Pan Xiu-cheng, Chen Zhi, Ji Fang, Guo Zhong-sheng, Chen Min, Fu Juan-juan
Department of Infectious Diseases, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Zhonghua Gan Zang Bing Za Zhi. 2008 Sep;16(9):683-7.
To investigate the roles of Kruppel-like factor 6 (KLF6) and its splice variant KLF6V on suppressing growth and inducing differentiation of human hepatocellular carcinoma hepG2 cells.
KLF6V cDNA was amplificated by RT-PCR from human hepatocellular carcinoma (HCC) tissue and then sequenced. The recombinant vectors expressing KLF6 variant (KLF6V) were constructed using molecular clone technology based on established plasmid pcDNA3.1A(-)/wtKLF6. KLF6V or KLF6-transfected HepG2 cells were established after being screened with G418. Growth activity of HepG2/KLF6 or HepG2/KLF6V cells was detected by in vitro MTT assay. Expression of p21WAF1 or cyclin D1 protein was detected by Western blot, and expressions of AFP or ALB protein were measured by radioimmunoassay.
A novel alternatively spliced transcript of the human KLF6 gene was found and its sequencing revealed that the variant form of KLF6 lacked 126nt and its encoded protein products had a deletion of 42 aa near the COOH-terminal amino acid in comparison with full-length KLF6. Although KLF6 alternative splicing was present in both normal and cancerous tissues, expression of the KLF6 splice variants seemed to be up-regulated in HCCs tissues. The isoform of KLF6 proteins antagonized the ability of wild-type KLF6 to up-regulate p21 expression or down-regulate cyclin D1 expression and suppress HepG2 cell proliferation. KLF6 gene increased albumin production and decreased alpha fetoprotein production of the cells.
The isoform of KLF6 protein, present in HCC tissue, antagonizes the ability of wild-type KLF6 to suppress cell proliferation and induce cellular differentiation.
研究Kruppel样因子6（KLF6）及其剪接变体KLF6V在抑制人肝癌HepG2细胞生长及诱导其分化中的作用。
采用RT-PCR从人肝癌组织中扩增KLF6V cDNA并测序。基于已有的质粒pcDNA3.1A(-)/wtKLF6，利用分子克隆技术构建表达KLF6变体（KLF6V）的重组载体。经G418筛选后建立KLF6V或KLF6转染的HepG2细胞。采用体外MTT法检测HepG2/KLF6或HepG2/KLF6V细胞的生长活性。通过蛋白质免疫印迹法检测p21WAF1或细胞周期蛋白D1蛋白的表达，采用放射免疫分析法检测甲胎蛋白（AFP）或白蛋白（ALB）蛋白的表达。
发现了人KLF6基因一种新的可变剪接转录本，测序显示KLF6的变体形式缺失126个核苷酸，其编码的蛋白质产物与全长KLF6相比，在COOH末端氨基酸附近缺失42个氨基酸。虽然KLF6可变剪接在正常组织和癌组织中均存在，但KLF6剪接变体在肝癌组织中的表达似乎上调。KLF6蛋白的异构体拮抗野生型KLF6上调p21表达或下调细胞周期蛋白D1表达以及抑制HepG2细胞增殖的能力。KLF6基因增加细胞白蛋白的产生并减少甲胎蛋白的产生。
肝癌组织中存在的KLF6蛋白异构体拮抗野生型KLF6抑制细胞增殖和诱导细胞分化的能力。
