Identification of miRNAs that specifically target tumor suppressive KLF6-FL rather than oncogenic KLF6-SV1 isoform

Abstract

The Krüppel like factor 6 (KLF6) gene encodes multiple protein isoforms derived from alternative mRNA splicing, most of which are intimately involved in hepatocarcinogenesis and tumor progression. Recent bioinformatics analysis shows that alternative mRNA splicing of the KLF6 gene produces around 16 alternatively spliced variants with divergent or even opposing functions. Intriguingly, the full-length KLF6 (KLF6-FL) is a tumor suppressor gene frequently inactivated in liver cancer, whereas KLF6 splice variant 1 (KLF6-SV1) is an oncogenic isoform with antagonistic function against KLF6-FL. Compelling evidence indicates that miRNA, the small endogenous non-coding RNA (ncRNA), acts as a vital player in modulating a variety of cellular biological processes through targeting different mRNA regions of protein-coding genes. To identify the potential miRNAs specifically targeting KLF6-FL, we utilized bioinformatics analysis in combination with the luciferase reporter assays and screened out two miRNAs, namely miR-210 and miR-1301, specifically targeted the tumor suppressive KLF6-FL rather than the oncogenic KLF6-SV1. Our in vitro experiments demonstrated that stable expression of KLF6-FL inhibited cell proliferation, migration and angiogenesis while overexpression of miR-1301 promoted cell migration and angiogenesis. Further experiments demonstrated that miR-1301 was highly expressed in liver cancer cell lines as well as clinical specimens and we also identified the potential methylation and histone acetylation for miR-1301 gene. To sum up, our findings unveiled a novel molecular mechanism that specific miRNAs promoted tumorigenesis by targeting the tumor suppressive isoform KLF6-FL rather than its oncogenic isoform KLF6-SV1.

Keywords: Alternative splicing; KLF6; miRNA.

Figure 1. Ectopic KLF6 expression impaired in vitro growth of liver cancer cells. (A) Equal numbers of KLF6 and vector-transfected stable cells were seeded into 96 well plates and cell growth was quantified by XTT assays for 4 d. Reduced cell proliferation was observed in KLF6 stable transfectants. *P < 0.05, **P < 0.01. (B) KLF6 stable transfectants and respective parental cells were plated in 6 well plate and colonies were stained by crystal violet after 10 d. Impaired colony formation ability was found in KLF6 stable transfectants. **P < 0.01. (C&D) After serum starvation treatment, KLF6 stable transfectants and respective parental cells were harvested and subjected to cell cycle analysis. Repression on G1/S phase transition was observed in PLC/PRF/5-KLF6 and HepG2-KLF6 cells. ***P < 0.001. (E) To monitor anchorage-independent growth of liver cancer cells, low intensity of KLF6 stable transfectants and respective parental cells were seeded into soft agar and visualized by crystal violet staining. The representative PLC/PRF/5-KLF6 and HepG2-KLF6 cells formed significantly less colonies in soft agar, as compared with parental cells. **P < 0.01 ***P < 0.001.

Figure 2. Ectopic KLF6 expression inhibited tube formation and cell motility. (A) Equal numbers of KLF6 and vector-transfected HUVEC cells were seeded into 96 well plates and the ability of endothelial cell tube formation was quantified by measuring the length of the capillary-like structures by Image J. Impaired tube formation was observed in HUVEC cells after overexpression of KLF6. *P < 0.05. (B&C) Quantitative RT-PCR was used to determine the expression profiles of angiogenesis marker genes. Reduced expression of angiogenesis marker genes was observed in PLC/PRF/5-KLF6 (B) and HepG2-KLF6 (C) cells. *P < 0.05, **P < 0.01, ***P < 0.001. (D&E) Cell motility was assessed by wound healing assays. KLF6 stable transfectants migrated slower toward the central as compared with the parental cells. *P < 0.05, **P < 0.01.

