghai 200032, P.R. China.
Int J Mol Med. 2012 Dec;30(6):1321-6. doi: 10.3892/ijmm.2012.1140. Epub 2012 Sep 25.
In the present study, we constructed a lentivirus vector encoding the miR-29a precursor and established two stably infected cell lines, PLC-29a and 97L-29a. The overexpression of miR-29a was confirmed by TaqMan RT-PCR and significantly suppressed the growth of the hepatocellular carcinoma cell lines MHCC-97L and PLC. Dual-luciferase reporter assays indicated that the SPARC mRNA 3'UTR was directly targeted by miR-29a since the mutated 3'UTR was not affected. Silencing SPARC expression by RNAi knockdown resulted in a similar effect as miR-29a overexpression on hepatocellular carcinoma (HCC) cell growth regulation. Anti-miR-29a oligonucleotides (AMOs) upregulated the levels of SPARC in the HCC cells. The phosphorylation of AKT/mTOR downstream of SPARC was inhibited in miR-29a-overexpressing HCC cells. We further examined and compared the expression levels of miR-29a in HCC tissues and the corresponding nearby non-cancerous liver tissues of 110 patients with HCC by qRT-PCR, and significantly lower expression of miR-29a was observed in the tissues affected by HCC. Our findings demonstrate that the expression of miR-29a is important in the regulation of the SPARC-AKT pathway and HCC growth.
在本研究中，我们构建了一个编码 miR-29a 前体的慢病毒载体，并建立了两个稳定感染的细胞系，PLC-29a 和 97L-29a。通过 TaqMan RT-PCR 证实 miR-29a 的过表达显著抑制了肝癌细胞系 MHCC-97L 和 PLC 的生长。双荧光素酶报告基因实验表明，SPARC mRNA 3'UTR 是被 miR-29a 直接靶向的，因为突变的 3'UTR 不受影响。通过 RNAi 敲低沉默 SPARC 表达会对肝癌（HCC）细胞生长调节产生与 miR-29a 过表达相似的效果。抗 miR-29a 寡核苷酸（AMOs）上调 HCC 细胞中 SPARC 的水平。SPARC 下游的 AKT/mTOR 磷酸化在 miR-29a 过表达的 HCC 细胞中受到抑制。我们进一步通过 qRT-PCR 检测和比较了 110 例 HCC 患者的 HCC 组织和相应的邻近非癌性肝组织中 miR-29a 的表达水平，结果发现 miR-29a 在受 HCC 影响的组织中的表达明显降低。我们的研究结果表明，miR-29a 的表达在调节 SPARC-AKT 通路和 HCC 生长中很重要。
