中文摘要：

目的构建人microRNA-338-3p（has-miR-338-3p）慢病毒表达载体并初步筛选鉴定miR-338-3p的靶基因。方法由miRBase数据库查找获得miR-338-3p前体序列，设计目的基因片段并人工合成；将miR．338．3p前体序列和pLV-THM载体经双酶切后连接，产生pLV-THM．miR-338-3p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、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的SW-620亚细胞系，利用Real-time RT-PCR检测miR-338-3p表达，Western-blot检测SMO蛋白表达水平，Transwell小室穿透实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miR-338-3p的慢病毒表达载体pLV-THM—miR-338-3p，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的SW-620亚细胞系中，miR-338-3p表达水平显著高于阴性对照组和未处理组，SMO蛋白表达水平明显下降，且肿瘤细胞表现出侵袭转移能力的减弱。结论成功构建了has-miR-338-3p慢病毒表达载体和稳定过表达miR-338-3p的SW-620亚细胞系，初步证实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，此为进一步深入研究miR-338-3p在结直肠癌中生物学功能奠定了基础。

英文摘要：

Objective To construct a lentiviral expression vector of has-miR-338-3p and verify its target gene. Methods The pre-miR-338-3p was synthesized and inserted into pLV-THM, and the recombinant plasmid pLV-THM-miR-338-3p was confirmed by restriction endonuclease analysis and DNA sequencing. 293T cells were co-transfected with the lentiviral vector pLV-THM-miR-338-3p, psPAX2 and pMD2.G, and 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-positive cells. The expression of miR-338-3p was determined using real-time RT-PCR and the expression of SMO protein was detected with Western blotting in the infected SW-620 cells. The invasiveness of the infected SW-620 cells was assessed using Transwell assay. Results Restriction enzyme digestion and DNA sequencing demonstrated successful construction of the lentiviral vector pLV-THM-miR-338-3p. SW-620 cells infected with pLV-THM-miR-338-3p showed a significantly increased expression of miR-338-3p, and the overexpression of miR-338-3p suppressed the expression of SMO protein and the invasiveness of the cells. Conclusion The successful construction of the lentiviral vector pLV-THM-miR-338-3p and the establishment of a SW-620 cell line with miR-338-3p overexpression provide the basis for further study of the molecular function of miR-338-3p in colorectal carcinoma. MiR-338-3p can suppress SMO gene expression to inhibit the invasiveness of colorectal carcinoma cells.