摘要
目的探讨肿瘤来源的外泌体(TDEs)微小RNA-4644(miR-4644)驱动胰腺癌(PC)增殖的机制。方法2022年9月至2024年12月利用超速离心法富集AsPC-1和BxPC-3细胞来源的外泌体;基因表达综合数据库(GEO)和实时定量聚合酶链式反应(RT-qPCR)实验验证miR-4644在PC来源外泌体(PC TDEs)中高表达;双荧光素酶报告基因实验验证miR-4644与软脂酰化磷蛋白3(SPRY3)的3’-非翻译区(3’UTR)的结合;利用共培养和转染miR-4644模拟物[分组:阴性对照(NC)组,SPRY3过表达(OE)组,OE+miR-4644组],以及细胞计数试剂盒-8(CCK8),集落形成和原位移植瘤试验(采集时间:21 d;分组:随机对照;方法:双因素方差分析)验证TDEs miR-4644靶向SPRY3对PC细胞的增殖的影响。两组间采用非参数检验或独立样本t检验。多重比较采用Bonferroni或Dunnettt检验。结果MiR-4644是PC的促癌分子。AsPC-1细胞和BxPC-3细胞的miR-4644组与NC组的吸光度组间比较,差异有统计学意义(1.86±0.11比1.47±0.08,F=10.21,P<0.0001;0.96±0.08比0.86±0.02,F=3.90,P<0.05)。集落形成试验结果证明AsPC-1细胞和BxPC-3细胞中miR-4644组的集落形成数量高于NC组[(155.70±15.50)个比(117.70±6.66)个,t=3.90,P<0.05;(41.67±4.51)个比(17.00±3.00)个,t=3.46,P<0.05]。双荧光素酶报告基因实验证实WT中miR-4644组SPRY3的表达量低于NC组(1.00±0.14比0.50±0.12,t=4.61,P<0.01),MT中miR-4644组SPRY3的表达量与NC组差异无统计学意义(1.00±0.18比0.87±0.05,t=1.23,P>0.05),证实SPRY3是miR-4644的直接下游靶基因;CCK-8试验结果提示AsPC-1细胞和BxPC-3细胞中NC组与OE组、OE+miR-4644组的吸光度组间比较,差异有统计学意义(1.87±0.08比1.48±0.07比1.76±0.08,F=5.88,P<0.01;1.23±0.03比1.00±0.03比1.30±0.07,F=19.42,P<0.05)。集落形成实验结果显示,AsPC-1细胞和BxPC-3细胞中OE组与NC组、OE+miR-4644组的集落形成数量组间比较,差异有统计学意义[(51.67±8.02)个比(161.00±8.54)个比(103.00±1.73)个,F=191.90,P<0.001;(45.67±4.04)个比(74.33±10.79)个比(65.00±4.58)个,F=12.52,P<0.001]。PC原位移植瘤结果显示AsPC-1细胞和BxPC-3细胞中OE组与NC组、OE+miR-4644组的原位移植瘤重量组间比较,差异有统计学意义[(0.16±0.06)g比(0.50±0.12)g比(0.28±0.06)g,F=20.150,P<0.001;(0.15±0.01)g比(0.30±0.02)g比(0.24±0.03)g,F=78.950,P<0.001]。结论MiR-4644显著富集在TDEs中,通过靶向SPRY3促进PC增殖。
Objective To explore the roles of tumor-derived exosomes(TDEs)microRNA-4644(miR-4644)in progression of pancreatic cancer(PC)and its mechanism.Methods Ultracentrifugation was performed to enrich for exosomes from AsPC-1 and BxPC-3 cells.Gene Expression Omnibus(GEO)and real-time quantitative polymerase chain reaction(RT-qPCR)assays verified that miR-4644 was highly expressed in PC TDEs.A dual-luciferase reporter gene assay verified that miR-4644 bond to the 3’-untranslated region(3’UTR)of Sprouty 3(SPRY3).The co-culture and genes transfection were performed[subgroups:SPRY3 negative control(NC)group,SPRY3 overexpression(OE)group,OE+miR-4644 group],and cell counting kit-8(CCK-8),plate cloning and in-situ tumor transplantation assays(acquisition time:21 days;grouping:randomized controlled;method:two-way analysis of variance)validated that TDEs targeting SPRY3 via miR-4644 affected the proliferative capacity of PC cells.Results MiR-4644 acts as an oncogenic molecule in PC.The comparison of absorbance between the miR-4644 group and the NC group in AsPC-1 and BxPC-3 cells showed statistically significant differences(1.86±0.11 vs.1.47±0.08,F=10.21,P<0.0001;0.96±0.08 vs.0.86±0.02,F=3.90,P<0.05).Results from the colony formation assay demonstrated that the number of colonies in the miR-4644 group was higher than that in the NC group in both AsPC-1 and BxPC-3 cells[(155.70±15.50)colonies vs.(117.70±6.66)colonies,t=3.90,P<0.05;(41.67±4.51)colonies vs.(17.00±3.00)colonies,t=3.46,P<0.05].Dual-luciferase reporter assay confirmed that the expression of SPRY3 in the miR-4644 group was lower than that in the negative control(NC)group in wild-type(WT)construct(1.00±0.14 vs.0.50±0.12,t=4.61,P<0.01),while no significant difference was observed between the miR-4644 and NC groups in mutant-type(MT)construct(1.00±0.18 vs.0.87±0.05,t=1.23,P>0.05),confirming that SPRY3 is a direct downstream target gene of miR-4644.CCK-8 assay indicated significant differences in absorbance among the NC,OE group,as well as the OE miR-4644 group in AsPC-1 and BxPC-3 cells(1.87±0.08 vs.1.48±0.07 vs.1.76±0.08,F=5.88,P<0.011.23±0.03 vs.1.00±0.03 vs.1.30±0.07,F=19.42,P<0.05).Colony formation assay results demonstrated that the number of colonies formed by the OE group compared with the NC group and the OE miR-4644 group in AsPC-1 and BxPC-3 cells was statistically significant[(51.67±8.02)colonies vs.(161.00±8.54)colonies vs.(103.00±1.73)colonies,F=191.90,P<0.001;(45.67±4.04)colonies vs.(74.33±10.79)colonies vs.(65.00±4.58)colonies,F=12.52,P<0.001].The results of the orthotopic transplantation of PC tumors showed that the tumor weights in the OE group compared with the NC group and the OE miR-4644 group in AsPC-1 and BxPC-3 cells were statistically significant[(0.16±0.06)g vs.(0.50±0.12)g vs.(0.28±0.06)g,F=20.150,P<0.001;(0.15±0.01)g vs.(0.30±0.02)g vs.(0.24±0.03)g,F=78.950,P<0.001].Conclusion MiR-4644 is significantly enriched in TDEs and promotes PC proliferation by targeting SPRY3.
作者
李东奇
褚翔宇
马永蔌
张富生
田孝东
杨尹默
Li Dongqi;Chu Xiangyu;Ma Yongsu;Zhang Fusheng;Tian Xiaodong;Yang Yinmo(Department of Hepatobiliary and Pancreatic Surgery,Peking University First Hospital,Beijing 100034,China;Department of General Surgery,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)
出处
《中华实验外科杂志》
2026年第1期23-26,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金项目(82171722、82271764)。
