CAJ | 학술논문

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,Western blot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(0.92±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.
목적 구건만병독개도적구유특정이급결구적인microRNA-338-3p (miR-338-3p)억제제병관찰기대결직장암세포침습전이능력적영향.방법 유miRBase수거고사조획득miR-338-3p성숙서렬,설계목적기인편단병인공합성；장miR-338-3p억제제서렬화pLV-THM재체경쌍매절후련접,산생pLV-THM-miR-338-3p억제제만병독표체재체,쌍매절후측서감정,사선양성극륭；이pLV-THM-miR-338-3p억제제、psPAX2화pMD2.G질립공전염포장세포293T,포장산생만병독병측정적도.류식세포의사선건립은정과표체miR-338-3p억제제적SW-620아세포주,이용실시정량역전록취합매련반응(RT-qPCR)검측miR-338-3p표체,Western blot검측기파기인Smoothened (SMO)단백표체수평,Transwell소실천투시험검측종류세포전이능력.결과 경쌍매절감정화측서증실,성공구건료포함miR-338-3p억제제적만병독표체재체,도치형광현미경하관찰가견포장세포293T표체록색형광.은정과표체miR-338-3p억제제적SW-620아세포주중,miR-338-3p표체수평현저저우음성대조조(0.92±0.43비3.71 ±0.22,P＜0.01),SMO단백표체수평명현승고,차종류세포표현출침습전이능력적증강(세포수78.6±6.9비23.7±4.2,P＜0.01).결론 성공구건료miR-338-3p억제제적만병독표체재체화은정저표체miR-338-3p적SW-620아세포주,증실miR-338-3p가통과억제결직장암세포중SMO단백표체이억제종류세포전이.
Objective To construct a lentiviral expression vector of has-miR-338-3p-inhibitor and verify its influence on migration ability of colorectal 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 supernatant containing the lentivirus particles was harvested to determine the virus titer and used to infect SW-620 cells.Flow cytometry 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 cells.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 upexpression 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 seconda,structure provides the basis for the further study on molecular function of miR-338-3p in colorectal carcinoma.MiR-338-3p can inhibit colorectal carcinoma migration probably by suppressing SMO gene expression.