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目的构建过表达miR-335的稳定细胞株SW620并筛选和初步鉴定miR-335的靶基因。方法扩增包含hsa-miR-335前体序列在内的基因片段,并将其亚克隆至PLVTHM慢病毒载体;扩增包含RASA1 3’UTR种子区域的基因片段并将其亚克隆至psiCHECK-2载体,并对此重组载体进行定点突变构建psiCHECK-2-RASA1-Mut;检测萤火虫荧光素值(F)和海肾荧光素值(R),以R/F表示相对荧光素酶活性。流式细胞仪筛选建立稳定表达miR-335的细胞株,利用荧光定量PCR检测miR-335及其靶基因RASA1的表达,Western Blot检测RASA1蛋白水平的表达。结果质粒双酶切以及测序鉴定PLVTHM-miR335、psiCHECK-2-RASA1、psiCHECK-2-RASA1-Mut重组质粒构建成功。荧光定量PCR显示结直肠癌细胞中miR-335与RASA1表达趋势成负相关。双荧光素酶报告基因分析证实hsa-miR-335能够作用于RASA1的3’UTR。稳定过表达miR-335的细胞中,miR-335的表达明显高于对照组和未处理组,蛋白质水平有所下降。结论成功构建了PLVTHM-miR-335慢病毒重组质粒,构建过表达miR-335的细胞株SW620,初步证实RASA1是miR-335的靶基因。
Objective To construct a stable cell line SW620 overexpressing miR-335 and screen and identify the target gene of miR-335. Methods The gene fragment containing hsa-miR-335 precursor sequence was amplified and subcloned into PLVTHM lentiviral vector. The gene fragment containing the RASA1 3’UTR seed region was amplified and subcloned into psiCHECK-2 PsiCHECK-2-RASA1-Mut was constructed by site-directed mutagenesis of the recombinant vector. Firefly luciferin (F) and Renilla fluorescein (R) were detected and the relative luciferase activity was expressed as R / F. Flow cytometry was used to establish a cell line stably expressing miR-335. The expression of miR-335 and its target gene RASA1 was detected by real-time PCR. The protein expression of RASA1 was detected by Western Blot. Results Plasmid digestion and DNA sequencing confirmed that the constructed recombinant plasmid pVTHM-miR335, psiCHECK-2-RASA1 and psiCHECK-2-RASA1-Mut were successfully constructed. Fluorescent quantitative PCR showed that there was a negative correlation between miR-335 and RASA1 expression in colorectal cancer cells. Dual luciferase reporter gene analysis confirmed that hsa-miR-335 can act on the 3’UTR of RASA1. In miR-335-overexpressing cells, the expression of miR-335 was significantly higher than that of the control and untreated groups, and the protein level decreased. Conclusion The PLVTHM-miR-335 lentiviral vector was successfully constructed and the cell line SW620 overexpressing miR-335 was constructed. RASA1 is the target gene of miR-335.