人CyclinD3基因启动子区的分子克隆及在A2780细胞中的功能分析

Molecular Cloning and Function Analysis of Human Cyclin D3 Gene Promoter Region in A2780 Cells

目的对Cyclin D3启动子的结构和功能进行分析。方法用PCR的方法扩增不同长度的Cyclin D3启动子片段。将PCR产物克隆入荧光素酶报道载体pGL3-Basic Vector,构建含有Cyclin D3启动子片段的重组子pD3-。双荧光素酶报道系统检测重组子pD3-在A2780细胞中的启动子活性,筛选关键转录调控区域。TFSEARCH 1.3 v分析该区域,结合文献复习筛选可能的转录因子。结果用PCR方法扩增出了不同长度的Cyclin D3启动子片段。上述片段克隆到pGL3-Basic中,构建了重组子pD3-。双荧光素酶报道系统发现在Cyclin D3启动子上游-1044 bp至-766 bp之间、-362 bp至-197 bp之间,分别存在一个正性调控区域。-766 bp至-551 bp之间存在一负性调控区域。TFSEARCH发现在上述调控区中存在C/EBP、SP1、AP1、HSF和MZF1等转录因子的结合基序。结论在Cyclin D3启动子片段上游-1044 bp至-766 bp之间、-362 bp至-197 bp之间,分别存在一个正性调控区域;-766 bp和-551 bp之间存在一负性调控区域。转录因子C/EBP、SP1、AP1、HSF和MZF1等可能在Cyclin D3的转录调控中发挥重要作用。

Objective To analyze the structure and function of human Cyclin D3 promoter. Methods Cyclin D3 promoter sequences were amplified by PCR method and the PCR products were inserted into pGL3- Basic vectors to construct the Cyclin D3 promoter-pGL3-Basic vectors(pD3-). The pD3- plasmids were transiently transfected into A2780 cells with the liposome. The promoter activities of pD3- were detected by dual luciferase reporter assay to screen the key area of transcription and regulation. The regulation regions were assayed by TFSEARCH 1.3v to presume the key transcription factors. Results Cyclin D3 promoter sequences with different length were amplified successfully by PCR and the cyclin D3 promoter-pGL3-Basic vectors(pD3-)were well constructed. The dual luciferase reporter assay results implicated two positive(-1044bp to-766bp,-362bp to-197bp) and one negative(-766bp to-551bp) regulatory sequences located in Cyclin D3 promoter. TFSEARCH displayed that binding motifs of some transcription factors, such as C/EBP, SP1, AP1, HSF and MZF1, were contained in the regions. Conclusion Cyclin D3 promoter sequences have two positive(-1044bp to-766bp,-362bp to-197bp) and one negative(-766bp to-551bp) regulatory sequences which contain binding motifs of some transcription factors, such as C/EBP, SP1, AP1, HSF and MZF1, etc., and these transcription factors could regulate the promoter activity through binding motifs in A2780 cells.