Long noncoding RNA ILF3-AS1 aggravates papillary thyroid carcinoma progression via regulating the miR-4306/PLAGL2 axis

Abstract

Background: It have been proven that long non-coding RNAs (lncRNAs) serve as regulators in carcinogenesis. Interleukin enhancer binding factor 3 antisense RNA 1 (ILF3-AS1) has been illuminated as a prognostic factor in some cancers. Nevertheless, its expression pattern and possible functions in papillary thyroid carcinoma (PTC) have not been studied.

Methods: The expression of ILF3-AS1 was measured by RT-qPCR and ISH. Colony formation assay and EdU assay were used to probe cell proliferation. TUNEL assay was used for analysis of cell apoptosis. Immunofluorescence and western blot were conducted to evaluate the expression change of E-cadherin and N-cadherin. The RNA interaction was demonstrated by mechanism experiments, including pull down assay and dual luciferase reporter assay.

Results: ILF3-AS1 expression was evidently upregulated in PTC cell lines. ILF3-AS1 knockdown restrained the proliferation, migration and invasion of PTC cells. Mechanical investigation revealed that miR-4306 could interact with ILF3-AS1. PLAGL2 was a downstream target of miR-4306. The effects of ILF3-AS1 knockdown on the cellular processes were abrogated by miR-4306 downregulation or pleiomorphic adenoma gene-like 2 (PLAGL2) overexpression.

Conclusion: ILF3-AS1 plays tumor-promoting role in PTC via targeting miR-4306/PLAGL2 axis.

Keywords: ILF3-AS1; MiR-4306; PLAGL2; Papillary thyroid carcinoma.

Fig. 1

ILF3-AS1 is expressed at a high level in PTC cell lines. a The expression level of ILF3-AS1 in five PTC cell lines and one normal thyroid cell line was analyzed by RT-qPCR. b ILF3-AS1 expression was interfered by transfection of sh-ILF3-AS1#1/2 into IHH-4 and TPC-1 cells. c, d Colony formation assay and EdU assay were conducted to probe into the effects of sh-ILF3-AS1#1/2 on PTC cell proliferation. e TUNEL assay was used to evaluate the influence of sh-ILF3-AS1#1/2 on apoptosis. f IF assay explored the expressions of EMT markers after the ILF3-AS1 knockdown. ^**P < 0.01

Fig. 2

ILF3-AS1 interacts with miR-4306 in PTC cells. a, b The distribution of ILF3-AS1 in IHH-4 and TPC-1 cells was detected by subcellular fractionation assay and FISH assay. c Bioinformatics tool screened out 6 miRNAs that may have the ability to interact with ILF3-AS1. d RNA pull down assay was performed to test the interaction between 6 candidate miRNAs and ILF3-AS1. e The expression level of miR-4306 in PTC cell lines was assessed by RT-qPCR. f RNA pull down assay showed that ILF3-AS1 was pulled down abundantly by biotinylated miR-4306-WT. g The putative binding sites of miR-4306 with ILF3-AS1 were predicted. h The overexpressing efficacy of miR-4306 mimics was examined in IHH-4 and TPC-1 cells. i Dual Luciferase reporter assay was applied to prove the mutual interaction between ILF3-AS1 and miR-4306. ^*P < 0.05, ^**P < 0.01

Fig. 3

MiR-4306 suppresses PTC cell proliferation and migration. a, b The impacts of miR-4306 mimics on PTC cell proliferation were investigated through colony formation assay and EdU assay. c The effects of miR-4306 upregulation on PTC cell apoptosis were detected by TUNEL assay. d EMT process was observed in PTC cells with miR-4306 upregulation through IF assay. ^**P < 0.01

Fig. 4

PLAGL2 is a downstream target of miR-4306. a Venn diagram showed 3 common mRNA candidates for miR-4306 predicted by four databases. b, c RT-qPCR assay probed into the effects of miR-4306 mimics or sh/ILF3-AS1#1/2 on the expression of candidate mRNAs. d The expression level of PLAGL2 in PTC cells was identified by RT-qPCR. e Ago2-RIP assay demonstrated the enrichment of ILF3-AS1, miR-4306 and PLAGL2 in anti-Ago2 group or anti-IgG group. f The interaction between miR-4306 and PLAGL2 was proven by RNA pull down assay. g, h The predicted binding sites between miR-4306 and PLAGL2 was shown and verified by dual luciferase reporter assay. i Western blot analysis was conducted to detect the protein level of PLAGL2 after the overexpression or knockdown of miR-4306. ^*P < 0.05, ^**P < 0.01

Fig. 5

ILF3-AS1 expedites PTC progression via sponging miR-4306 to elevate PLAGL2 expression. a The expression of PLAGL2 was analyzed in response to pcDNA3.1/ PLAGL2. b, c The effects of miR-4306 and PLAGL2 on ILF3-AS1-mediated cell proliferation were demonstrated by colony formation assay and EdU assay. d–f The changes in apoptosis were observed in transfected PTC cells through TUNEL assay, flow cytometry analysis and caspase 3 activity detection. g, h The rescuing effects of miR-4306 inhibitor and pcDNA3.1/PLAGL2 on suppressed migratory and invasive capacities by sh-ILF3-AS1 were detected by wound healing assay and transwell assay. ^**P < 0.01