miR-338-3p靶向调控RNF121促进人肺癌A549的增殖及侵袭能力

miR-338-3p Targets and Regulates RNF121 to Promote the Proliferation and Invasion of Human Lung Cancer A549 Cells

近期研究表明,miR-338-3p对肺癌的增殖和侵袭具有重要作用,但其通过靶向调控环指蛋白121(ring finger protein 121, RNF121)在肺癌增殖和侵袭中的研究尚不清楚。为探究其机制,体外培养正常肺细胞系MRC-5和非小细胞肺癌系A549,采用qRT-PCR和Western印迹检测发现,A549肺癌细胞较MRC-5细胞中miR-338-3p表达下降,而RNF121表达增加(P<0.05)。通过TargetScan(http://www. targetscan. org/mamm31/)靶基因预测软件预测与RNF121的3′UTR结合的miR-338-3p。双荧光素酶报告证实了两者互补结合。构建靶向干扰RNF121的shRNA载体并转染细胞。CCK8检测各组细胞增殖情况;Annexin V-FITC/PI双染检测细胞凋亡;Transwell实验和细胞集落形成检测各组细胞侵袭和克隆增殖能力;将各组细胞接种于裸鼠皮下,绘制肿瘤生长曲线并测定瘤体大小;之后获取肿瘤组织通过TUNEL染色检测组织中细胞凋亡情况。结果显示:与Blank组比较,miR-338-3p mimic和sh-RNF121组中细胞增殖、侵袭以及细胞克隆形成能力被抑制但凋亡显著增加。体内实验显示,上述组成瘤能力降低,肿瘤组织中凋亡增强(P<0.05)。而miR-338-3p抑制组中细胞增殖活力、侵袭以及细胞克隆形成能力增强,且凋亡被显著抑制,同时裸鼠成瘤能力提高,肿瘤组织中凋亡减弱(P<0.05)。NC组、miR-338-3p inhibitor+sh-RNF121组部分指标与Blank组比较未见显著性差异(P>0.05)。综上所述,miR-338-3p能够靶向抑制RNF121的表达进而抑制A549细胞的增殖和侵袭,抑制裸鼠皮下移植瘤的生长,RNF121有望成为非小细胞肺癌治疗的新靶点。

Recent studies have shown that miR-338-3 p plays an important role in the proliferation and invasion of lung cancer, but whether miR-338-3 p regulates lung cancer proliferation and invasion through targeting ring finger protein 121(RNF121) is still unclear. In order to explore its mechanism, the normal lung cell line MRC-5 and the non-small cell lung cancer line A549 were cultured in vitro. Using qRT-PCR and Western blotting detection, we found that the expression of miR-338-3 p in A549 lung cancer cells was lower than that in MRC-5 cells, while RNF121 expression increased(P<0.05). The miR-338-3 p bound to the 3′UTR of RNF121 was predicted by TargetScan(http://www. targetscan. org/mamm31/) target gene prediction software. The dual luciferase reporter confirmed the complementary combination. We constructed a shRNA vector targeting RNF121 and transfected cells, used CCK8 experiments to detect cell proliferation in each group, employed Annexin V-FITC/PI double staining to detect cell apoptosis, performed Transwell experiments and cell colony formation experiments to detect cell invasion and clonal proliferation ability of each group. Cells of each group were inoculated under the skin of nude mice and we drew the tumor growth curve and the size of the tumor were measured. Then the tumor tissue was obtained and TUNEL staining was used to detect apoptosis in the tissue. Compared with the blank group, the cell proliferation, invasion, and cell cloning ability in the miR-338-3 p mimic and sh-RNF121 groups were inhibited, but apoptosis was significantly induced. At the same time, in vivo experiments found that the ability to form tumors was reduced, and apoptosis in tumor tissues was increased(P<0.05). The miR-338-3 p group had a higher cell proliferation activity, invasion and colony-forming ability but lower tumorigenicity and apoptosis in tumor tissues(P<0.05). Compared with the blank group, no significant difference was found in the NC group and miR-338-3 p inhibitor+sh-RNF121 group(P>0.05). In summary, miR-338-3 p can target the expression of RNF121 to inhibit the proliferation and invasion of A549 cells and inhibit the growth of subcutaneous transplanted tumors in nude mice. RNF121 is expected to become a new target for the treatment of non-small cell lung cancer.