LncRNA ARAP1‐AS2 promotes high glucose‐induced human proximal tubular cell injury via persistent transactivation of the EGFR by interacting with ARAP1

Figures

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  Figure 1. The expression and distribution of ARAP1‐AS2 in HK‐2 cells. A, qRT‐PCR analysis of the expression of ARAP1‐AS2 in HK‐2 cells of the normal‐glucose (NG) group and high‐glucose (HG) group (n = 3); the data are representative of three independent experiments. Data are presented as the mean ± SD. *P < .05, **P < .01, NS, no significant difference. B, Localization of ARAP1‐AS2 (red) in HK‐2 cells that were stimulated by high or normal glucose determined by FISH. ARAP1‐AS2 was mainly distributed in the cell nuclei (×400). Bar = 50 μmol/L. All probes were labelled with Cy3. HK‐2 cells in the control group did not exhibit self‐illumination. C, Coding Potential Assessment Tool (CPAT) was used to test the protein‐coding ability of ARAP1‐AS2. D, ORF finder was used to analyse the protein‐coding ability of ARAP1‐AS2. E, 5′ and 3′ rapid amplification of cDNA ends (RACE) assays in HK‐2 cells were used to detect the whole sequence of ARAP1‐AS2. Left: an image of PCR products from the 5′‐RACE and 3′‐RACE assays separated by gel electrophoresis. Right: sequencing of PCR products indicated the boundary between the universal anchor primer and ARAP1‐AS2 sequences

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  Figure 2. ARAP1‐AS2 regulated fibrosis and HK‐2 cell proliferation. A, The protein expression levels of collagen I, collagen IV and fibronectin (FN) in HK‐2 cells of the normal‐glucose (NG) group and high‐glucose (HG) group (n = 3) were detected by Western blot analysis. B, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid (3000 ng) in the NG group or ARAP1‐AS2 siRNAs (50 nmol/L) in the HG group in 6‐well plates, ARAP1‐AS2 overexpression and knockdown efficiencies were assessed by qRT‐PCR. C, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the protein expression levels of collagen I, collagen IV and FN were detected by Western blot analysis. D, After transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, cell proliferation was detected by CCK‐8 analysis at 24, 48 and 72 h. In all panels, the data are representative of three independent experiments. Data are presented as the mean ± SD. *P < .05, **P < .01, NS, not significant

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  Figure 3. ARAP1‐AS2 interacts with ARAP1. A, The 12th exon of ARAP1 (which is 183 bp in length) overlaps the second exon of ARAP1‐AS2. B, RNA pulldown assays were performed to detect whether ARAP1‐AS2 could bind ARAP1 in HK‐2 cells. ARAP1 was pulled down by ARAP1‐AS2, and the level of ARAP1 was detected by qRT‐PCR. C, The mRNA expression levels of ARAP1 in HK‐2 cells of the normal‐glucose (NG) group and high‐glucose (HG) group (n = 3) were detected by qRT‐PCR. D, The protein expression levels of ARAP1 in HK‐2 cells of the NG group and HG group (n = 3) were detected by Western blot analysis. E, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the mRNA expression level of ARAP1 was detected by qRT‐PCR. F, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the protein expression level of ARAP1 was detected by Western blot analysis. In all panels, the data are representative of three independent experiments. Data are presented as the mean ± SD. *P < .05, **P < .01, NS, not significant

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  Figure 4. ARAP1 interacts with CIN85 and regulates the ubiquitination of EGFR. A, Bioinformatic analysis of protein‐protein interaction networks by STRING v.10. A screenshot from STRING shows a network associated with ARAP1 and CIN85 and EGFR. B, The physical interaction between ARAP1 and CIN85 was detected by coimmunoprecipitation analysis. IgG was used as a negative control. C, RNA pulldown‐seq of ARAP1‐AS2. Fold change value = Log2((FPKM in Positive + 1)/(FPKM in Negative + 1)). A higher fold change value indicates more enrichment. D, Forty‐eight hours after transfection of ARAP1 shRNA (3000 ng) in the HG group in 6‐well plates, the ARAP1 knockdown efficiency was detected by Western blot analysis. E, Forty‐eight hours after transfection of ARAP1 shRNA (3000 ng) in the HG group in 6‐well plates, ARAP1 knockdown efficiency was assessed by qRT‐PCR. F, ARAP1 knockdown increased EGFR ubiquitination in the NG group and HG group. In all panels, the data are representative of three independent experiments. Data are presented as the mean ± SD. *P < .05, **P < .01, NS, not significant

