MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生

李雪; 曹浪; 甘敏; 周善璧

doi:10.16016/j.1000-5404.201909141

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MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生

基础医学 | 更新时间：2026-01-09

- MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生
- MiR-200b inhibits corneal angiogenesis in rats by regulating PIK3CA/AKT pathway
- 第三军医大学学报 2020年42卷第4期页码：407-413
- 作者机构：
  
  1.400016 重庆, 重庆医科大学附属第一医院眼科，重庆市眼科重点实验室
  2.401331 重庆, 重庆医科大学附属大学城医院眼科
- 作者简介：
  
  [周善璧，E-mail: zhoushanbi@aliyun.com ZHOU Shanbi, E-mail: zhoushanbi@aliyun.com
- 基金信息：
  
  重庆市医学科研计划资助项目(2013-1-033)
- DOI：10.16016/j.1000-5404.201909141
  中图分类号： R394.6; R364.33; R772.2
- 收稿：2019-09-18，
  
  修回：2019-12-03，
  
  纸质出版：2020-02-28
- 稿件说明：
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李雪, 曹浪, 甘敏, 等. MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生[J]. 第三军医大学学报, 2020,42(4):407-413.

Xue LI, Lang CAO, Min GAN, et al. MiR-200b inhibits corneal angiogenesis in rats by regulating PIK3CA/AKT pathway[J]. Journal of Third Military Medical University, 2020, 42(4): 407-413.
李雪, 曹浪, 甘敏, 等. MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生[J]. 第三军医大学学报, 2020,42(4):407-413. DOI： 10.16016/j.1000-5404.201909141.

Xue LI, Lang CAO, Min GAN, et al. MiR-200b inhibits corneal angiogenesis in rats by regulating PIK3CA/AKT pathway[J]. Journal of Third Military Medical University, 2020, 42(4): 407-413. DOI： 10.16016/j.1000-5404.201909141.

摘要

目的

探讨microRNA-200b(miR-200b)在大鼠角膜新生血管(corneal neovascularization

CNV)形成中的作用及其调节机制。

方法

24只雌性SD大鼠采用一眼经缝线法构建CNV模型(实验组)，另一眼作为正常对照组，于裂隙灯下观察建模后第4、7、14天角膜新生血管情况，采用HE染色观察角膜组织结构改变及炎性细胞浸润情况，RT-PCR检测造模后第14天各组大鼠角膜组织中miR-200b以及血管内皮生长因子(vascular endothelial growth factor

VEGF)的表达。利用脂质体转染人脐静脉内皮细胞(human umbilical vein endothelial cells

HUVECs)，分为miR-200b模拟物(miR-200b mimic)组、阴性对照(miR-NC)组、空白对照(CON)组，利用RT-PCR验证转染效果，分别采用CCK-8检测HUVECs增殖能力，划痕实验检测HUVECs迁移能力，Matrigel胶成管实验检测HUVECs管腔形成能力。并通过Western blot检测VEGF及PIK3CA、AKT蛋白的表达。

结果

成功构建大鼠CNV模型，HE染色显示与正常组相比，缝线后第4、7、14天，大鼠角膜组织排列紊乱，炎性细胞浸润逐渐加重(

0.05)，RT-PCR结果显示缝线后第14天角膜组织中miR-200b表达明显降低，而VEGF表达明显升高(

0.01)。与CON组和miR-NC组相比

miR-200b mimic组中miR-200b相对表达量显著升高(

0.01)

而增殖、迁移及管腔形成能力显著下降(

0.05)，转染miR-200b mimic后，HUVECs中VEGF、PIK3CA、p-AKT蛋白表达均明显下降(

0.05)。

结论

miRNA-200b可显著抑制HUVECs的增殖、迁移及成管能力，可能通过抑制PIK3CA/AKT通路参与CNV的发生和发展。

Abstract

Objective

To investigate the role of microRNA-200b (miR-200b) in corneal neovascularization (CNV) in rats and explore the possible mechanisms.

