circSNRK alleviates ischemia-reperfusion injury in renal tubular epithelial cell by up-regulating Akt pathway
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摘要:
目的 探讨环状RNA SNRK（circSNRK）在缺血-再灌注损伤（IRI）中的作用及机制。 方法 构建缺氧-复氧（IRI）细胞模型，检测IRI处理后circSNRK的表达情况及过表达circSNRK对细胞增殖和细胞凋亡的影响。分析circSNRK的作用靶点。将HK2细胞分为空白组（Mock组）、IRI组、对照质粒+IRI组（IRI+NC组）、过表达人circSNRK+IRI组（IRI+circSNRK组）、过表达人circSNRK+IRI+蛋白激酶B（Akt）抑制剂组（IRI+circSNRK+MK2206组）、对照质粒组（NC组）。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞增殖及凋亡情况。进行circSNRK作用靶点的基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）聚类分析。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞CDKN1A、Akt、B细胞淋巴瘤（Bcl）-2、半胱氨酸天冬氨酸蛋白酶（Caspase）-9信使RNA（mRNA）表达水平，p21、Bcl-2、Caspase-9、Akt、p-Akt蛋白表达水平。检测NC组、IRI+NC组、IRI+circSNRK组、IRI+circSNRK+MK2206组细胞增殖及凋亡情况。 结果 与Mock组比较，IRI组circSNRK表达水平较低，HK2细胞增殖能力下降，细胞凋亡增多。IRI+circSNRK组细胞增殖能力较IRI+NC组升高，细胞凋亡较IRI+NC组减少。circSNRK可通过51个微小RNA（miRNA）作用于648个靶点。GO富集分析显示，circSNRK作用靶点主要富集于细胞过程和生物调节等生物学过程，细胞部分、细胞和细胞外部分等细胞成分，以及结合、结合蛋白和酶等分子功能。KEGG聚类分析显示，circSNRK作用靶点主要富集在癌症信号通路、磷脂酰肌醇-3-激酶（PI3K）-Akt信号通路和癌症miRNA等相关通路。与Mock组比较，IRI组CDKN1A和Caspase-9 mRNA相对表达量较高，miR-99a-5p RNA表达水平较高，Akt和Bcl-2 mRNA相对表达量较低；与IRI+NC组比较，IRI+circSNRK组CDKN1A和Caspase-9 mRNA相对表达量较低，Akt和Bcl-2 mRNA相对表达量较高，miR-99a-5p RNA表达水平较低，差异均有统计学意义（均为P < 0.05）。IRI组p21和Caspase-9蛋白表达较Mock组增多，p-Akt、Akt和Bcl-2蛋白表达较Mock组减少；IRI+circSNRK组p21和Caspase-9蛋白表达较IRI+NC组减少，p-Akt、Akt和Bcl-2蛋白表达较IRI+NC组增多。circSNRK和Akt上存在miR-99a-5p结合位点。与NC组比较，IRI+NC组细胞增殖能力下降；与IRI+NC组比较，IRI+circSNRK组细胞增殖能力升高；与IRI+circSNRK组比较，IRI+circSNRK+MK2206组细胞增殖能力下降（均为P < 0.05）。IRI+NC组细胞凋亡水平较NC组高；IRI+circSNRK组细胞凋亡水平较IRI+NC组低；IRI+circSNRK+MK2206组细胞凋亡水平较IRI+circSNRK组高。 结论 在IRI条件下，circSNRK可影响HK2细胞增殖和凋亡，可能是通过Akt通路发挥作用。 Abstract:Objective To investigate the role and mechanism of circular RNA SNRK (circSNRK) in ischemia-reperfusion injury (IRI). Methods A hypoxia-reoxygenation (IRI) cell model was established. The expression level of circSNRK after IRI treatment and the effect of overexpression of circSNRK on cell proliferation and apoptosis were detected. The targets of circSNRK were identified. HK2 cells were divided into the blank group (Mock group), IRI group, control plasmid+IRI group (IRI+NC group), human circSNRK overexpression+IRI group (IRI+circSNRK group), human circSNRK overexpression+IRI+protein kinase B (Akt) inhibitor group (IRI+circSNRK+MK2206 group) and control plasmid group (NC group). Cell proliferation and apoptosis were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the target of circSNRK were carried out. The expression levels of CDKN1A, Akt, B-cell lymphoma (Bcl)-2, cysteinyl aspartate specific proteinase (Caspase)-9 messenger RNA (mRNA), and those of p21, Bcl-2, Caspase-9, Akt and p-Akt proteins were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups, respectively. Cell proliferation and apoptosis were determined in the NC, IRI+NC, IRI+circSNRK and IRI+circSNRK+MK2206 groups. Results Compared with the Mock group, the expression level of circSNRK was lower, and cell proliferation capability of HK2 cells was decreased and cell apoptosis was increased in the IRI group. In the IRI+circSNRK group, cell proliferation capability was higher, whereas cell apoptosis was lower than those in the IRI+NC group. circSNRK could act on 648 targets through 51 microRNAs (miRNAs). GO enrichment analysis revealed that the targets of circSNRK were mainly enriched in biological processes (such as cell process and biological regulation), cell components (such as cell parts, cells and extracellular parts), and molecular functions (such as binding, binding proteins and enzymes). KEGG enrichment analysis showed that the targets of circSNRK were mainly enriched in cancer signaling pathway, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, miRNA