血浆外泌体源性miR-20a-5p通过靶向PIK3R1影响雌激素受体阳性乳腺癌细胞骨转移

Plasma exosome-derived miR-20a-5p affects bone metastasis in estrogen receptor-positive breast cancer cells by targeting PIK3R1

目的探讨血浆外泌体源性miR-20a-5p通过靶向PIK3R1对雌激素受体阳性的乳腺癌(estrogen receptor-positive breast cancer,ER(+)BC)骨转移的影响。方法借助生物信息学网站检索与ER(+)BC骨转移相关的数据集,选择miR-20a-5p纳入研究。收集90例ER(+)BC患者与相对应的健康人的血浆,检测血浆中PIK3R1的表达,从血浆中提取外泌体,检测外泌体中miR-20a-5p的表达。双荧光素酶报告实验验证miR-20a-5p与PIK3R1的靶向调控关系。分别将转染NC-inhibitor、miR-inhibitor的外泌体或转染si-NC、si-PIK3R1的ER(+)BC细胞注射到小鼠左心室,Micro-CT扫描骨组织并进行骨组织TRAP染色。将转染NC-inhibitor、miR-inhibitor的外泌体与转染si-NC、si-PIK3R1的ER(+)BC细胞共培养后,使用Western blot检测破骨细胞分子c-fos、NFATc1的表达。结果qRT-PCR检测发现较正常血浆外泌体(1±0.26)或细胞(1±0.13),miR-20a-5p在ER(+)BC血浆外泌体(1.49±0.27)(t=12.40,P<0.001)及BC细胞系MCF-7(1.64±0.13)(t=6.03,P=0.004)、BT474(1.49±0.11)(t=4.98,P=0.008)、T47D(1.98±0.15)(t=8.55,P=0.001)中均表达升高。而与正常血浆外泌体(1±0.25)或细胞(1±0.10)比较,PIK3R1在ER(+)BC血浆外泌体(0.69±0.24)(t=8.48,P<0.001)及ER(+)BC细胞系MCF-7(0.73±0.05)(t=4.18,P=0.014)、BT474(0.61±0.05)(t=6.04,P=0.004)、T47D(0.34±0.04)(t=10.61,P<0.001)中则表达降低。PIK3R1在血浆(t=8.48,P<0.001)及ER(+)BC细胞系MCF-7(t=4.18,P=0.014)、BT474(t=6.04,P=0.004)、T47D(t=10.61,P<0.001)中则表达降低。PIK3R1被证实为miR-20a-5p的靶基因。与NC-inhibitor组小鼠相比,miR-inhibitor组小鼠的骨小梁组织体积(t=3.32,P=0.029)、骨小梁区域体积(t=6.24,P=0.003)、骨体积分数(t=7.35,P=0.002)和骨矿物密度(t=13.72,P<0.001)均增加,成熟破骨细胞数量减少。与NC-inhibitor组比较,miR-inhibitor组细胞中c-fos(t=9.04,P=0.001)和NFATc1(t=13.42,P<0.001)的表达减少。与miR-inhibitor+si-NC组相比,miR-inhibitor+si-PIK3R1组中小鼠的骨小梁组织体积(t=3.03,P=0.039)、骨小梁区域体积(t=6.37,P=0.003)、骨体积分数(t=3.36,P=0.028)和骨矿物密度(t=6.92,P=0.002)均减少,成熟破骨细胞数量增加,细胞中c-fos(t=7.75,P=0.002)和NFATc1(t=9.65,P=0.001)的表达增加。结论ER(+)BC患者血浆外泌体源性miR-20a-5p通过抑制PIK3R1表达来促进ER(+)BC骨转移。

Objective To investigate the effects of plasma exosome-derived miR-20a-5p on bone metastases in estrogen receptor-positive breast cancer(ER(+)BC)by targeting PIK3R1.Methods The data sets related to ER(+)BC bone metastasis were retrieved with the help of bioinformatics website,miR-20a-5p was included in the study.The plasma of 90 ER(+)BC patients and the corresponding healthy people were collected to detect the expression of PIK3R1 in the plasma,and exosomes were extracted from the plasma to detect expression of miR-20a-5p in exosomes.The dual-luciferase reporter assay was used to verify the targeting regulation relationship between miR-20a-5p and PIK3R1.The exosomes transfected with NC-inhibitor and miR-inhibitor or ER(+)BC cells transfected with si-NC and si-PIK3R1 were injected into the left ventricle of mice,respectively,and the bone tissue was scanned by Micro-CT and bone tissue TRAP staining was performed.After co-culturing NC-inhibitor and miR-inhibitor-transfected exosomes with si-NC and si-PIK3R1-transfected ER(+)BC cells,Western blot was used to detect the expression of osteoclast molecules c-fos and NFATc1.Results qRT-PCR assay showed that compared with normal plasma exosomes(1±0.26)or cells(1±0.13),miR-20a-5p was significantly increased in ER(+)BC plasma exosomes(1.49±0.27)(t=12.40,P<0.001)and BC cell line MCF-7(1.64±0.13)(t=6.03,P=0.004),BT474(1.49±0.11)(t=4.98,P=0.008),T47D(1.98±0.15)(t=8.55,P=0.001).But compared with normal plasma exosomes(1±0.25)or cells(1±0.10),expression of PIK3R1 in ER(+)BC plasma exosomes(0.69±0.24)(t=8.48,P<0.001)and ER(+)BC cell lines MCF-7(0.73±0.05)(t=4.18,P=0.014),BT474(0.61±0.05)(t=6.04,P=0.004),and T47D(0.34±0.04)(t=10.61,P<0.001)was inhibited.PIK3R1 was confirmed as a target gene of miR-20a-5p.Compared with mice in NC-inhibitor group,trabecular bone tissue volume(t=3.32,P=0.029),trabecular bone area volume(t=6.24,P=0.003),bone volume fraction(t=7.35,P=0.002)and bone mineral density(t=13.72,P<0.001)of mice in miR-inhibitor group were both increased,the number of mature osteoclasts was decreased.Compared with NC inhibitor group,expression of c-fos(t=9.04,P=0.001)and NFATc1(t=13.42,P<0.001)in miR-inhibitor group was decreased.Compared with miR-inhibitor+si-NC group,trabecular bone tissue volume(t=3.03,P=0.039),trabecular bone area volume(t=6.37,P=0.003),bone volume fraction(t=3.36,P=0.028)and bone mineral density(t=6.92,P=0.002)were decreased,the number of mature osteoclasts was increased,and expressionof c-fos(t=7.75,P=0.002)and NFATc1(t=9.65,P=0.001)was increased in miR-inhibitor+si-PIK3R1 group.Conclusion PlasmaExosome-derived miR-20a-5p from ER(+)BC patients promotes ER(+)BC bone metastasis by inhibiting the expression of PIK3R1.