Long Non-Coding RNA LINC00239 Functions as a Competitive Endogenous RNA by Sponging microRNA-484 and Enhancing KLF12 Expression to Promote the Oncogenicity of Colorectal Cancer

Abstract

Background: Long intergenic non-protein coding RNA 239 (LINC00239) is an oncogenic long non-coding RNA in acute myeloid leukemia. We aimed to determine LINC00239 expression in colorectal cancer (CRC) and examine the influences of LINC00239 on tumor behaviors of CRC cells. Furthermore, the mechanism underlying the actions of LINC00239 in CRC was unveiled in detail.

Materials and methods: Quantitative real-time polymerase chain reaction was used to detect LINC00239 expression in CRC tissues and cell lines. CRC cell proliferation, apoptosis, migration, and invasion were investigated by cell counting kit-8 assays, flow cytometry, and cell migration and invasion assays, respectively. Tumor xenograft experiments were performed to evaluate the tumor growth of CRC cells in vivo. The interactions among LINC00239, microRNA-484 (miR-484), and kruppel-like factor 12 (KLF12) were analyzed by bioinformatics prediction, RNA immunoprecipitation and luciferase reporter assay.

Results: LINC00239 was upregulated in CRC tissues and cell lines. LINC00239 knockdown impaired CRC cell proliferation, migration, and invasion and promoted apoptosis in vitro. Additionally, LINC00239 deficiency inhibited CRC growth in vivo. Mechanistically, LINC00239 functioned as a competing endogenous RNA by directly sponging miR-484, thereby enhancing KLF12 expression. Rescue experiments further corroborated that miR-484 inhibition or KLF12 overexpression reversed the inhibitory actions of LINC00239 knockdown in CRC cells.

Conclusion: The LINC00239/miR-484/KLF12 pathway executed critical roles in CRC oncogenicity and may provide potential targets for CRC treatments.

Keywords: CRC; kruppel-like factor 12; long intergenic non-protein coding RNA 239; miRNA sponge.

Figure 1

LINC00239 is upregulated in CRC. (A) LINC00239 expression in COAD and READ samples from TCGA and GTEx databases. (B) qRT-PCR was performed to detect LINC00239 expression in 63 pairs of CRC tissues and matched adjacent normal tissues. (C) LINC00239 expression in CRC cell lines was determined by qRT-PCR. A normal human colon epithelium cell line was used as the control. (D) The correlation between LINC00239 expression and tumor stage in COAD and READ patients data from TCGA and GTEx databases. (E, F) Kaplan–Meier overall survival and disease-free survival curves according to LINC00239 expression in TCGA and GTEx. **P < 0.01.

Figure 2

LINC00239 exerts oncogenic roles during CRC progression. (A, B) Relative LINC00239 expression in SW480 and HCT116 cells after si-LINC00239 or pcDNA3.1-LINC00239 transfection was measured by qRT-PCR. (C, D) The proliferation of si-LINC00239-transfected or pcDNA3.1-LINC00239-transfected SW480 and HCT116 cells was analyzed via the CCK-8 assay. (E, F) Flow cytometry analysis of SW480 and HCT116 cell apoptosis after LINC00239 knockdown or overexpression. (G, H) The migratory capacity of SW480 and HCT116 cells after LINC00239 knockdown or overexpression were examined by cell migration assay. (I, J) Cell invasion assay was employed for determining cell invasion in SW480 and HCT116 cells after si-LINC00239 or pcDNA3.1-LINC00239 transfection. **P < 0.01.

Figure 3

LINC00239 functions as a miR-484 sponge in CRC. (A) The prediction of LINC00239 subcellular localization by lncATLAS. (B) Nuclear and cytoplasmic fractionations of SW480 and HCT116 cells were separated by subcellular fractionation, and the distribution of LINC00239 was analyzed. (C) The potential miRNAs sequestered by LINC00239 were predicted by miRDB. (D) qRT-PCR was applied to detect the expression of miRNAs in SW480 and HCT116 cells transfected with si-LINC00239 or si-NC. (E) The expression of miR-484 in 63 pairs of CRC tissues and matched adjacent normal tissues was measured by qRT-PCR. (F) Pearson’s correlation analysis was applied to assess the correlation between LINC00239 and miR-484 in 63 CRC tissues. (G) The wild-type miR-484 binding site in the sequences of LINC00239. The mutated sequences were also displayed (LINC00239-MUT). (H) SW480 and HCT116 cells were cotransfected with miR-484 mimic or NC mimic and luciferase reporter vectors LINC00239-WT or LINC00239-MUT. Transfected cells were collected at 48 h post-transfection, and luciferase activity was determined. (I) RIP assays were performed in SW480 and HCT116 cells to reveal the enrichment of LINC00239 and miR-484 in the immunoprecipitates conjugated to Ago2. **P < 0.01.

