摘要
目的探讨微小RNA(miR)-325-3p在乳腺癌阿霉素(ADR)耐药中的机制。方法收集2024年1月至2024年12月湖南省人民医院就诊的62例乳腺癌患者组织样本,根据肿瘤组织对阿霉素(ADR)耐药或敏感,分为阿霉素耐药组(21例),阿霉素敏感组(29例)。逐步增加ADR浓度(5~60 nmol/L)建立耐药细胞系MCF-7/ADR和MDA-MB-231/ADR。采用原位杂交(ISH)、实时定量聚合酶链反应(RT-qPCR)检测miR-325-3p表达,通过转染miR-325-3p抑制剂调控其表达水平。流式细胞术分析细胞凋亡,Transwell实验评估迁移和侵袭能力。蛋白质印迹法(Western blot)检测第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因(PTEN)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)通路蛋白,双荧光素酶报告基因实验验证miR-325-3p与PTEN的靶向关系。符合正态分布的计量资料采用独立样本t检验或单因素方差分析。结果miR-325-3p在阿霉素耐药组乳腺癌组织和耐药细胞系的表达量高于敏感组乳腺癌组织和敏感细胞系,差异有统计学意义(乳腺癌组织:611.79±34.26比101.72±11.84,MCF-7/ADR:630.86±13.74比99.87±17.98;MDA-MB-231/ADR:877.20±36.69比96.99±30.79,t=37.23、62.08、43.10,P<0.05)。转染miR-325-3p抑制剂的细胞的MCF-7/ADR与MDA-MB-231/ADR细胞半数抑制浓度(IC_(50))低于转染miR-NC的细胞及未转染的空白组细胞,差异有统计学意义(137.77±20.28比240.63±23.52比243.25±19.64、77.73±20.79比266.95±22.08比240.63±23.52,F=56.3、178.4,P<0.05)。miR-325-3p抑制表达MCF-7/ADR细胞组与MDA-MB-231/ADR细胞的细胞凋亡率高于阴性对照组和空白对照组,差异有统计学意义[(15.76±1.38)%比(1.63±0.72)%比(2.60±0.80)%、(17.66±1.00)%比(1.04±0.27)%比(1.47±1.04)%,F=428.8、876.3,P<0.05]。miR-325-3p抑制剂处理的MCF-7/ADR与MDA-MB-231/ADR细胞迁移细胞数和侵袭细胞数低于空白对照组及阴性对照组的迁移细胞数和侵袭细胞数,差异有统计学意义[细胞迁移数:(88.02±19.15)个比(256.59±25.95)个比(265.19±20.08)个,(52.67±20.46)个比(240.80±16.60)个比(242.65±16.16)个,侵袭细胞数:(38.11±6.31)个比(190.15±6.02)个比(186.16±8.24)个、(33.94±8.15)个比(210.51±5.96)个比(205.87±11.60)个,F=145.2、261.8、109.6、899.1,P<0.05)。miRNA-325-3p抑制剂与pGLO(质粒载体)-PTEN-WT(野生型)共转染导致荧光活性高于对照组,差异有统计学意义(2.58±0.10比0.99±0.11,F=182.9,P<0.05)。miRNA-325-3p抑制剂处理后的PTEN mMRA表达及蛋白表达显著高于对照组,差异有统计学意义(579.24±10.21比96.29±21.37、258.88±5.21比45.00±9.87,t=53.94、50.70,P<0.05)。结论miR-325-3p通过调控PTEN/Akt/mTOR通路影响乳腺癌阿霉素耐药。
Objective To investigate the mechanism of microRNA(miR)-325-3p in doxorubicin(ADR)resistance of breast cancer.Methods Breast cancer tissue samples were collected from 62 patients(21 ADR-resistant and 29 ADR-sensitive)at Hunan Provincial People’s Hospital from January 2024 to December 2024.Based on tumor tissue response to Doxorubicin(ADR),patients were stratified into ADR-resistant(n=21)and ADR-sensitive(n=29)groups.ADR-resistant cell lines(MCF-7/ADR and MDA-MB-231/ADR)were established through stepwise ADR concentration escalation(5-60 nmol/L),designated as the ADR-resistant cell line group.Parental cell lines(MCF-7/MDA-MB-231)without ADR treatment were designated as the parental/ADR-sensitive cell group.ADR-resistant cell lines transfected with miR-325-3p inhibitor were designated as the miR-325-3p inhibition group,while those transfected with scrambled sequence inhibitor served as the negative control group(miR-NC).Untransfected ADR-resistant cell lines were designated as the blank control group.The miR-325-3p expression was detected by ISH and RT-qPCR,and its levels were modulated using miR-325-3p inhibitors.Cell apoptosis was analyzed by flow cytometry,while migration and invasion abilities were assessed via Transwell assays.Western blotting was performed to examine phosphatase and tensin homologue deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway proteins,and the targeting relationship between miR-325-3p