The Molecular Mechanism of miR-205-5p Targeting ERBB3 to Regulate PI3K/AKT/mTOR Pathway and Inhibit Angiogenesis in Hemorrhoids
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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 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，miR-205-5p与ERBB3 mRNA的3'UTR靶向结合。miR-205-5p mimic组痔疮大鼠和HUVEC细胞中ERBB3表达量显著降低，VEGFR2、Cyclin D1、Bcl-2、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平显著下调，Cleaved-caspase 3和Bax水平显著上调，大鼠肛周组织病理状况改善，HUVEC细胞增殖、迁移和侵袭力降低，凋亡率升高，血管生成数量及分支减少，pcDNA-ERBB3逆转了这一效应。 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。 -
关键词:
- 痔疮 /
- miR-205-5p /
- ERBB3 /
- PI3K/AKT/mTOR通路 /
- 血管生成
Abstract: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 expressions of miR-205-5p and ERBB3 in clinicopathological samples were detected by qRT-PCR, and the targeting relationship was verified by dual-luciferase. 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 low expressed and ERBB3 was highly expressed in clinical samples of hemorrhoids, and miR-205-5p targeted the 3'UTR of ERBB3 mRNA. The expression of ERBB3 in hemorrhoidal rats and HUVEC cells in miR-205-5p mimic group was significantly decreased, and the levels of VEGFR2, Cyclin D1, Bcl-2, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR were significantly down-regulated, the levels of cleaved-caspase 3 and Bax were significantly up-regulated, the perianal histopathology of rats was improved, the proliferation, migration and invasion activities of HUVEC cells were reduced, the apoptosis rate was increased, and the number and branches of angiogenesis were reduced, and these effects were eventually reversed by pcDNA-ERBB3. Conclusion MiR-205-5p can reduce angiogenesis and relieve hemorrhoids by targeting the inhibition of ERBB3 expression, down-regulating the activity of PI3K/AKT/mTOR pathway, inhibiting cell proliferation, migration and invasion, and promoting apoptosis. -
Key words:
- Hemorrhoids /
- miR-205-5p /
- ERBB3 /
- PI3K/AKT/mTOR pathway /
- Angiogenesis
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图 1 miR-205-5p和ERBB3在临床病理样本中的表达水平
A～B：qRT-PCR检测miR-205-5p和ERBB3的相对表达量；C：Western blot检测ERBB3蛋白表达（“H”：正常组，n=12；“P”：疾病组，n=12）；D：免疫荧光染色检测VEGFR2蛋白表达（200 ×，标尺50 μm）；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 1. The expression of miR-205-5p and ERBB3 in clinicopathological samples
图 2 miR-205-5p与ERBB3的靶向关系
A：TargetScan数据库预测miR-205-5p和ERBB3的结合位点；B：双荧光素酶报告基因实验验证miR-205-5p与ERBB3的靶向关系；C：Western blot检测过表达miR-205-5p后ERBB3蛋表达水平，n=3； **P < 0.01，***P < 0.001。
Figure 2. The targeting relationship between miR-205-5p and ERBB3
图 3 过表达miR-205-5p对痔疮大鼠的影响
A：qRT-PCR检测各组大鼠肛周组织中miR-205-5p的相对表达量；B：HE染色观察各组大鼠肛周组织病理变化（200 ×，标尺50 μm），n=5；C：TUNEL染色检测各组大鼠肛周组织细胞凋亡率和凋亡阳性细胞定量；D：免疫组化检测ERBB3和VEGFR2蛋白表达情况（200 ×，标尺50 μm），n=5；E：Western blot检测ERBB3、VEGFR2、Cyclin D1、Cleaved-caspase 3、Bax和Bcl-2蛋白水平，n=5；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 3. The effect of overexpression of miR-205-5p on hemorrhoidal rats
图 4 ERBB3受miR-205-5p调控影响痔疮的进展
A：qRT-PCR检测各组大鼠肛周组织中miR-205-5p的相对表达量；B：HE染色观察肛周组织病理变化（200 ×，标尺50 μm），n=5；C：TUNEL染色检测细胞凋亡率和阳性细胞定量；D：免疫组化检测ERBB3和VEGFR2蛋白表达情况（200 ×，标尺50 μm），n=5；E：Western blot检测ERBB3、VEGFR2、Cyclin D1、Cleaved-caspase 3、Bax和Bcl-2蛋白水平，n=5；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 4. ERBB3 is regulated by miR-205-5p to affect the progression of hemorrhoids
图 5 过表达ERBB3对痔疮大鼠的影响
A：qRT-PCR检测各组大鼠肛周组织中miR-205-5p的相对表达量；B：HE染色观察肛周组织病理变化（200 ×，标尺50 μm），n=5；C：TUNEL染色检测细胞凋亡率和阳性细胞定量；D：免疫组化检测ERBB3和VEGFR2蛋白表达情况（200 ×，标尺50 μm），n=5；E：Western blot检测ERBB3、VEGFR2、Cyclin D1、Cleaved-caspase 3、Bax、Bcl-2、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白水平，n=5；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 5. The effect of overexpression of ERBB3 on hemorrhoidal rats
图 6 过表达ERBB3促进血管生成与PI3K/AKT/mTOR通路相关
A：免疫共沉淀法验证ERBB3与PI3K的相互作用，n=3；B：qRT-PCR检测转染效率；C：血管生成实验；D：Western blot检测相关蛋白水平，n=3；E：MTT法检测细胞增殖能力；F：划痕愈合实验检测细胞迁移能力；G-H：Transwell实验检测细胞迁移侵袭能力；I：AnnexinV-FITC/PI检测细胞凋亡率；J：流式细胞仪检测细胞周期；*P < 0.05，**P < 0.01，***P < 0.001，与NC组相比，##P < 0.01。
Figure 6. Overexpression of ERBB3 promotes angiogenesis through PI3K/AKT/mTOR pathway
图 7 miR-205-5p靶向ERBB3调控PI3K/AKT/mTOR通路对HUVEC细胞的影响
A：qRT-PCR检测各组miR-205-5p表达量；B：Western blot检测相关蛋白表达水平，n=3；C：MTT法检测HUVEC细胞增殖能力；D：划痕愈合实验检测HUVEC细胞迁移能力；E～F：Transwell实验检测HUVEC细胞迁移侵袭能力；G: AnnexinV-FITC/PI检测HUVEC细胞凋亡率；H：流式细胞仪检测HUVEC细胞周期；I：血管生成实验；*P < 0.05，**P < 0.01，***P < 0.001。
Figure 7. The effect of miR-205-5p targeting ERBB3 to regulate PI3K/AKT/mTOR pathway on HUVEC cells
表 1 引物序列
Table 1. Primer Sequence
Primer Sequence（5′-3′） miR-205–5p Forward： CGTCCTTCATTCCACCGG Reverse： AGTGCAGGGTCCGAGGTATT ERBB3 Forward： CAAGATTCCAGTCTGCATTAAAGTC Reverse： CAGCATATGATCTGTCACAGCTTG U6 Forward： CTCGCTTCGGCAGCACATA Reverse： AACGCTTCACGAATTTGCGT β-actin Forward： TGACGTGGACATCCGCAAAG Reverse： CTGGAAGGTGGACAGCGAGG 
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