摘要
目的构建慢病毒介导的具有特定二级结构的人microRNA-338-3p(miR.338.3p)抑制剂并观察其对结直肠癌细胞侵袭转移能力的影响。方法由miRBase数据库查找获得miR-338-3p成熟序列,设计目的基因片段并人工合成;将miR-338-3p抑制剂序列和pLV-THM载体经双酶切后连接,产生pLV-THM-miR-338-3p抑制剂慢病毒表达载体,双酶切后测序鉴定,筛选阳性克隆;以pLV-THM-miR-338-3p抑制剂、psPAX2和pMD2.G质粒共转染包装细胞293T,包装产生慢病毒并测定滴度。流式细胞仪筛选建立稳定过表达miR-338-3p抑制剂的SW-620亚细胞株,利用实时定量逆转录聚合酶链反应(RT-qPCR)检测miR-338-3p表达,Westernblot检测其靶基因Smoothened(SMO)蛋白表达水平,Transwell小室穿透试验检测肿瘤细胞转移能力。结果经双酶切鉴定和测序证实,成功构建了包含miR-338-3p抑制剂的慢病毒表达载体,倒置荧光显微镜下观察可见包装细胞293T表达绿色荧光。稳定过表达miR-338-3p抑制剂的SW-620亚细胞株中,miR-338.3p表达水平显著低于阴性对照组(O.92±0.43比3.71±0.22,P〈0.01),SMO蛋白表达水平明显升高,且肿瘤细胞表现出侵袭转移能力的增强(细胞数78.6±6.9比23.7±4.2,P〈0.01)。结论成功构建了miR-338-3p抑制剂的慢病毒表达载体和稳定低表达miR-338-3p的SW-620亚细胞株,证实miR-338-3p可通过抑制结直肠癌细胞中SMO蛋白表达而抑制肿瘤细胞转移。
Objective To construct a lentiviral expression vector of has-miR-338-3p-inhibitor and verify its influence on migration ability of eoloreetal carcinoma cells. Methods The miR-338-3p inhibitor sequence was synthesized artificially and inserted into pLV-THM plasmid consequently. The recombinant plasmid pLV-THM-miR-338-3p-inhibitor was confirmed by restriction endonuclease analysis and DNA sequencing. HEK-293T cells were co-transfected with lentivirus vector pLV-THM-miR-338-3p-inhibitor, psPAX2 and pMD2. G. The superuatant containing the lentivirus particles was harvested to determine the virus titer and used to infect SW-620 cells. Flow eytometry was employed for sorting the GFP( + ) cells. The expression of miR-338-3p was detected by using real-time quantitative reverse transcription-polymerase chain reaction ( RT-qPCR), and Western blotting was used to detect the expression of Smoothened (SMO) protein in SW-620 ceils. Moreover, the migration ability of the transfected SW-620 cells was assessed by Transwell assay. Results Restriction enzyme digestion and DNA sequencing demonstrated that the lentivirus vector pLV-THM-miR-338-3p-inhibitor was constructed successfully. The expression of miR-338-3p in SW-620 cells infected with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased (0. 92 ± 0. 43 vs 3.71 ± 0. 22,P 〈 0.01 ). Moreover, the down-expression of miR-338-3p caused the up- expression of SMO protein in SW-620 cells, which showed obviously enhanced migration ability in Transwell assay (78.6 ±6. 9 vs 23.7± 4. 2, P 〈 0.01 ). Conclusion The successful construction of lentivirus vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides the basis for the further study on molecular function of miR-338-3p in eolorectal carcinoma. MiR-338-3p can inhibit colorectal carcinoma migration probably by suppressing SMO gene expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第1期80-83,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金青年基金资助项目(81101896)