小鼠CYP2R1的异源表达及其对癌细胞增殖的差异性作用

Heterologous Expression of Mouse CYP2R1 and Its Differential Effect on Cancer Cells Proliferation

细胞色素P450(cytochrome P450,CYP450)是一类血红素氧化酶。细胞色素P450家族2亚家族R成员1(cytochrome P450 family 2 subfamily R member 1,CYP2R1)是一种主要在肝组织中表达的维生素D羟化酶。目前,对于小鼠CYP2R1蛋白质的结构、物化特性和病理机制的理了解仍十分有限。本研究结合基因克隆和生物信息学分析,获得小鼠CYP2R1基因CDS序列及其生物学特征。随后,利用pcDNA3.1-CYP2R1真核表达载体,通过细胞划痕、MTT分析、real-time qPCR方法检测异源表达CYP2R1对肺癌细胞A549、胃癌细胞7901、肝癌细胞HepG2以及正常细胞HEK293T细胞的迁移、增殖和细胞周期基因表达的影响,探明其对癌细胞增殖的作用。结果显示,由C57BL/6小鼠肝组织的CYP2R1基因,序列长度1506 bp,其中,CDS 468 bp。其编码的155个氨基酸,与其他11个物种间的同源性均较高,其三级结构与人CYP2R1略有不同。构建的pcDNA3.1-CYP2R1真核表达载体,可在体外培养细胞中提高CYP2R1基因mRNA表达105倍以上,蛋白质水平提高约30倍。值得注意的是,异源表达CYP2R1在癌细胞增殖中的作用具有差异性,其中,CYP2R1通过显著降低细胞周期蛋白基因CyclinD1(P<0.05)和Caspase3(P<0.01),而抑制7901细胞的增殖。该研究结果为进一步探索CYP2R1的生物学作用,特别是在分析其对癌细胞增殖方面提供了基础研究数据,并为进一步明确CYP2R1在癌症相关治疗中的重要意义奠定了理论基础。

Cytochrome P450(CYP450)is a kind of heme enzyme.Cytochrome P450 family 2 subfamily R member 1(CYP2R1)is an important vitamin D 25-hydroxylation enzyme and mainly expresses in the liver.However,the understanding of mouse CYP2R1 protein structure,physicochemical properties and pathology are unclear.The gene clone and bio-informatics analysis were performed to obtain the mouse CYP2R1 CDs sequence and its biological characteristics.Then,using the eukaryotic expression vector pcDNA3.1-CYP2R1,the effects of heterologous expression CYP2R1 on lung,gastric,liver cancer cell and normal cell proliferation were discovered by cell scratch assay,MTT and real-time qPCR in A549 lung cancer cells,7901 gastric cancer cell,HepG2 hepatocellular carcinoma cell and normal cell HEK293T,respectively.The results showed that the length of CYP2R1 gene from C57BL/6 mouse liver was 1506 bp,including CDs 468 bp.The sequence of CYP2R1 was responsible for coding 155 amino acids.The homology among mouse and other 11 different species was relatively high and the CYP2R1 tertiary structure was distinctive between mice and human.Comparing with the negative control,the pcDNA3.1-CYP2R1 vector increased expression levels of CYP2R1 mRNA above 105 times and its protein about 30 times in vitro.Noticeably,the effect of heterologous expression of CYP2R1 on cancer cell proliferation was differential.It suggested that CYP2R1 could inhibit the proliferation ability of 7901 cells through suppressing Cyclin D1(P<0.05)and Caspase 3(P<0.01)mRNA expression.The present study provides further understanding of the biological activity and function of mouse CYP2R1 in proliferation of different types of cancer cells.