Transcription Factor E2F1 Exacerbates Papillary Thyroid Carcinoma Cell Growth and Invasion via Upregulation of LINC00152

Abstract

Background: Papillary thyroid carcinoma (PTC) is the most common thyroid neoplasm, whereas transcription factor E2F1 has been previously implicated in PTC progression. The current study sought to elucidate the underlying mechanism of E2F1 in PTC cell biological activities via regulation of long intergenic noncoding RNA 152 (LINC00152).

Methods: Firstly, the expression patterns of LINC00152 and E2F1 in PTC were determined. Besides, TPC-1 and IHH-4 cells were adopted to carry out a series of experiments. Cell proliferation was detected by means of a cell counting kit-8 assay and colony formation assay, while cell migration and invasion abilities were assessed using a Transwell assay. Next, the interaction between E2F1 and LINC00152 was certified. Lastly, xenograft transplantation was carried out to validate the effects of E2F1 depletion on PTC.

Results: Both LINC00152 and E2F1 were highly expressed in PTC cells. Knockdown of LINC00152 led to reduced cell activity, while LINC00152 overexpression brought about the opposing trends. Likewise, E2F1 knockdown quenched cell proliferation, migration, and invasion. However, the combination of E2F1 knockdown and LINC00152 overexpression resulted in augmented cell growth. In addition, E2F1 induced LINC00152 overexpression, which accelerated cell proliferation, migration, and invasion by activating the PI3K/AKT axis, whereas the administration of LY294002, the inhibitor of PI3K, led to reversal of the same. Finally, xenograft transplantation validated that E2F1 inhibition could suppress LY294002, thereby discouraging tumor growth.

Conclusion: Our findings highlighted that E2F1 augmented PTC cell proliferation and invasion by upregulating LINC00152 and the PI3K/AKT axis. Our discovery provides therapeutic implications for PTC alleviation.

Figure 1

LINC00152 promotes PTC cell growth and invasion. (a) LINC00152 expression patterns in PTC cell lines measured by RT-qPCR. (b, c) Transfection efficiency of LINC00152 in (b) TPC-1 and (c) IHH-4 cells verified by RT-qPCR. (d, e) Proliferation of (d) TPC-1 and (e) IHH-4 cells detected by CCK-8. (f) Proliferation of TPC-1 and IHH-4 cells detected by colony formation assay. (g, h) Migration and invasion of (g) TPC-1 and (h) IHH-4 cells assessed via Transwell assay. Repetitions = 3. Data are expressed as mean ± standard deviation. The t-test was used for pairwise comparison. One-way ANOVA was used to determine statistical significance. Tukey's multiple comparisons test was applied for post hoc test. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 2

E2F1 induces the upregulation of LINC00152 expression. (a) E2F1 expression patterns in PTC cell lines measured by RT-qPCR. (b) Binding site between E2F1 and LINC00152 promoter predicted by JASPAR. (c, d) The interaction between E2F1 and LINC00152 in (c) TPC-1 and (d) IHH-4 cells verified by RIP. (e, f) The interaction between E2F1 and LINC00152 in (e) TPC-1 and (f) IHH-4 cells detected by dual-luciferase reporter gene assay. (g, h) Expression patterns of E2F1 and LINC00152 in (g) TPC-1 and (h) IHH-4 cells after silencing E2F1 assessed by RT-qPCR. Repetitions = 3. Data are expressed as mean ± standard deviation. The t-test was used for pairwise comparison. One-way ANOVA was used to determine statistical significance. Tukey's multiple comparisons test was applied for post hoc test. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 3

LINC00152 overexpression diminishes the repressive role of E2F1 knockdown on PTC cell growth and invasion. (a) Expression patterns of E2F1 and LINC00152 in TPC-1 cells assessed via RT-qPCR. (b) E2F1 expression patterns in TPC-1 cells calculated by Western blot analysis. (c, d) Cell proliferation detected by (c) CCK-8 and (d) colony formation assay. (e) Cell migration and invasion measured through Transwell assay. Repetitions = 3. Data are expressed as mean ± standard deviation. One-way ANOVA was used to determine statistical significance. Tukey's multiple comparisons test was applied for post hoc test. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

LINC00152 regulates PTC cell growth and invasion via the PI3K/AKT axis. Initially, 10 μM LY294002 was added into IHH-4 cells to conduct pretreatment for 2 h. (a) Levels of the PI3K/AKT axis-related proteins (PI3K, p-PI3K, AKT, and p-AKT) verified through Western blot analysis. (b, c) Cell proliferation detected by (b) CCK-8 and (c) colony formation assay. (d) Cell migration and invasion measured through Transwell assay. Repetitions = 3. Data are expressed as mean ± standard deviation. The t-test was used for pairwise comparison. One-way ANOVA was used to determine statistical significance. Tukey's multiple comparisons test was applied for post hoc test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 5

E2F1 knockout inhibits PTC tumor growth in vivo. (a) Tumor volume assessment. (b) Images of transplanted tumors in nude mice. (c) Weights of transplanted tumor in nude mice. (d) Expression patterns of LINC00152 and E2F1 mRNA detected by RT-qPCR. (e) Level of E2F1 protein verified through Western blot analysis. (f) Ki67 positive expression patterns measured by immunohistochemical staining. N = 6. Data are expressed as mean ± standard deviation. The t-test was used for pairwise comparison. ^∗∗p < 0.01; ^∗∗∗p < 0.001.