and PTEN was validated using a dual-luciferase reporter assay.Continuous variables were analyzed using independent samples t-test(for normally distributed data with homogeneity of variance)or one-way ANOVA,followed by LSD post hoc test for pairwise comparisons.Results The expression level of miR-325-3p was significantly higher in doxorubicin-resistant breast cancer tissues and resistant cell lines than their sensitive counterparts,and the difference was statistically significant(breast cancer tissues:611.79±34.26 vs.101.72±11.84;MCF-7/ADR:630.86±13.74 vs.99.87±17.98;MDA-MB-231/ADR:877.20±36.69 vs.96.99±30.79,t=37.23,62.08,43.10,P<0.05).Transfection with a miR-325-3p inhibitor significantly reduced the half-maximal inhibitory concentration(IC_(50))of MCF-7/ADR and MDA-MB-231/ADR cells compared to miR-NC-transfected and untransfected control cells,and the difference was statistically significant(137.77±20.28 vs.240.63±23.52 vs.243.25±19.64,77.73±20.79 vs.266.95±22.08 vs.240.63±23.52,F=56.3,178.4,P<0.05).The miR-325-3p suppression significantly increased the apoptosis rate in both MCF-7/ADR and MDA-MB-231/ADR cells compared to the negative control(miR-NC)and blank control groups,and the difference was statistically significant[(15.76±1.38)%vs.(1.63±0.72)%vs.(2.60±0.80)%,(17.66±1.00)%vs.(1.04±0.27)%vs.(1.47±1.04)%,F=428.8,876.3,P<0.05].The miR-325-3p inhibition markedly decreased the migratory and invasive capacities of MCF-7/ADR and MDA-MB-231/ADR cells.The number of migratory cells and invasive cells in MCF-7/ADR and MDA-MB-231/ADR cells treated with miR-325-3p inhibitor was lower than that in the blank control group and the negative control group,and the difference was statistically significant[cell migration number:(88.02±19.15)cells vs.(256.59±25.95)cells vs.(265.19±20.08)cells,(52.67±20.46)cells vs.(240.80±16.60)cells vs.(242.65±16.16)cells,invasive cell number:(38.11±6.31)cells vs.(190.15±6.02)cells vs.(186.16±8.24)cells,(33.94±8.15)cells vs.(210.51±5.96)cells vs.(205.87±11.60)cells,F=145.2,261.8,109.6,899.1,P<0.05].Luciferase reporter assays demonstrated that co-transfection of the miR-325-3p inhibitor with pGLO-PTEN-WT(wild-type)resulted in significantly higher fluorescence activity than the control group,and the difference was statistically significant(2.58±0.10 vs.0.99±0.11,F=182.9,P<0.05).The miR-325-3p inhibition significantly upregulated both mRNA and protein expression of PTEN(579.24±10.21 vs.96.29±21.37,258.88±5.21 vs.45.00±9.87,t=53.94,50.70,P<0.05).Conclusion MiR-325-3p affects doxorubicin resistance in breast cancer by regulating the PTEN/Akt/mTOR pathway.
作者
王慧玲
武亚琴
孙瑾
张振宇
张超杰
段伊榕
Wang Huiling;Wu Yaqin;Sun Jin;Zhang Zhenyu;Zhang Chaojie;Duan Yirong(Department of Breast and Thyroid Surgery,Hunan Provincial People’s Hospital(The First Affiliated Hospital of Hunan Normal University),Changsha 410005,China;Clinical Medical College of Hunan Normal University,Changsha 410005,China)
出处
《中华实验外科杂志》
2025年第7期1252-1256,共5页
Chinese Journal of Experimental Surgery
基金
湖南省自然基金面上项目(2022JJ30345)
湖南省卫健委一般指导课题(202104012788)。