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  Figure 5. Dual immunofluorescent staining for ARAP1 and CIN85. A, Dual immunofluorescent staining for ARAP1 and CIN85 in HK‐2 cells of the normal‐glucose (NG) group and high‐glucose (HG) group (×400). Bar = 50 μmol/L. B, Dual immunofluorescent staining for ARAP1 and CIN85 in the renal tissues of healthy control and DN patients (×400). Bar = 50 μmol/L. C, Dual immunofluorescent staining for ARAP1 and CIN85 in the renal tissues of normal control and db/db mice (×400). Bar = 50 μmol/L

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  Figure 6. ARAP1 regulates activation of the EGFR/TGF‐β/Smad3 pathway in HK‐2 cells. A, The protein expression levels of ARAP1, total EGFR, EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173), TGF‐β1, p‐Smad3, collagen I, collagen IV and fibronectin (FN) in the normal‐glucose (NG) group, high‐glucose (HG) group and HG group administered the EGFR tyrosine kinase inhibitor AG1478 were detected by Western blot analysis. B, After the addition of ag1478 to the hg group, cell proliferation was detected by cck‐8 assay at 24, 48 and 72 h. C, Hk‐2 cells were pre‐treated with arap1 shrna for 24 h and then divided into the ng group and hg group. The protein expression levels of ARAP1, total EGFR, and EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173) were detected at 12, 24, 36 and 48 h after stimulation by high glucose. 0 h means that the HK‐2 cells were only transfected with ARAP1 shRNA for 24 h under normal‐glucose and were not stimulated with high glucose. High‐glucose stimulation was carried out for 12‐48 h. D, Forty‐eight hours after transfection of ARAP1 shRNA in the NG group and HG group, the protein expression of TGF‐β1, p‐Smad3, collagen I, collagen IV and FN…

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  Figure 7. ARAP1‐AS2 promotes the expression of fibrosis and cell proliferation via the EGFR/TGF‐β/Smad3 pathway. A, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the normal‐glucose (NG) group and high‐glucose (HG) group, the protein expression levels of total EGFR, EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173), TGF‐β1 and p‐Smad3 were detected by Western blot analysis. B, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the mRNA expression level of total EGFR was detected by qRT‐PCR. C, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the mRNA expression level of TGF‐β1 was detected by qRT‐PCR. D, Forty‐eight hours after transfection of the ARAP1‐AS2 overexpression plasmid or ARAP1‐AS2 siRNA in the NG group and HG group, the mRNA expression level of Smad3 was detected by qRT‐PCR. E, Forty‐eight hours after cotransfection of the ARAP1‐AS2 overexpression plasmid and ARAP1 shRNA or transfection of the ARAP1‐AS2 overexpression plasmid accompanied by treatment with the…

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  Figure 8. Schematic representation of the proposed model: possible mechanism by which ARAP1‐AS2 is involved in high glucose‐induced proximal tubular cell injury and fibrosis via the EGFR/TGF‐β/Smad3 pathway. Increased ARAP1‐AS2 expression in a high‐glucose environment can up‐regulate ARAP1 expression. Subsequently, ARAP1 competes with Cbl for CIN85 binding in early endosomes and reduces the ubiquitination of EGFR, thus providing more total EGFR proteins to maintain the persistent activation of EGFR in diabetic nephropathy. The persistent activation of EGFR then maintains persistent activation of the TGF‐β/Smad3 pathway and aggravates proximal tubular cell injury and fibrosis in diabetic nephropathy