Int J Stem Cells.  2019 Jul;12(2):347-359. 10.15283/ijsc19041.

Long Non-Coding RNA LINC00525 Promotes the Stemness and Chemoresistance of Colorectal Cancer by Targeting miR-507/ELK3 Axis

Affiliations
  • 1Department of Colorectal Surgery, Yidu Central Hospital of Weifang City, Qingzhou, China. wangshunsheng728@126.com

Abstract

BACKGROUND AND OBJECTIVES
This study aims to explore the effects of a long non-coding RNA, LINC00525, on colorectal cancer (CRC) and its underlying molecular mechanisms.
METHODS
The qPCR, MTT, colony formation, Western blotting, Luciferase reporter and biotin pull-down, shRNA knockdown and DNA fragmentation assays were performed in this study.
RESULTS
High expressions of LINC00525 were associated with poor prognosis of CRC patients. LINC00525 knockdown decreased stemness properties and increased sensitivities to oxaliplatin. MiR-507 was a direct target of LINC00525 and overexpression of miR-507 significantly decreased abilities of tumorsphere formation and cell growth. Overexpression of miR-507 resulted in a decrease of expression of cancer stem cell markers and the increase of apoptosis rates. MiR-507 regulated the expression of ELK3. In addition, LINC00525 knockdown decreased the expression of ELK3. Restoration of ELK3 expression abrogated the effects of LINC00525 knockdown. LINC00525 could be served as prognostic marker of CRC.
CONCLUSIONS
LINC00525 enhanced stemness properties and increased sensitivities of CRC cells to oxaliplatin by targeting miR-507/ELK3 axis.

Keyword

Long non-coding RNA; LINC00525; Colorectal cancer; miR-507; ELK3

MeSH Terms

Apoptosis
Biotin
Blotting, Western
Colorectal Neoplasms*
DNA Fragmentation
Humans
Luciferases
Neoplastic Stem Cells
Prognosis
RNA, Long Noncoding*
RNA, Small Interfering
Biotin
Luciferases
RNA, Long Noncoding
RNA, Small Interfering

Figure

  • Fig. 1 LINC00525 was highly expressed in CRC and increased LINC00525 expression predicted poor prognosis in CRC patients. (a) The expression level of LINC00525 was analyzed in CRC tissues compared with normal tissues from GSE21510. (b) qRT-PCR analysis of LINC00525 expression in CRC tissues (n=60) and normal human intestinal tissues (n=60) (shown as log10 (2−ΔΔCT)). (c) The LINC00525 expression level was quantified by qRT-PCR analysis in CRC tissues of oxaliplatin sensitive patients (OxS) and oxaliplatin resistant patients (OxR). (d) qRT-PCR analysis of the relative expressions of LINC00525 in five CRC cell lines (Caco2, SW480, SW620, HCT116, and HT29) and a normal human intestinal epithelial cell line (HIEC). (e) qRT-PCR analysis of the relative expressions of LINC00525 in sphere-forming HCT116 and HT29 compared to parental HCT116 and HT29 cells. (f) Kaplan–Meier’s analysis of the correlation between LINC00525 expression and the overall survival of CRC patients. *p<0.05, **p<0.01, ***p<0.001.

  • Fig. 2 Silencing of LINC00525 inhibited the stemness and oxaliplatin resistance of colorectal cancer. (a) The relative expression levels of LINC00525 in HCT116 and HT29 cells transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR) were detected by qRT-PCR. (b) Knockdown of LINC00525 in HCT116 and HT29 cells significantly decreased the capacity to form tumorspheres. (c~e) The levels of CD44, SOX2 and OCT4 were determined by qRT-PCR and western blot in HCT116 and HT29 cells transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR). (f) The relative expression levels of LINC00525 in oxaliplatin resistant HCT116 (HCT116-OxR) and HT29 (HT29-OxR) cells transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR) were detected by qRT-PCR. (g~i) Knockdown of LINC00525 significantly inhibited CRC-OxR cells growth under oxaliplatin treatment. Cell viabilities were determined by MTT and colony formation assays. (j) Inhibition of LINC00525 significantly promoted CRC-OxR cell apoptosis. Cell apoptosis was determined by DNA fragmentation assay. (k) The apoptotic markers p53, Caspase-3, Bcl-2 in oxaliplatin resistant HCT116 (HCT116-OxR) and HT29 (HT29-OxR) cells transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR) were detected by western blotting. The data represent as mean±SD from three independent experiments. *p<0.05, **p<0.01.

