L1(ORF1)基因通过编码和非编码RNA表达对细胞增殖的影响

Contributions of L1(ORF1) gene to cell proliferation via coding and non-coding RNA with full-length sequence in HEK293 cells

目的探索L1(ORF1)基因过量表达对细胞增殖的影响。方法 PCR方法制备L1(ORF1)全长以及5'端不同截短的天然序列以及各种突变体,构建真核重组表达载体,利用脂质体方法瞬时转染HEK293细胞,Western印迹方法验证蛋白表达,利用活细胞计数试剂盒(CCK-8)进行细胞增殖能力分析。结果过表达天然全长蛋白[L1(ORF1)]的重组体能明显促进细胞增殖,终止密码位于第109个氨基酸位置的全长序列重组体[L1(ORF1)109]对细胞增殖同样具有非常明显的促进作用,但仅含有氨基端109个氨基酸的基因片段的重组体对细胞增殖没有促进作用。另外,终止密码位于其他位置的全长序列重组体[L1(ORF1)48,终止突变在第48个氨基酸位置]以及含有多处突变的全长序列重组体[L1(ORF1)m]对细胞的增殖也未见有显著影响。结论 L1(ORF1)全长序列编码蛋白具有显著地促进细胞增殖作用,同时在特殊位点含有终止突变的全长序列转录产物对细胞的增殖也具有显著促进作用,L1(ORF1)全长序列可能通过其编码的蛋白以及非编码RNA两种形式对细胞增殖进行调控,位于序列中的关键位点在此过程中起着重要的作用,为探索L1(ORF1)序列在细胞增殖中的调控机制奠定了基础。

ObjectiveTo explore the mechanism of L1（ORF1） gene for cell proliferation.MethodsL1（ORF1） gene in the full-length form and with different mutants as well as 5＇truncated sequence was prepared by PCR amplification and constructed into eukaryotic expression vectors.The different recombinants were transfected into HEK293 cells by cationic liposome.The expression of L1（ORF1） protein was detected by Western blotting and cell proliferation was detected by CCK-8 approach.ResultsThe growth capacity of transfected HEK293 cells with overexpression of full-length protein [L1（ORF1）] and L1（ORF1）109,which encompassed a stop code in 109th amino acid of full-length sequence,was significantly enhanced compared with the control.However,the overexpression of the recombinant construct encompassed the gene fragment of N-terminal 109 amino acids didn＇t exhibit any contribution to cell proliferation,neither did the other recombinants,L1（ORF1）48 which encompassed a stop code in 48th amino acid of full-length sequence and L1（ORF1） which encompassed many mutant codes in full-length sequence.ConclusionThe full-length L1（ORF1）can regulate cell proliferation through either its encoding RNA or non-coding RNA.The 5＇truncated sequence and full-length sequence that encompass the inappropriate mutations fail to promote cell proliferation.