Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2

Abstract

Objective: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis.

Methods: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer.

Results: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer.

Conclusion: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.

Keywords: Apoptosis; LncRNA LINC00488; PON2; Proliferation; Thyroid cancer; miR-376a-3p.

Figure 1. Knockdown of LINC00488 inhibits proliferation and promotes apoptosis of thyroid cancer cells

BCPAP and TPC-1 cells were transfected with either LINC00488 shRNAs or negative control (NC) shRNAs. (A) The expression level of lncRNA LINC00488 in normal thyroid cells (Nthy-ori3-1) and thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) were determined by RT-qPCR. *P<0.05, **P<0.01 vs. the Nthy-ori3-1 cell line. (B) RT-qPCR analysis of the cell transfection. (C) CCK-8 assay for the cell viability. (D) EdU assay for the cell proliferation. (E) Flow cytometry for the cell cycle distribution. (F) Flow cytometry for the cell apoptosis. (G) Western blot analysis for the protein expression levels of proliferation-related proteins. (H) Western blot analysis for the protein expression levels of apoptosis-related proteins. *P<0.05, **P<0.01 vs. the sh-NC group. The data were presented as mean ± SD and the experiments were repeated three times.

Figure 2. Knockdown of LINC00488 represses migration and invasion of thyroid cancer cells

Cells were treated similarly as in Figure 1. (A) Cell capacity of migration (scratch test). (B) Transwell chamber assay for cell migration and invasion. (C) Western blot analysis of the expression level of migration related proteins (MMP-2 and MMP-9). **P<0.01 vs. the sh-NC group. The data were presented as mean ± SD and the experiments were repeated three times.

Figure 3. LINC00488 directly binds to miR-376a-3p and down-regulates the expression of miR-376a-3p

(A) The predicted LINC00488 binding sites in the region of miR-376a-3p and the corresponding mutant sequence were shown. (B) The expression level of miR-376a-3p both in BCPAP and TPC-1 cells after transfected with NC mimic and miR-376a-3p mimic, respectively. ***P<0.001 vs. the NC mimic group. (C) Relative values of luciferase signal. **P<0.01 vs. the NC mimic group. (D) The expression level of miR-376a-3p both in BCPAP and TPC-1 cells after transfected with sh-NC and LINC00488 shRNAs, respectively. **P<0.01 vs. the sh-NC group. (E) The expression level of miR-376a-3p in normal thyroid cell and thyroid cancer cell lines. **P<0.01 vs. the Nthy-ori3-1 cell line. The data were presented as mean ± SD and the experiments were repeated three times.

Figure 4. MiR-376a-3p targets PON2 and causes post-transcriptional suppression

(A) The predicted miR-376a-3p binding sites in the region of PON2 and the corresponding mutant sequence were shown. (B) Relative values of luciferase signal. (C) The expression level of PON2 both in BCPAP and TPC-1 cells was determined by RT-qPCR after transfected with NC mimic and miR-376a-3p mimic, respectively. (D) Western blot analysis of the expression level of PON2 protein. **P<0.01 vs. the NC mimic group. (E) Immunocytochemistry assay. (F) The expression level of PON2 in normal thyroid cell and thyroid cancer cell lines. **P<0.01 vs. the Nthy-ori3-1 cell line. The data were presented as mean ± SD and the experiments were repeated three times.

Figure 5. MiR-376a-3p/PON2 mediates the inhibitory effects of sh-LINC00488 on the thyroid cancer cell progression

Either miR-376a-3p inhibitor or PON2 shRNAs were transfected into the LINC00488 knockdown BCPAP cells. (A) RT-qPCR was performed to evaluate the efficiency of miR-376a-3p inhibitor and LINC00488 knockdown. **P<0.01 vs. the NC inhibitor group and the sh-NC group, respectively. (B) CCK-8 assay for the cell viability. (C) EdU assay for the cell proliferation. (D) Flow cytometry for the cell apoptosis. (E) Cell capacity of migration (scratch test). (F) Transwell chamber assay for cell migration and invasion. **P<0.01 vs. the sh-NC + NC inhibitor group, ^#P<0.05, ^##P<0.01 vs. the sh-LINC00488 + NC inhibitor group, and ^ΔP<0.05 vs. the sh-LINC00488 + miR-376a-3p inhibitor group. The data were presented as mean ± SD and the experiments were repeated three times.