  • Fig. 3 MiR-507 was a direct target of LINC00525. (a) The prediction for miR-507 binding sites on LINC00525 transcript and schematic of luciferase reporter vector constructs LINC00525 wild-type (LINC00525 wt) and the miR-507-binding-site mutated (LINC00525 mt) one. (b, c) The luciferase activities in HCT116 and HT29 cells co-transfected with miR-507 or miR-NC mimics and luciferase reporters containing LINC00525 wt or LINC00525 mt. Data are presented as the relative ratio of hRluc luciferase activity to hluc+ luciferase activity. (d) The relative expression levels of miR-507 in HCT116 and HT29 cells transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR) were detected by qRT-PCR. (e) The relative expression levels of miR-507 in HCT116 and HT29 cells transfected with LINC00525 plasmid (pSin-LINC00525) or empty vector (pSin-vec) were detected by qRT-PCR. (f, g) Lysates from HCT116 and HT29 cells were incubated with in vitro-synthesized biotin-labeled sense or antisense DNA probes against LINC00525 for biotin pull-down assay, followed by qRT-PCR analysis. (h) The relative expression levels of LINC00525 in HCT116 and HT29 cells transfected with miR-507 mimics (miR-507) or mimics negative control (miR-NC) were detected by qRT-PCR. The data represent as mean±SD from three independent experiments. *p<0.05, **p<0.01, ns indicates no significance.

  • Fig. 4 MiR-507 suppressed the stemness and oxaliplatin resistance of colorectal cancer. (a) The relative expression levels of miR-507 in HCT116 and HT29 cells transfected with miR-507 mimics (miR-507) or mimics negative control (miR-NC) were detected by qRT-PCR. (b) Overexpression of miR-507 in HCT116 and HT29 cells significantly decreased the capacity to form tumorspheres. (c~e) The levels of CD44, SOX2 and OCT4 were determined by qRT-PCR and western blotting in HCT116 and HT29 cells transfected with miR-507 mimics (miR-507) or mimics negative control (miR-NC). (f) The relative expression levels of miR-507 in oxaliplatin resistant HCT116 (HCT116-OxR) and HT29 (HT29-OxR) cells transfected with miR-507 mimics (miR-507) or mimics negative control (miR-NC) were detected by qRT-PCR. (g~i) Overexpression of miR-507 significantly inhibited CRC-OxR cells growth. Cell viabilities were determined by MTT and colony formation assays. (j) Overexpression of miR-507 increased CRC-OxR cells apoptosis. Cell apoptosis was determined by DNA fragmentation assay. (k) The apoptotic markers p53, Caspase-3, Bcl-2 in oxaliplatin resistant HCT116 (HCT116-OxR) and HT29 (HT29-OxR) cells transfected with miR-507 mimics (miR-507) or mimics negative control (miR-NC) were detected by western blotting. The data represent as mean±SD from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

  • Fig. 5 ELK3 was a direct target of miR-507. (a) The sequences of binding sites between miR-507 and ELK3 3′UTR, including wild-type (ELK3 3′UTR (WT)) and mutant (ELK3 3′UTR (MT)). (b, c) Luciferase reporter assay was performed in the cells co-transfected with miR-507 or miR-NC mimics and plasmids containing ELK3 3′UTR (WT) or ELK3 3′UTR (MT). (d) ELK3 mRNAs in biotinylated miRNA/target mRNA complex was detected using qRT-PCR. Biotinylated miR-507 (bio-miR-507) or biotinylated control random RNA (bio-miR-NC) was used to pulldown ELK3 mRNA in the complex. (e~g) The relative expression levels of ELK3 in HCT116 and HT29 cells transfected with indicated microRNA mimics or microRNA inhibitors were detected by qRT-PCR and western blotting. (h~j) The relative expression levels of ELK3 were detected by qRT-PCR and western blot. HCT116 and HT29 cells were transfected with LINC00525 shRNAs (sh-LINC00525-1, sh-LINC00525-2) or empty vector (sh-CTR) and LINC00525 plasmid (pSin-LINC00525) or empty vector (pSin-vec). Data are representative of three or more independent experiments. The data represent as mean±SD from three independent experiments. *p<0.05, **p<0.01, ns indicates no significance.

  • Fig. 6 Restoration of ELK3 expression abrogated the effects of LINC00525 knockdown. (a, b) Expression levels of ELK3 were determined by qRT-PCR and western blot in the parental HCT116 cells. (c) The capacity to form tumorspheres of these parental HCT116 cells was determined by tumorsphere formation assay. (d, e) mRNA and protein levels of CD44, SOX2 and OCT4 in these parental HCT116 cells were determined by qRT-PCR and western blotting. (f~j) Oxaliplatin resistant HCT116 (HCT116-OxR) cells subjected to co-transfection with empty plasmids (sh-CTR+pSin-vec), LINC00525 shRNA and empty plasmid (sh-LINC00525-1+pSin-vec) or LINC00525 shRNA and ELK3 overexpression plasmid (sh-LINC00525-1+pSin-ELK3). (f, g) Expression levels of ELK3 were determined by qRT-PCR and western blotting in the oxaliplatin resistant HCT116 (HCT116-OxR) cells. (h, i) MTT and colony formation assays were used to determine cell growth of the oxaliplatin resistant HCT116 (HCT116-OxR) cells. (j) Apoptosis levels of these oxaliplatin resistant HCT116 (HCT116-OxR) cells were measured by DNA fragmentation assay. (k) The apoptotic markers p53, Caspase-3, Bcl-2 in these oxaliplatin resistant HCT116 (HCT116-OxR) cells were detected by western blotting. The Data represent as mean±SD from three or more independent experiments. *p<0.05, **p<0.01, ***p<0.001.


Reference

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