Methods

Twenty-four female SD rats were subjected to corneal suture to induce CNV in one eye

with the other eye as the normal control. On the 4th

7th and 14th days after the suture

CNV was observed under a slit lamp

and the structure and inflammatory cell infiltration in the corneal tissue were observed using HE staining. On day 14 after the suture

the expression levels of miR-200b and vascular endothelial growth factor (VEGF) mRNA in the cornea were detected using qRT-PCR. Human umbilical vein endothelial cells (HUVECs) were transfected

via

liposome with a miR-200b mimic or a negative control sequence (miR-NC)

and the cell proliferation

migration and tube formation abilities were evaluated using CCK-8 assay

Transwell chamber assay and Matrigel assay

respectively. The expression levels of VEGF and PIK3CA/AKT proteins in the cells were detected by Western blotting.

Results

CNV models were successfully established by corneal suture in the rats. HE staining showed that corneal suture resulted in disordered alignment of the corneal tissue and progressively increased inflammatory cell infiltration on days 4

7 and 14 after the suture (

0.05). RT-PCR showed that miR-200b expression decreased and VEGF expression increased significantly in the corneal tissue on day 14 after the suture (

0.01). Compared with the cells without transfection or transfected with miR-NC

the HUVECs transfected with the miR-200b mimic exhibited significantly attenuated proliferation

migration and lumen formation abilities (

0.05) with also significantly lowered expression of VEGF

PIK3CA and p-AKT proteins (

0.05).

Conclusion

miRNA-200b can significantly inhibit the proliferation

migration and tube formation of HUVECs

and may participate in the occurrence and development of CNV by inhibiting the PIK3CA/AKT pathway.

关键词

Keywords

references

BACHMANN B, TAYLOR R S, CURSIEFENV C. Corneal neovascularization as a risk factor for graft failure and rejection after keratoplasty: an evidence-based meta-analysis[J]. Ophthalmology, 2010, 117(7): 1300-5.e7. DOI: 10.1016/j.ophtha.2010.01.039.

HOU P F, LI H L, YONG H M, et al . PinX1 represses renal cancer angiogenesis via the mir-125a-3p/VEGF signaling pathway[J ] . Angiogenesis, 2019, 22(4): 507-519. DOI: 10.1007/s10456-019-09675-z..

ZHUANG Z, XIAO-QIN, HU H, et al . Down-regulation of microRNA-155 attenuates retinal neovascularization via the PI3K/Akt pathway[J ] . Mol Vis, 2015, 21: 1173-1184..

HOU J Y, LIU L, ZHU Q, et al . MicroRNA-185 inhibits angiogenesis in humanmicrovascular endothelial cells through targeting stromal interaction molecule 1[J ] . Cell Biol I nt, 2016, 40(3): 318-328. DOI: 10.1002/cbin.10572.

LEWIS A, FELICE C, KUMAGAI T, et al . The miR-200 family is increased in dysplastic lesions in ulcerative colitis patients[J ] . PLoS ONE, 2017, 12(3): e0173664. DOI: 10.1371/journal.pone.0173664.

FU Y X, LIU X H, ZHOU N T, et al . MicroRNA-200b stimulatestumour growth in TGFBR2-null colorectal cancers by negatively regulating p27/kip1[J ] . J Cell Physiol, 2014, 229(6): 772-782. DOI: 10.1002/jcp.24497.

WANG Y C, YIN X H, ZHAO L, et al . MicroRNA-200b inhibits pituitary tumor cell proliferation and invasion by targeting PKCα[J ] . Exp Ther Med, 2017, 14(2): 1706-1714. DOI: 10.3892/etm.2017.4681.

LI D F, WANG H, SONG H M, et al . The microRNAs miR-200b-3p and miR-429-5p target the LIMK1/CFL1 pathway to inhibit growth and motility of breast cancer cells[J ] . Oncotarget, 2017, 8(49): 85276-85289. DOI: 10.18632/oncotarget.19205.

毛旖旎, 胡雁, 侯胜平, 等.大鼠角膜缘新生血管微小RNA与血管内皮生长因子相关性分析[J].重庆医科大学学报, 2014, 39(8): 1090-1094. DOI: 10.13406/j.cnki.cyxb.000324.

MAO Y N, HU Y, HOU S P, et al . Correlation between microRNA expression profiles and VEGF of corneal neovascularization in rats[J ] . J Chongqing Med Univ, 2014, 39(8): 1090-1094. DOI: 10.13406/j.cnki.cyxb.000324.