Figure 4

KLF12 is a direct target of miR-484 in CRC cells. (A) MiR-484 mimic or NC mimic was introduced into SW480 and HCT116 cells. After transfection, qRT-PCR was performed to detect the expression of miR-484 and evaluate the overexpression efficiency. (B, C) CCK-8 assays and flow cytometry analyses were used to detect the proliferation and apoptosis of SW480 and HCT116 cells following miR-484 mimic or NC mimic transfection. (D, E) Cell migration and invasion assays displayed the effects of miR-484 upregulation on the migration and invasion of SW480 and HCT116 cells. (F) KLF12 3ʹ-UTR containing the miR-484 binding sequences. The mutated KLF12 3ʹ-UTR containing the mutant miR-484 binding site was also shown. (G) A luciferase reporter assay was conducted in SW480 and HCT116 cells transfected with miR-484 mimic or NC mimic and KLF12-WT or KLF12-MUT. (H) The mRNA level of KLF12 in 63 pairs of CRC tissues and matched adjacent normal tissues was detected by qRT-PCR. (I) The correlation between KLF12 and miR-484 levels in 63 CRC tissues was explored by Pearson’s correlation analysis. (J, K) qRT-PCR and Western blotting were carried out to quantify KLF12 mRNA and protein levels in SW480 and HCT116 cells after miR-484 overexpression. **P < 0.01.

Figure 5

LINC00239 sequesters miR-484 and consequently enhances KLF12 expression. (A, B) KLF12 mRNA and protein levels were analyzed in LINC00239-depleted SW480 and HCT116 cells using qRT-PCR and Western blotting, respectively. (C) RIP assays were conducted in SW480 and HCT116 cells to reveal the enrichment of LINC00239, miR-484, and KLF12 in the immunoprecipitates conjugated to Ago2. (D) Pearson’s correlation analysis identified the correlation between LINC00239 and KLF12 expression in 63 CRC tissues. (E, F) MiR-484 inhibitor or NC inhibitor was transfected into LINC00239-depleted SW480 and HCT116 cells. The mRNA and protein levels of KLF12 were measured in different groups using qRT-PCR and Western blotting, respectively. **P < 0.01.

Figure 6

Inhibition of miR-484 counteracts the suppressive effects of LINC00239 knockdown in CRC cells. (A) qRT-PCR analysis was used to evaluate the silencing efficiency of miR-484 inhibitor in SW480 and HCT116 cells. (B–E) MiR-484 inhibitor or NC inhibitor, in combination with si-LINC00239, was transfected into SW480 and HCT116 cells. CCK-8 assay, flow cytometry analysis, and cell migration and invasion assays were performed to evaluate cell proliferation, apoptosis, migration, and invasion, respectively. *P < 0.05 and **P < 0.01.

Figure 7

The effects of LINC00239 silencing on the malignant phenotypes of CRC cells are abolished by KLF12 overexpression. (A) The protein level of KLF12 was detected by Western blotting in SW480 and HCT116 cells after pcDNA3.1 or pcDNA3.1-KLF12 transfection. (B–E) SW480 and HCT116 cells were transfected with pcDNA3.1 or pcDNA3.1-KLF12 and si-LINC00239. Cell proliferation, apoptosis, migration, and invasion in different groups were analyzed by CCK-8 assay, flow cytometry analysis, and cell migration and invasion assays, respectively. **P < 0.01.

Figure 8

LINC00239 silencing inhibits the tumor growth of CRC cells in vivo. (A) Representative images of tumor xenografts originating from SW480 cells stably expressing sh-LINC00239 or sh-NC. (B) Tumor volume was detected weekly using the formula: volume = 1/2 (length × width²). (C) All mice were euthanized, and tumor xenografts were weighed. (D, E) qRT-PCR analysis of LINC00239 and miR-484 expression in tumor xenografts in the sh-LINC00239 and sh-NC groups. (F) The protein level of KLF12 in tumor xenografts in the sh-LINC00239 and sh-NC groups was detected by Western blotting. **P < 0.01.