Alu RNA accumulation induces epithelial-to-mesenchymal transition by modulating miR-566 and is associated with cancer progression

Abstract

Alu sequences are the most abundant short interspersed repeated elements in the human genome. Here we show that in a cell culture model of colorectal cancer (CRC) progression, we observe accumulation of Alu RNA that is associated with reduced DICER1 levels. Alu RNA induces epithelial-to-mesenchymal transition (EMT) by acting as a molecular sponge of miR-566. Moreover, Alu RNA accumulates as consequence of DICER1 deficit in colorectal, ovarian, renal and breast cancer cell lines. Interestingly, Alu RNA knockdown prevents DICER1 depletion-induced EMT despite global microRNA (miRNA) downregulation. Alu RNA expression is also induced by transforming growth factor-β1, a major driver of EMT. Corroborating this data, we found that non-coding Alu RNA significantly correlates with tumor progression in human CRC patients. Together, these findings reveal an unexpected DICER1-dependent, miRNA-independent role of Alu RNA in cancer progression that could bring mobile element transcripts in the fields of cancer therapeutic and prognosis.

Figure 1

Alu RNA levels in CRC cells lines. (a) Northern blot analysis shows Alu RNA abundance in SW480 and SW620 cells. Densitometric values normalized against U6 small nucleolar RNA are shown in parentheses. (b) Western blot analysis shows DICER1 protein abundance in SW480 and SW620 cells. Densitometric values normalized against β-tubulin are shown in parentheses. (c) Levels of DICER1 mRNA and Alu transcripts in SW480 and SW620 as evaluated by quantitative reverse transcriptase (qRT–PCR). Transfection of DICER1 antisense oligonucleotide (DICER1 AS) downregulates DICER1 protein (d) and mRNA, and increases the abundance of Alu RNA transcripts (e) in SW480 cells as compared with control antisense oligonucleotide (Ctrl AS). Alu RNA overexpression (f) and DICER1 reduction (g) do not alter SW480 cell viability. (h) Alu RNA induces a reorganization of the cytoskeleton. Representative images of Alu RNA- (right) or vehicle- (Mock, left) transfected SW480 cells stained for F-actin with rhodamine-phalloidin (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 25 μm. For all panels: n=3; *P<0.05. Error bars denote s.e.m.

Figure 2

Alu RNA induces EMT. (a) Alu RNA reduces E-cadherin and increase N-cadherin, Vimentin and Fibronectin mRNAs as evaluated by quantitative reverse transcriptase (qRT–PCR). (b) Representative images of Alu RNA- (right) or vehicle- (Mock, left) transfected SW480 cells stained for F-actin (red), E-cadherin (green) and Vimentin (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 50 μm. (c) Alu RNA increases the level of the mesenchymal markers (Fibronectin and Vimentin) and of the EMT-TFs TWIST1 and ZEB1, and reduces the epithelial marker E-cadherin, as evaluated by western blot. Densitometric values normalized against β-Tubulin are shown in parentheses. (d and e) Alu RNA increases SW480 cell migration and invasion. Data are expressed as fold of induction with respect to vehicle-transfected cells (Mock). (f) Alu RNA increases SW480 ability to grow in anchorage-dependent manner. The bars indicate the average of colony numbers in each well. Representative pictures of two conditions are shown. For all panels: n=3; *P<0.05. Error bars denote s.e.m.

Figure 3

Antisense oligonucleotide (Alu AS) targeting Alu RNA sequences rescues the DICER1 deficit-induced EMT. (a) Alu AS reduces Alu RNA accumulation induced by DICER1 deficit in SW480 cells as evaluated by quantitative reverse transcriptase (qRT–PCR). (b) Evaluation of DICER1, Vimentin, Fibronectin, E-cadherin, TWIST1 and ZEB1 protein levels by western blot analysis. Densitometric values normalized against β-Tubulin are shown in parentheses. (c) Representative images of F-actin (red), E-cadherin (green) and Vimentin (red) staining. Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 50 μm. (d and e) Alu AS inhibits DICER1-deficit induced SW480 cell migration and invasion. Data are expressed as fold of induction with respect to Ctrl AS. (f) DICER1 AS induced global miRNA expression deficits in SW480 cells compared with Ctrl AS. No significant difference in miRNA abundance between Alu AS and Ctrl AS-treated DICER1-depleted cells as evaluated by qRT–PCR. For all panels: n=3; *P<0.05. Error bars denote s.e.m.

Figure 4

Alu RNA acts as a molecular decoy for miR-566 inducing EMT. (a) Alu RNA transfection in SW480 cells reduces miR-566 level as evaluated by quantitative reverse transcriptase (qRT–PCR). (b) SW620 cells show less miR-566 level compared with SW480 cells, as evaluated by qRT–PCR. (c) The activity of Renilla luciferase (RLuc) was downregulated by miR-566 and not by NT miRNA. SW480 cells were co-transfected with psiCHECK2 and/or psiCHECK2/Alu, and miR-566 and/or NT miRNA. The bars indicate Firefly luciferase (FLuc) activities normalized against Renilla. Each experiment was repeated at least three times and each sample was assayed in triplicate. (d) miR-566 prevents the Alu-induced EMT, as shown by Fibronectin, E-cadherin, TWIST1 and ZEB1 levels evaluated by western blot. Densitometric values normalized against β-Tubulin are shown in parentheses. (e) Representative images of F-actin (red) and E-cadherin (green) staining. Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 75 μm.

Figure 5

TGF-β1 stimulation induces Alu RNA accumulation. (a) The stimulation of SW480 cells with 5 ng/ml TGF-β1 induces EMT. Representative images (upper) of TGF-β1 (right) or not- (NT, left) treated SW480 cells stained for F-actin (red) and E-cadherin (green). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 75 μm. Western blot (lower) shows abundance of Fibronectin, E-cadherin, TWIST1 and ZEB1. Densitometric values normalized against β-Tubulin are shown in parentheses. (b) TGF-β1 increases Alu RNA accumulation as evaluated by northern blot (upper) and quantitative reverse transcriptase (qRT–PCR) (lower). (c) TGF-β1 decreases miR-566 in SW480 cells, as evaluated by qRT–PCR. (d and e) miR-566 and Alu AS rescues the TGF-β1-induced EMT. Western blots show abundance of Fibronectin, E-cadherin, TWIST1 and ZEB1. Densitometric values normalized against β-Tubulin are shown in parentheses. For all panels: n=3; *P<0.05. Error bars denote s.e.m.

Figure 6

Alu RNA accumulation in CRC patients. (a) Alu RNA expression in non-tumoral tissues (NT), primary colon tumor (T) and liver metastasis (M) of 13 matched patients, as evaluated by quantitative reverse transcriptase (qRT–PCR) and normalized them to 18S mRNA. *P=0.05; **P=0.049; ***P=0.001. (b) Northern blot analysis shows Alu RNA abundance in six patients. U6 small nucleolar RNA was used as loading control.