慢病毒介导的具有特定二级结构的microRNA-338-3p抑制剂的构建

Establishment of lentivirus-mediated microRNA-338-3p inhibitor with specific secondary structure

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.