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目的研究胃癌相关miR-148a与胃泌素受体CCKBR的调控关系,并分析其调控结合位点。方法生物信息学预测人CCKBR 3’UTR上miR-148a的结合位点;利用PCR扩增miR-148a前体构建真核表达载体;Northern Blot检测miR-148a真核表达载体的表达;构建CCKBR 3’UTR野生型和突变型荧光素酶报告载体,并利用双荧光素酶活性分析检测分析miR-148a对CCKBR基因表达的调控和结合位点;Western Blot检测miR-148a过表达对CCKBR蛋白表达的作用。结果在人CCKBR 3’UTR上找到3个miR-148a的潜在结合位点;miR-148a真核表达载体构建成功,转染胃癌细胞后可显著过表达;miR-148a通过人CCKBR 3’UTR上423bp处的结合位点抑制CCKBR的基因表达;miR-148a过表达显著抑制胃癌细胞中CCKBR的蛋白表达。结论 CCKBR是胃癌相关miR-148a的靶基因,miR-148a通过其3’UTR上的结合位点抑制CCKBR的基因表达和蛋白合成,提示miR-148a可能通过调控CCKBR参与胃癌的发生发展。
Objective To study the regulation of gastric cancer related miR-148a and gastrin receptor CCKBR, and to analyze its regulatory binding sites. Methods The bioinformatics method was used to predict the binding site of miR-148a on human 3’UTR of human CCKBR. The miR-148a precursor was amplified by PCR to construct the eukaryotic expression vector. Northern Blot was used to detect the expression of miR-148a. CCKBR 3 ’UTR wild-type and mutant luciferase reporter vector, and using dual luciferase activity assay analysis of miR-148a CCKBR gene expression regulation and binding sites; Western Blot detection miR-148a over-expression of CCKBR protein expression effect. Results Three potential miR-148a binding sites were found on the human CCKBR 3’UTR. The miR-148a eukaryotic expression vector was successfully constructed and transfected into gastric cancer cells and was overexpressed. MiR-148a was transfected into human CCKBR 3’UTR The binding site at 423bp inhibited the gene expression of CCKBR. Overexpression of miR-148a significantly inhibited the expression of CCKBR in gastric cancer cells. Conclusion CCKBR is the target gene of gastric cancer related miR-148a. MiR-148a inhibits the gene expression and protein synthesis of CCKBR through its 3’UTR binding site, suggesting that miR-148a may be involved in the development of gastric cancer by regulating CCKBR.