CAJ | 학술논문

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。
목적：연구위암상관miR-148a여위비소수체CCKBR적조공관계，병분석기조공결합위점。방법생물신식학예측인CCKBR 3’ UTR상miR-148a 적결합위점；이용 PCR확증 miR-148a 전체구건진핵표체재체；Northern Blot검측miR-148a진핵표체재체적표체；구건CCKBR 3’UTR야생형화돌변형형광소매보고재체，병이용쌍형광소매활성분석검측분석miR-148a대CCKBR기인표체적조공화결합위점；Western Blot검측miR-148a과표체대CCKBR단백표체적작용。결과재인CCKBR 3’UTR상조도3개miR-148a적잠재결합위점；miR-148a진핵표체재체구건성공，전염위암세포후가현저과표체；miR-148a통과인CCKBR 3’UTR상423bp처적결합위점억제CCKBR적기인표체；miR-148a과표체현저억제위암세포중CCKBR적단백표체。결론 CCKBR시위암상관miR-148a적파기인，miR-148a통과기3’UTR상적결합위점억제CCKBR적기인표체화단백합성，제시miR-148a가능통과조공CCKBR삼여위암적발생발전。
Objective To investigate the regulation role of gastric cancer related miR-148a on gastrin receptor CCKBR expression, and find the correct binding sites of miR-148a in CCKBR 3’UTR.Methods The potential binding sites of miR-148a in the CCKBR 3’UTR were predicted with the bioinformatic tools;The miR-148a expressing plasmid was constructed by PCR, and miR-148a expression was verified by Northern Blot;The luciferase report plasmids containing the wild type and mutated binding sites of CCKBR 3’ UTR were constructed, and were used to study the regulation mechanism and identify the binding sites of miR-148a by luciferase activity analysis; The regulation effect of miR-148a on CCKBR protein expression was checked by Western Blot.Results Three potential binding sites of miR-148a in the CCKBR 3’ UTR were found; The miR-148a expressing plasmid was constructed successfully, and highly expressed miR-148a after transfected to gastric cancer cells;The inhibitory effect of miR-148a on CCKBR protein expression was checked by Western Blot.Over-expression of miR-148a inhibited CCKBR expression by directly binding to the binding site in CCKBR 3’UTR 423bp.Conclusion CCKBR is a target of miR-148a, and its expression is inhibited by the binding of miR-148a on its 3’ UTR, indicating that miR-148a may participates in the progression of gastric cancer by regulating CCKBR expression.