miR-205-5p靶向ERBB3调控PI3K/AKT/mTOR通路抑制血管生成在痔疮中的分子机制  

The Molecular Mechanism of miR-205-5p Targeting ERBB3 to Regulate PI3K/AKT/mTOR Pathway and Inhibit Angiogenesis in Hemorrhoids

作　　者：李志霄 郑霞 李春玲 刘庆圣[2] 张衡[2] Zhixiao LI;Xia ZHENG;Chunling LI;Qingsheng LIU;Heng ZHANG(The 3rd Affiliated Hospital of Yunnan University of Traditional Chinese Medicine,Kunming Yunnan 650500;Dept.of Anorectal,Kunming Municipal Hospital of Traditional Chinese Medicine,Kunming Yunnan 650500,China)

机构地区：[1]云南中医药大学第三附属医院,云南昆明650500 [2]昆明市中医医院肛肠科,云南昆明650500

出　　处：《昆明医科大学学报》2024年第6期22-35,共14页Journal of Kunming Medical University

基　　金：云南省教育厅科学研究基金资助项目(2023Y0436);云南省科技厅科技计划基金资助项目(202101AZ070001-245)。

摘　　要：目的 探讨miR-205-5p靶向ERBB3调控PI3K/AKT/mTOR通路抑制血管生成在痔疮中的分子机制。方法 收集2021年07月至2022年06月临床12例患者的痔核和正常肛周组织,通过qRT-PCR检测临床病理样本中miR-205-5p和ERBB3的表达情况,双荧光素酶报告基因实验验证miR-205-5p与ERBB3的靶向关系。将miR-205-5p mimic、pcDNA-ERBB3、miR-205-5p inhibitor、sh-ERBB3、oe-ERBB3及阴性对照质粒分别或共同转染至痔疮模型大鼠和HUVEC细胞。HE、TUNEL染色观察组织病理和细胞凋亡情况,免疫组化检测ERBB3、VEGFR2蛋白定位及表达水平,Western blot检测ERBB3、VEGFR2、Cyclin D1、Cleaved-caspase 3、Bax、Bcl-2和PI3K/AKT/mTOR通路相关蛋白的表达水平。MTT、划痕愈合、Transwell实验、流式细胞术、血管形成实验检测各组HUVEC细胞增殖、迁移、侵袭能力、凋亡率和血管生成量。结果 痔疮临床样本中miR-205-5p呈低表达,ERBB3呈高表达(P <0.001),miR-205-5p与ERBB3(WT)的3'UTR靶向结合(P <0.001)。miR-205-5p mimic组痔疮大鼠和HUVEC细胞中ERBB3表达量显著降低(P <0.01,P <0.001),VEGFR2(P <0.001)、Cyclin D1(P <0.01)、Bcl-2(P <0.001)、p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR(P <0.001)水平显著下调,Cleaved-caspase 3和Bax水平显著上调(P <0.01),大鼠肛周组织病理状况改善,HUVEC细胞增殖、迁移和侵袭力均降低(P <0.001),凋亡率升高(P <0.001),血管生成数量及分支减少(P <0.001),pcDNAERBB3逆转了这一效应(P <0.001)。结论 miR-205-5p能通过靶向抑制ERBB3表达,下调PI3K/AKT/mTOR通路活性,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,减少血管生成,从而缓解痔疮进展。Objective To explore the molecular mechanism of miR-205-5p targeting ERBB3 to regulate PI3K/AKT/mTOR pathway and inhibit angiogenesis in hemorrhoids.Methods The hemorrhoidal nucleus and normal perianal tissues of 12 patients from July 2021 to June 2022 were collected,the expression of miR-205-5p a nd ERBB3 in the samples was detected by qRT-PCR,and the targeting relationship was verified by dualluciferase.miR-205-5p mimic,pcDNA-ERBB3,miR-205-5p inhibitor,sh-ERBB3,oe-ERBB3 and negative control plasmids were respectively or co-transfected into hemorrhoidal model rats and HUVEC cells.The pathological changes and apoptosis of perianal tissue in each group were observed by HE and TUNEL staining,and the localization of expression of ERBB3 and VEGFR2 proteins were detected by immunohistochemistry,and the expression levels of ERBB3,VEGFR2,Cyclin D1,cleaved-caspase 3,Bax,Bcl-2 and PI3K/AKT/mTOR pathway related proteins were detected by Western blot.The proliferation,migration,invasion,apoptosis and angiogenesis of HUVEC were detected by MTT,wound healing,transwell,flow cytometry and angiogenesis assay.Results MiR-205-5p was lowly expressed and ERBB3 was highly expressed in clinical samples of hemorrhoids(P < 0.001),and miR-205-5p targeted the 3'UTR of ERBB3(WT)(P < 0.001).The expression of ERBB3 in hemorrhoidal rats and HUVEC cells in miR-205-5p mimic group was significantly decreased(P < 0.01,P < 0.001),and the levels of VEGFR2(P < 0.001),Cyclin D1(P < 0.01),Bcl-2(P < 0.001),p-PI3K/PI3K,p-AKT/AKT,pm TOR/mTOR( P < 0.001) were significantly down-regulated,the levels of Cleaved-caspase 3 and Bax were significantly up-regulated(P < 0.01),the perianal histopathology of rats was improved,the proliferation,migration and invasion activities of HUVEC cells were reduced(P < 0.001),the apoptosis rate was increased(P < 0.001),and the number and branches of angiogenesis were reduced(P < 0.001),and these effects were eventually reversed by pcDNA-ERBB3( P < 0.001).Conclusion MiR-205-5p can reduce angiogenesis and relieve hemorrhoids by

关 键 词：痔疮 miR-205-5p ERBB3 PI3K/AKT/mTOR通路 血管生成 

分 类 号：R657.18[医药卫生—外科学]