Livin is involved in TGF-β1-induced renal tubular epithelial-mesenchymal transition through lncRNA-ATB

Abstract

Background: Renal interstitial fibrosis is accepted as a crucial component of chronic kidney diseases (CKD). Epithelial-mesenchymal transition (EMT) is an important factor contributing to renal interstitial fibrosis. Livin, due to its ability to induce EMT, is an important regulator of many types of tumors and might also be involved in human renal tubular EMT.

Methods: We confirmed that Livin and lncRNA-ATB could aggravate EMT in vivo and in vitro, lncRNA-ATB could be suppressed by the silencing of Livin whereas Livin expression was nearly stable when lncRNA-ATB was overexpressed or knocked out.

Results: Livin was upregulated in vivo and in vitro at the similar rate as the occurrence of EMT, which could be relieved when Livin was silenced. LncRNA-ATB, which is another important regulator of EMT, was also found highly expressed during this process. The silencing of lncRNA-ATB could lessen the severity of EMT, and the overexpression of lncRNA-ATB could aggravate EMT without affecting the expression of Livin.

Conclusions: Livin promotes EMT through the regulation of lncRNA-ATB. The silencing of Livin might be an effective targeted therapy for renal fibrosis.

Keywords: Livin; epithelial-mesenchymal transition (EMT); lncRNA activated by TGF-β (lncRNA-ATB); renal fibrosis; transforming growth factor beta 1 (TGF-β1).

Figure 1

Livin highly expressed in UUO model. (A) Immunohistochemistry analysis of E-cadherin, α-SMA, Vimentin and Livin expression, magnification 200×. Compared with sham-operated group, E-cadherin expression was decreased in renal epithelial cells membranes, α-SMA, Vimentin and Livin were significantly highly expressed in cytoplasm and part of nucleus in UUO group. (B) qPCR showed that the relative expression of E-cadherin was downregulated, α-SMA, Vimentin and Livin were upregulated in UUO group. *P<0.05, **P<0.01, compared to sham group. UUO, unilateral ureteral obstruction.

Figure 2

EMT was induced by TGF-β1 in HK2 cells. (A) HK-2 cells were treated with 2 ng/mL TGF-β1 for 48 h. The morphological change was observed with inverted microscope (10×); (B) Western blot analysis showed that the expression of E-cadherin was decreased and α-SMA and Vimentin were increased; (C) qPCR showed parallel changes with WB results. *P<0.05, **P<0.01, compared to control group; (D) immunofluorescence staining showed E-cadherin was downregulated, α-SMA and Vimentin were upregulated in 2 ng/mL groups; (E) transwell assay showed that migration ability of HK2 cells enhanced significantly in 2 ng/mL group, cell counting result was statistically significant, **P<0.01 compared to control group. EMT, epithelial-mesenchymal transition; TGF-β1, transforming growth factor beta 1.

Figure 3

Livin was involved in TGF-β1 induced EMT. (A) WB showed that Livin expression was increased after HK2 cells were treated by TGF-β1 especially in 2 ng/mL group; (B) immunofluorescence indicated that Livin was highly expressed in cytoplasm and nucleus; (C) qPCR showed that the relative mRNA expression of Livin was highest in 2 ng/mL group, *P<0.05 compared to 0 ng/mL group. EMT, epithelial-mesenchymal transition; TGF-β1, transforming growth factor beta 1.

Figure 4

Silence of Livin could alleviate TGF-β1 induced EMT. (A) qPCR showed siRNAs were transfected and si-Livin1 was the most effective; (B) WB verified that si-Livin could inhibit Livin expression; (C) compared with TGF-β1 groups, E-cadherin expression elevated, α-SMA and Vimentin were reduced in si-Livin + TGF-β1 groups; (D) qPCR showed parallel changes with WB results. *P<0.05, **P<0.01; (E) immunofluorescence staining showed E-cadherin was upregulated, α-SMA and Vimentin were downregulated in si-Livin + TGF-β1 groups; (F) transwell assay showed that migration ability of HK2 cells significantly depressed in si-Livin + TGF-β1 group, cell counting result was statistically significant, **P<0.01. EMT, epithelial-mesenchymal transition; TGF-β1, transforming growth factor beta 1.

Figure 5

Livin regulated TGF-β1 induced EMT by lncRNA-ATB. (A) lncRNA-ATB was increased in TGF-β1 group; (B) lncRNA-ATB was increased in UUO group; ***P<0.001; (C,D) qPCR verified that lncRNA-ATB was successfully overexpressed or knockout. **P<0.01; (E) compared to the control groups, E-cadherin was significantly decreased in ATB-OE group and α-SMA and Vimentin were increased. In si-ATB+TGF-β1 group, the expression of E-cadherin, α-SMA and Vimentin were close to the control group; (F) qPCR showed parallel changes with WB results. **P<0.01; (G) The expression of lncRNA-ATB was decreased as Livin knockout; (H) the expression of Livin were not affected in ATB-OE group or si-ATB group; (I) qPCR showed that mRNA expression of Livin were not significant different. EMT, epithelial-mesenchymal transition; TGF-β1, transforming growth factor beta 1.