周小军. DATS对缝线诱导的大鼠角膜新生血管的影响及机制研究[J].国际眼科杂志, 2017, 17(11): 2015-2018.

ZHOU X J. Effect of DATS on experimental corneal neovascularization induced by corneal suture in rats cornea[J]. Int Eye Sci, 2017, 17(11): 2015-2018.

ZONG R R, ZHOU T, LIN Z R, et al . Down-regulation of MicroRNA-184 is associated with corneal neovascularization[J ] . Invest Ophthalmol Vis Sci, 2016, 57(3): 1398-1407. DOI: 10.1167/iovs.15-17417.

SHEN J K, YANG X R, XIE B, et al . MicroRNAs regulate ocular neovascularization[J ] . Mol Ther, 2008, 16(7): 1208-1216. DOI: 10.1038/mt.2008.104.

李化, 李彦, 周善璧. MiRNA-142-5p在大鼠角膜新生血管中的作用及机制研究[J].第三军医大学学报, 2018, 40(8): 699-704. DOI: 10.16016/j.1000-5404.201711061.

LI H, LI Y, ZHOU S B. Role of miRNA-142-5p in regulation of rat corneal neovascularization and related pathways[J]. J Third Mil Med Univ, 2018, 40(8): 699-704. DOI: 10.16016/j.1000-5404.201711061.

LIU C H, WANG Z X, HUANG S, et al . MicroRNA-145 regulates pathological retinal angiogenesis by suppression of TMOD3[J ] . Mol Ther Nucleic Acids, 2019, 16: 335-347. DOI: 10.1016/j.omtn.2019.03.001.

CHOI Y, YOON S, JEONG Y, et al . Regulation of vascular endothelial growth factor signaling by miR-200b[J ] . Mol Cells, 2011, 32(1): 77-82. DOI: 10.1007/s10059-011-1042-2.

MCARTHUR K, FENG B, WU Y X, et al . MicroRNA-200b regulates vascular endothelial growth factor-mediated alterations in diabetic retinopathy[J ] . Diabetes, 2011, 60(4): 1314-1323. DOI: 10.2337/db10-1557.

CHAN Y C, KHANNA S, ROY S, et al . MiR-200b targets Ets-1 and is down-regulated by hypoxia to induceangiogenic response of endothelial cells[J ] . J Biol Chem, 2011, 286(3): 2047-2056. DOI: 10.1074/jbc.M110.158790.

LIANG D L, WANG Z X, YAN Z Q, et al . Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module[J ] . Sci Rep, 2017, 7: 43798. DOI: 10.1038/srep43798.

CLAESSON-WELSH L, WELSH M. VEGFA andtumour angiogenesis[J]. J Intern Med, 2013, 273(2): 114-127. DOI: 10.1111/joim.12019.

YU Q Q, WU H, HUANG X, et al . MiR-1 targets PIK3CA and inhibits tumorigenic properties of A549 cells[J ] . Biomedecine Pharmacother, 2014, 68(2): 155-161. DOI: 10.1016/j.biopha.2014.01.005.

MA Y, WEI S, LIN Y C, et al . PIK3CA as an oncogene in cervical cancer[J ] . Oncogene, 2000, 19(23): 2739-2744. DOI: 10.1038/sj.onc.1203597.

ISAKOFF S J, ENGELMAN J A, IRIE H Y, et al . Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells[J ] . Cancer Res, 2005, 65(23): 10992-11000. DOI: 10.1158/0008-5472.CAN-05-2612.

朱晓志, 王冬梅, 王丽蓉. miR-200b-5p靶向ATAD2调控P13K/AKT信号通路参与宫颈癌血管生成的作用机制[J].中国性科学, 2019, 28(7): 42-46. DOI: 10.3969/j.issn.1672-1993.07.010.

ZHU X Z, WANG D M, WANG L R. The role of mir-200b-5p targeting ATAD2 in the regulation of P13K/Akt signaling pathway in the angiogenesis of cervical cancer[J]. Chin J Human Sexual, 2019, 28(7): 42-46. DOI: 10.3969/j.issn.1672-1993.07.010.

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