沉默RET基因表达对甲状腺髓样癌细胞增殖的影响

Effect of silencing RET gene expression on the proliferation of medullary thyroid carcinoma cells

目的研究沉默RET基因表达对人甲状腺髓样癌（medullary thyroid carcinoma，MTC）细胞系TT细胞增殖的影响及其机制。方法将含RET的双链DNA克隆人pSilence^TM 3．1-H1 neo真核载体，构建pSilencer—ret重组质粒，以脂质体转染高表达RET的TT细胞。实时定量PCR和Western印迹法检测RET mRNA和蛋白的表达，以及细胞外信号调节激酶（ERK）的磷酸化水平，CCK-8法测定细胞增殖。结果质粒测序证实重组质粒构建成功，转染后能明显抑制TT细胞RET mRNA和蛋白的表达，抑制细胞的增殖（P〈0．05），ERK磷酸化水平降低62．4％。结论构建的pSilencer—ret重组质粒能有效地降低人MTC细胞RET的表达，抑制细胞增殖，与其降低ERK通路的信号转导有关，为MTC的基因治疗提供新策略。

Objective To investigate the effect of silencing RET gene expression on TT（ human medullary thyroid carcinoma cell lines）cell proliferation and its mechanism. Methods The double strands DNA containing RET gene was cloned into the pSileneerTM3. 1-HI neo eukaryotic expression vector, and then was transfected into TT cells expressing high level of RET by lipofactamine. The levels of RET mRNA and protein expression and extracellular signal regulated kinase （ERK）phosphorylation were detected by real-time PCR and Western blotting. TT cell proliferation was tested by cell counting kit-8. Results The recombinant plasmid was successfully constructed as shown by sequencing. After the recombinant plasmid was transfected into TT cells, ret mRNA and RET protein expressions were reduced, along with decreased TT cell proliferation and ERK phosphorylation （ P 〈 0.05 ）. Conclusions The pSilencer-ret plasmid decreases the level of RET expression and inhibits TT cell proliferation ,which is involved in ERK pathway. This may provide a new gene therapy approach for medullary thyroid carcinoma.