MALAT-1基因种属同源序列重组质粒的构建及其与人鼻咽癌细胞株C666-1侵袭转移的关系

Recombination of Species homologous fragments of MALAT-1 gene eukaryotic expression vector and its relationship with invasive and metastatic ability of C666-1 nasopharyngeal carcinoma cell line

目的：运用基因重组技术构建人MAL-AT-1基因种属同源序列（Species homologous frag-ments/shf）重组真核荧光表达载体,并探讨其在MALAT-1高表达的人鼻咽癌细胞株C666-1中的表达和对其侵袭转移的影响。方法：应用RT-touchdown PCR的方法,从鼻咽癌患者正常切缘组织中扩增出MALAT-1基因的种属同源序列,将所得的cDNA序列定向克隆至真核表达载体pEGFP-C1质粒中,构建出重组真核荧光表达载体;采用脂质体Lipofectamine 2000介导重组质粒pEGFP-C1-MALAT-1/shf瞬时转染MALAT-1高表达的人鼻咽癌细胞株C666-1;通过实时荧光定量PCR方法检测MALAT-1基因种属同源序列转染组和空载体转染组细胞中MALAT-1/shf的表达水平;细胞基质胶侵袭实验研究转染组细胞侵袭能力的变化。结果：经酶切鉴定及测序分析,证实成功构建人MALAT-1/shf重组真核荧光表达载体。pEGFP-C1-MALAT-1/shf（6 918~8 441 bp）转染组平均侵袭细胞数（51.20±5.933）显著高于MALAT-1/mock组（13.40±3.847）,差异具有统计学意义（P=0.000）。这说明转染pEGFP-C1-MALAT-1/shf（6 918~8 441 bp）重组质粒后,细胞的侵袭能力明显增强。结论：成功构建了人MALAT-1基因种属同源序列真核荧光表达载体pEGFP-C1-MALAT-1/shf,并成功在MALAT-1高表达的人鼻咽癌细胞株C666-1中表达,MALAT-1/shf（6 918~8 441 bp）与人鼻咽癌C666-1细胞株的侵袭转移密切相关。

OBJECTIVE： To construct the recombinant plasmid pEGFP-C1-MALAT-1/shf with gene recombinant technique,and explore its expression and invasive ability in nasopharyngeal carcinoma C666-1 cell line of high expression MALAT-1 gene.METHODS： The species homologous fragments of MALAT-1 were amplified from human normal nasopharyngeal tissue of nasopharyngeal carcinoma patients by RT-touchdown PCR.The obtained cDNA was cloned into eukaryotic expression vector pEGFP-C1 to generate recombinant pEGFP-C1-MALAT-1/shf.pEGFP-C1-MALAT-1/shf expression plasmids were transfected into nasopharyngeal carcinoma C666-1 cell of high expression MALAT-1 gene by lipofectamine 2000.Real-time PCR analysis was performed to evaluate the expression of MALAT-1/shf at mRNA level,respectively.In vitro cell matrigel invasion assays were used to study the ability of MALAT-1/shf cell invasion.RESULTS： Recombinant pEGFP-C1-MALAT-1/shf was confirmed by restriction enzyme digestion and sequence analysis.The average number of invasive cells（51.20±5.933） of pEGFP-C1-MALAT-1/shf（6 918-8 441 bp） was significantly higher than MALAT-1/mock（13.40±3.847）,and the difference had Statistically significant（P=0.000）.This showed that MALAT-1/shf（6 918-8 441 bp） over-expression significantly increased the abilities of invasion of C666-1.CONCLUSION： Recombinant pEGFP-C1-MALAT-1/shf can be successfully constructed and expressed in C666-1 cell line of high expression MALAT-1 gene,and MALAT-1/shf（6 918-8 441 bp） expression correlates with the invasion and metastasis of nasopharyngeal carcinoma cell C666-1.