Figure 3. A screening assay identified potential miRNAs that target KLF6-FL rather than its spliced variant KLF6-SV1. (A) Schematic diagrams of the full-length KLF6-FL (4 679 bp) and spliced variant KLF6-SV1 (4 525 bp). KLF6-SV1 was generated by alternative 5′ splice sites and it lacked 154bp in the terminal of exon 2 when compared with KLF6-FL. (B) Bioinformatics prediction screened out some potential miRNAs that specifically targeted the 154bp region. (C) HEK293 cells were co-transfected with miRNAs listed above and pmirGLO Dual-luciferase carrying the specific 154bp region. Luciferase reporter assays showed that miR-210 and miR-1301 profoundly repressed the luciferase reporter activity. *P < 0.05, **P < 0.01.

Figure 4. MiR-210 and miR-1301 regulated KLF6-FL protein expression through interaction with its protein coding region. (A) Schematic diagrams of miRNA binding sites on KLF6-FL. Two miR-210 and miR-1301 binding sites in the protein-coding region of KLF6-FL were highly conserved across different species. Upper panel showed sequence alignment of the miR-210 binding sites across seven different species. Lower panel presented sequence alignment of the miR-1301 binding sites across seven different species. (B) RT-PCR analysis showed the relative expression of KLF6-FL after ectopic expression of miR-675, miR-210 or miR-1301. No significant change in mRNA level was observed after transfection with these miRNAs. (C) Immunoblotting of KLF6 in HepG2 cells transfected with miRNA mimics or negative control. MiR-210 and miR-1301 significantly suppressed KLF6-FL expression in protein level. (D) HEK293 cells were co-transfected with miRNA mimics together with Flag-KLF6-FL. Cell extracts were subjected to immunoblotting analysis to detect Flag tag. Western blot showed that both miR-210 and miR-1301 significantly attenuated the protein expression of KLF6-FL with Flag tag. (E) HEK293 cells were co-transfected with miRNA mimics combined with plasmid harboring the coding region of KLF6-FL. Luciferase reporter assays presented the repression of miR-210 and miR-1301 on the activity of luciferase reporter harboring KLF6-FL in HEK-293 cells. The Firefly luciferase activities were normalized to β-Galactosidase activity. *P < 0.05, **P < 0.01. (F) Site-directed mutagenesis of the binding sites for miR-210 and miR-1301 abolished the suppressive effect. (G) Immunoblotting of KLF6 in HepG2 cells transfected with miRNA mimics or negative control. Ectopic expression of miR-210 and miR-1301 did not affect KLF6-SV1 protein expression. (H) HEK293 cells were co-transfected with miRNA mimics combined with plasmid harboring coding region of KLF6-SV1. No significant change on luciferase activity was presented after ectopic expression of miR-210 and miR-1301.

Figure 5. Upregulation of miR-1301 was observed in human hepatoma cell lines. (A) Quantitative RT–PCR analysis of mature miR-1301 in serial human hepatoma cell lines. Elevated miR-1301 expression was observed in part of human hepatoma cell lines. **P < 0.01, ***P < 0.001. (B to F) Representative RT–PCR results showed relative miR-1301 expression levels in several liver cancer cell lines after treatments with DNMTs inhibitor 5-Azacytidine and HDACs inhibitor Trichostatin A. 5-Azacytidine and Trichostatin A decreased the miR-1301 expression in part of liver cancer cells. MiR-1301 expression level was normalized to housekeeping gene U6 and calculated using 2^−ΔΔCt. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Re-expression of miR-1301 or silence of KLF6 promoted tube formation and cell migration. (A&B) Equal numbers of miR-1301 or siKLF6-transfected HUVEC cells were seeded into 96 well plates and the tube formation capacity is quantified by measuring the length of the capillary-like structures. Enhanced tube formation capacity was observed in HUVEC cells with overexpression of miR-1301 (A) or silence of KLF6 (B). **P < 0.01, ***P < 0.001. (C&D) Cell migration was assessed by wound healing assays. Overexpression of miR-1301 or silence of KLF6 promoted liver cancer cell motility. *P < 0.05, **P < 0.01.

Figure 7. Expression analysis of miR-1301 and KLF6 isoforms in paired HCC and adjacent normal tissues by RT-PCR. (A) Elevated expression of miR-1301 was observed in 35 paired HCC specimens. (B&C) Downregulation of KLF6-FL (B) and upregulation of KLF6-SV1 (C) were displayed in in HCC specimens.