人KIAA1173基因的克隆、转染及生物学特性的实验研究

目的 建立表达KIAA1173基因的鼻咽癌6-10B细胞模型,并观察转染细胞的生物学活性。方法 从新鲜肌肉组织中提取总RNA,采取逆转录PCR得到人KIAA1173基因cDNA。获得的人KIAA1173基因克隆到真核表达载体pcDNA3.1(+),进行酶切和序列鉴定。将重组质粒pcDNA3.1(+)-KIAA1173和空载体利用阳离子脂质体介导转入鼻咽癌6-10B细胞,经G418筛选阳性克隆。用RT-PCR、原位杂交及免疫细胞化学等方法检测KIAA1173基因在6-10B细胞中的稳定表达情况,并通过MTT法、肿瘤细胞侵袭实验及裸鼠体内成瘤性实验方法,检测KIAA1173基因对鼻咽癌细胞的增殖、侵袭及成瘤能力的影响。以空白质粒转染组作为对照。结果 在mRNA和蛋白水平证实,转染细胞内有KIAA1173基因的高表达,表明细胞可表达人KIAA1173基因。6-10B在转染了pcDNA3.1(+)-KIAA1173后,较转染空载体pcDNA3.1(+),肿瘤细胞的增殖能力、侵袭能力及裸鼠体内成瘤能力明显降低(P<0.05)。结论 结果提示KIAA1173基因可能是一鼻咽癌肿瘤抑制基因。获得生物学活性稳定的KIAA1173基因高表达的鼻咽癌细胞模型,为进一步研究奠定了基础。

Cloning of human KIAA1173 gene and biological characterization of transfected 6-10B cells

Objective To establish a 6-10B cell line with stable expression of KIAA1173 gene and study the biological behaviors of the cells. Methods The total RNA was extracted from normal skeletal muscular tissues for cloning of KIAA1173 gene by means of RT-PCR which was subsequently introduced into pcDNA3.1 ( ) vector. The recombinant eukaryotic expression vector pcDNA 3.1( )-KIAA1173 was constructed and identified by endonuclease digestion and sequencing before transfection into 6-10B cells via lipofectamine with the empty vector as the control. The positive cell clones were obtained by G418 selection. Stable expression of KIAA1173 gene in the transfected 6-10B cells was determined by RT-PCR, in situ hybridization and immunocytochemistry, and the biological behaviors of the transfected cells were observed by MTT assay, cell invasion assay and tumorigenesis assay in nude mice. Results High expression of KIAA1173 at both mRNA and protein levels was observed in the transfected 6-10B cells. The capability of proliferation, invasion and tumorgenicity of the KIAA1173- transfected cells in nude mice was lowered in comparison with those of the cells transfected with pcDNA3.1 ( ) vector (P<0.05). Conclusions KIAA1173 genes may function as a potential tumor suppressor of nasopharyngeal carcinoma both in vitro and in vivo. The 6-10B cell line expressing KIAA1173 has been obtained, which can be helpful for further study of KIAA1173 gene.