非接触共培养体系下抑制ARPE-19中CAMKⅡ表达对HUVECs迁移和侵袭及管腔形成的影响

Effects of inhibiting CAMKⅡ expression in ARPE-19 cells on the migration, invasion, and tube formation of HUVECs in a non-contact co-culture system

摘要:

目的：探讨非接触共培养体系下抑制人视网膜色素上皮细胞(ARPE)中Ca²⁺/钙调蛋白依赖性蛋白激酶Ⅱ(CAMKⅡ)表达对人脐静脉内皮细胞(HUVECs)迁移、侵袭、管腔形成的影响。

方法：将过表达CAMKⅡ-δ的ARPE-19样本进行RNA测序，应用生物信息学分析差异基因参与的功能。使用transwell小室构建ARPE-19和HUVECs非接触共培养体系，根据实验干预措施分为：空白组：仅接种未共培养的HUVECs，无ARPE-19细胞； 对照组：ARPE-19和HUVECs细胞均使用完全培养基进行共培养； AIP组(CAMKⅡ抑制组)：ARPE-19使用含有AIP(160 nmol/L)的完全培养基，HUVECs使用完全培养基，进行共培养。检测HUVECs迁移、侵袭和管腔形成能力的变化，并通过Western blotting检测CAMKⅡ/AMPK/mTOR/VEGFA蛋白表达水平。

结果：生信分析发现差异基因参与细胞生长与死亡和细胞运动等生物学过程。划痕和transwell迁移实验均表明AIP组的HUVECs相对迁移率均明显低于对照组(均P<0.05)。而侵袭和小管形成实验表明，AIP组的相对侵袭率和相对管腔形成率较对照组无明显改变(均P>0.05)。Western blotting结果表明AIP组CAMKⅡ、P-mTOR、VEGFA蛋白表达较对照组均明显下调，而P-AMPK蛋白表达较明显上调(均P<0.05)。

结论：在非接触共培养体系下抑制ARPE-19细胞中CAMKⅡ表达可以显著降低HUVECs迁移能力，但不能改变侵袭和管腔形成能力，这可能是通过AMPK/mTOR/VEGFA信号通路实现的。

Abstract:

AIM: To investigate the effect of inhibiting Ca²⁺/calmodulin-dependent protein kinase Ⅱ(CAMKⅡ)expression in adult retinal pigment epithelial cell line-19(ARPE-19)cells on the migration, invasion, and tube formation of human umbilical vein endothelial cells(HUVECs)in a non-contact co-culture system.

METHODS: RNA sequencing was performed on ARPE-19 cells overexpressing CAMKⅡ-δ, and bioinformatics was used to analyze the biological functions of the differentially expressed genes. Transwell inserts was used to construct a non-contact co-culture system of ARPE-19 and HUVECs. The experimental groups included: blank group: only HUVECs were inoculated without ARPE-19 cells; control group: ARPE-19 and HUVECs cells were co-cultured with complete medium; AIP group(CAMKⅡ inhibition group): ARPE-19 cells in AIP(160 nmol/L)were co-cultured with HUVECs in complete medium. The migration, invasion and tube formation abilities of HUVECs were detected. The protein expression levels of CAMKⅡ/AMPK/mTOR/VEGFA were detected by Western blotting.

RESULTS:Bioinformatics analysis found that the differentially expressed genes could affect biological processes such as cell growth and death and cell movement. The scratch test and transwell migration test showed that the relative mobility of HUVECs in the AIP group was significantly lower than that in the control group(all P<0.05). However, the invasion and tube formation assay showed that the relative invasion rate and tube formation rate of the AIP group were not significantly different from those of the control group(both P>0.05). Western blotting results showed that the expression levels of CAMKⅡ, P-mTOR, and VEGFA proteins in the AIP group were significantly lower than those in the control group, while the expression level of the P-AMPK protein was significantly higher than that in the control group(all P<0.05).

CONCLUSION:In the non-contact co-culture system, inhibition of CAMKⅡ expression in ARPE-19 cells significantly reduced the migration ability of HUVECs, but it cannot change the invasion and tube formation ability, which may be achieved by AMPK/mTOR/VEGFA.