微小RNA-6791-5p靶向磷酸呋喃酸性簇分选蛋白2抑制结直肠癌增殖、迁移和侵袭

MicroRNA-6791-5p targeting phosphofurin acidic cluster sorting protein 2 inhibits the proliferation,migration and invasion of colorectal cancer

目的探讨微小RNA(miR)-6791-5p对人结直肠癌细胞增殖、迁移和侵袭功能的影响及其分子机制。方法采集培养人结直肠癌细胞系RKO、SW480、DLD1、HCT116以及人正常结肠上皮细胞NCM460。通过反转录实时荧光定量聚合酶链反应(RT-qPCR)检测miR-6791-5p在各细胞系中的表达水平(表达倍数为0.52±0.04、0.83±0.06、0.60±0.12、0.68±0.07),并构建miR-6791-5p模拟物(miR-6791-5p mimic)及其阴性对照Negative control,并将其转染至RKO细胞。使用RT-qPCR检测miR-6791-5p和PACS2 mRNA的表达水平(表达倍数为0.468±0.032)。利用蛋白质印迹法(Western blot)检测PACS2蛋白的表达水平[RKO:(51.590±6.018)%]。使用两样本t检验进行统计分析,结果以均数±标准差(±s)表示,以P<0.05为差异有统计学意义。结果miR-6791-5p在人结直肠癌细胞系表达水平显著低于人结肠上皮细胞,其中RKO和DLD1下调最明显(RKO组低于460组:0.52±0.04比1.00、DLD1组低于460组0.60±0.12比1.00),差异有统计学意义(RKO:t=17.811,P<0.0001;DLD1:t=5.372,P<0.01)。RKO转染后,mimic组miR-6791-5p表达水平高于mimic NC组(3.28±0.44比1.00),差异有统计学意义(RKO:t=8.764,P<0.01)。细胞计数试剂(CCK-8)检测表明mimic组24、48、72、96 h细胞吸光度值显著低于mimic NC组(24 h吸光度值为0.185±0.004比0.250±0.030,48 h吸光度值为0.275±0.040比0.376±0.020,72 h吸光度值为0.497±0.120比0.657±0.010,96 h吸光度值为0.691±0.004比0.905±0.019),差异有统计学意义(RKO:t=10.821,P<0.01)。平板克隆实验表明mimic组集落形成数低于mimic NC组(115.70±12.51比176.00±19.18),差异有统计学意义(RKO:t=4.581,P<0.05)。划痕愈合实验显示miR-6791-5p mimic组划痕愈合率低于NC组[(14.960±0.848)%比(36.480±1.340)%],差异有统计学意义(t=23.491,P<0.0001)。Transwell迁徙和侵袭实验显示穿透微孔膜的细胞数mimic组低于mimic NC组(迁移数目miR-6791-5p mimic组比NC组42.330±3.512比125.700±7.760,侵袭数目分别为54.33±4.16比159.30±7.76),差异有统计学意义(Transwell迁移t=16.931,P<0.01)、(Transwell侵袭t=20.641,P<0.01)。在线靶预测软件(miRWalk、mirtarbase、targetscan)预测miR-6791-5p可能的靶基因,并通过qRT-PCR及Western blot验证,mimic组PACS2的mRNA及蛋白表达水平低于mimic NC组[比例为(51.590±6.018)%比1.00],差异有统计学意义[mRNA(RKO:t=27.951,P<0.0001);Western blot(RKO:t=13.931,P<0.001)]。结论miR-6791-5p在结直肠癌细胞中低表达,过表达miR-6791-5p能抑制结直肠癌细胞增殖、迁移及侵袭能力,并且明显下调PACS2的mRNA和蛋白表达水平,说明miR-6791-5p可能通过PACS2调控结直肠癌增殖、迁移及侵袭能力。

Objective To investigate the impact of microRNA(miR)-6791-5p on the proliferation,migration and invasion of human colorectal cancer cells and its underlying molecular mechanism.Methods Human colorectal cancer cell lines RKO,SW480,DLD1,HCT116,and human normal colon epithelial cells NCM460 were cultured and collected.The expression levels of miR-6791-5p in different cell lines were detected using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)(relative expression 0.52±0.04,0.83±0.06,0.60±0.12,0.68±0.07).MiR-6791-5p mimic and its negative control(negative control)were constructed and transfected into RKO cells.The expression levels of miR-6791-5p and PACS2 mRNA were measured using RT-qPCR(relative expression 0.468±0.032).The expression level of PACS2 protein was detected using Western blotting[RKO:(51.590±6.018)%].Statistical analysis was performed using two-sample t-test,and the results were presented as mean±standard deviation(±s).A P-value less than 0.05 indicated statistically significant differences.Results MiR-6791-5p expression was significantly lower in human colorectal cancer cell lines compared to human normal colon epithelial cells,with the most prominent downregulation observed in RKO and DLD1 cells(RKO group lower than NCM460 group 0.52±0.04 vs.1.00,DLD1group lower than NCM460 group 0.60±0.12 vs.1.00),showing statistically significant differences(RKO:t=17.811,P<0.0001;DLD1:t=5.372,P<0.01).After transfection in RKO cells,the expression level of miR-6791-5p was higher in the mimic group compared to the mimic NC group(mimic group was higher than the NC group 3.28±0.44 vs.1.00),exhibiting statistical significance(RKO:t=8.764,P<0.01).cell counting kit-8(CCK-8)assay indicated that the A values of the mimic group were significantly lower than those of the mimic NC group at 24,48,72,and 96 h(24 h A value was 0.185±0.004 vs.0.25±0.03,48 h A value was 0.275±0.04 vs.0.376±0.02,72 h A value was 0.497±0.12 vs.0.657±0.01,96h A value was 0.691±0.004 vs.0.905±0.019),showing statistical significance(RKO:t=10.821,P<0.01).The plate clone formation experiment showed a significant reduction in colony formation in the mimic group compared to the mimic NC group(mimic group lower than the NC group 115.7±12.51 vs.176.0±19.18,RKO:t=4.581,P<0.05).Scratch healing assay demonstrated a significantly decreased healing rate in the mimic group[miR-6791-5p mimic to NC(14.96±0.848)%vs.(36.48±1.34)%],with statistical significance(t=23.491,P<0.0001).Transwell migration and invasion assays revealed a lower number of cells passing through the microporous membrane in the mimic group compared to the mimic NC group(The number of migration miR-6791-5p mimic group was 42.33±3.512 vs.125.7±7.76 in NC group and the number of invasion was 54.33±4.16 vs.159.3±7.76),showing statistical significance(Transwell migration t=16.931,P<0.01;Transwell invasion t=20.641,P<0.01).Prediction using online target prediction software(miRWalk,mirtarbase,targetscan)suggested potential target genes of miR-6791-5p.qRT-PCR and Western blot verification indicated that the mRNA and protein expression levels of PACS2 were significantly downregulated in the mimic group compared to the mimic NC group[mimic group lower than the NC group expression level(51.590±6.018)%vs.1.000 mRNA:RKO:t=27.951,P<0.0001;Western blotting:RKO:t=13.931,P<0.001].Conclusion MiR-6791-5p is downregulated in colorectal cancer cells.Overexpression of miR-6791-5p suppresses the proliferation,migration,and invasion abilities of colorectal cancer cells and significantly downregulates the mRNA and protein expression levels of PACS2,suggesting that miR-6791-5p may regulate colorectal cancer proliferation,migration,and invasion abilities through PACS2.