in cancer and other related signaling pathways. Compared with the Mock group, the relative expression levels of CDKN1A and Caspase-9 mRNA were higher, the expression level of miR-99a-5p RNA was higher and the relative expression levels of Akt and Bcl-2 mRNA were lower in the IRI group. Compared with the IRI+NC group, the relative expression levels of CDKN1A and Caspase-9 mRNA were lower, those of Akt and Bcl-2 mRNA were higher, and the expression level of miR-99a-5p RNA was lower in the IRI+circSNRK group, and the differences were statistically significant (all P < 0.05). Compared with the Mock group, the expression levels of p21 and Caspase-9 proteins were higher, while those of p-Akt, Akt and Bcl-2 proteins were lower in the IRI group. Compared with the IRI+NC group, the expression levels of p21 and Caspase-9 proteins were lower, whereas those of p-Akt, Akt and Bcl-2 proteins were higher in the IRI+circSNRK group. The miR-99a-5p binding sites were observed in circSNRK and Akt. Compared with the NC group, cell proliferation capability was declined in the IRI+NC group. Compared with the IRI+NC group, cell proliferation capability was elevated in the IRI+circSNRK group. Compared with the IRI+circSNRK group, cell proliferation capability was declined in the IRI+circSNRK+MK2206 group (all P < 0.05). The cell apoptosis level in the IRI+NC group was higher than that in the NC group. The cell apoptosis level in the IRI+circSNRK group was lower compared with that in the IRI+NC group. The cell apoptosis level in the IRI+circSNRK+MK2206 group was higher than that in the IRI+circSNRK group. Conclusions Under IRI conditions, circSNRK may affect the proliferation and apoptosis of HK2 cells probably via the Akt signaling pathway. -
Key words:
- Kidney transplantation /
- Ischemia-reperfusion injury /
- Circular RNA /
- Micro RNA /
- Protein kinase B /
- Cell apoptosis /
- Cell proliferation /
- Target
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图 1 人circSNRK来源、鉴定以及同源性分析
注：A图为人源circSNRK以及sanger测序验证环化拼接位点；B图为人和大鼠circSNRK序列Blast比对。
Figure 1. Sources, identification and homology analysis of human circSNRK
图 2 IRI对HK2细胞circSNRK表达水平的影响
注：A为Mock组和IRI组的circSNRK表达水平，与Mock组比较，aP < 0.05；B图为酶切验证circSNRK质粒构建；C图为过表达circSNRK效率验证，与NC组比较，aP < 0.05。
Figure 2. The effect of IRI on the circSNRK expression level of HK2 cells
图 3 过表达circSNRK促进细胞增殖和抑制细胞凋亡
注：A图为各组HK2细胞增殖情况，与Mock组比较，aP < 0.05，与IRI+NC组比较，bP < 0.05；B为各组HK2细胞凋亡情况。
Figure 3. Overexpression of circSNRK promotes cell proliferation and inhibits apoptosis
图 4 circSNRK作用靶点的GO富集和KEGG聚类分析
注：A图为GO富集分析结果；B图为KEGG聚类分析结果。
Figure 4. GO enrichment and KEGG clustering of circSNRK targets
图 5 circSNRK对Akt通路的影响
注：A图各组CDKN1A、Akt、Bcl-2、Caspase-9 mRNA相对表达量，与Mock组比较，aP < 0.05，与IRI+NC组比较，bP < 0.05；B图为各组p21、Bcl-2、Caspase-9、p-Akt、Akt蛋白相对表达量；C图为各组miR-99a-5p和miR-100-5p RNA相对表达量，与Mock组比较，aP < 0.05，与IRI+NC组比较，bP < 0.05；D图为miR-99a-5p和circSNRK以及Akt的相互作用位点。
Figure 5. Effect of circSNRK on Akt pathway
图 6 circSNRK通过Akt通路影响HK2细胞增殖和凋亡
注：A图为各组细胞增殖情况，与NC组比较，aP < 0.05，与IRI+NC组比较，bP < 0.05，与IRI+circSNRK组比较，cP < 0.05；B图为各组细胞凋亡情况。
Figure 6. circSNRK affects HK2 cell proliferation and apoptosis via the Akt pathway
表 1 引物序列
Table 1. Primer sequences
基因 序列（5’→3’） circSNRK 上游TGATGCACCTGCAGTAGGAAA 下游GGCCTCGACCCAAGGTTTTA CDKN1A 上游ACTCTCAGGGTCGAAAACGG 下游CAAGAAAGTTGGGTAGGGCTTC Akt 上游GTCATCGAACGCACCTTCCAT 下游AGCTTCAGGTACTCAAACTCGT Bcl-2 上游TCATGTGTGTGGAGAGCGTC 下游CAGCCCAGACTCACATCACC Caspase-9 上游CACGGCTTTGATGGAGATGG 下游TCTCTCGATGGACACAGAGC GAPDH 上游GAGTCAACGGATTTGGTCGT 下游GACAAGCTTCCCGTTCTCAG U6 上游CTCGCTTCGGCAGCACATA 下游CGAATTTGCGTGTCATCCTTG miR-99a-5p 上游AACCCGTAGATCCGATCTT 上游AACCCGTAGATCCGAAC miRNA Universe 通用下游GTGCAGGGTCCGAGGT 
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