Long Non-Coding RNA MAPK8IP1P2 Inhibits Lymphatic Metastasis of Thyroid Cancer by Activating Hippo Signaling via Sponging miR-146b-3p

Abstract

The principal issue derived from thyroid cancer is its high propensity to metastasize to the lymph node. Aberrant exprssion of long non-coding RNAs have been extensively reported to be significantly correlated with lymphatic metastasis of thyroid cancer. However, the clinical significance and functional role of lncRNA-MAPK8IP1P2 in lymphatic metastasis of thyroid cancer remain unclear. Here, we reported that MAPK8IP1P2 was downregulated in thyroid cancer tissues with lymphatic metastasis. Upregulating MAPK8IP1P2 inhibited, while silencing MAPK8IP1P2 enhanced anoikis resistance in vitro and lymphatic metastasis of thyroid cancer cells in vivo. Mechanistically, MAPK8IP1P2 activated Hippo signaling by sponging miR-146b-3p to disrupt the inhibitory effect of miR-146b-3p on NF2, RASSF1, and RASSF5 expression, which further inhibited anoikis resistance and lymphatic metastasis in thyroid cancer. Importantly, miR-146b-3p mimics reversed the inhibitory effect of MAPK8IP1P2 overexpression on anoikis resistance of thyroid cancer cells. In conclusion, our findings suggest that MAPK8IP1P2 may serve as a potential biomarker to predict lymphatic metastasis in thyroid cancer, or a potential therapeutic target in lymphatic metastatic thyroid cancer.

Keywords: Hippo signaling; MAPK8IP1P2; anoikis resistance; lymph node metastasis; thyroid cancer.

Figure 1

MAPK8IP1P2 is downregulated in thyroid cancer with lymphatic metastasis. (A) MAPK8IP1P2 expression in 59 paired thyroid cancer tissues and the matched adjacent normal tissues in the thyroid cancer dataset from TCGA. (B) Real-time PCR analysis of MAPK8IP1P2 expression in our 24 paired thyroid cancer tissues and their matched adjacent normal tissues, including 9 thyroid cancer tissues without lymphatic metastasis and 15 thyroid cancer tissues with lymphatic metastasis. The number on the abscissa indicated the patient number according to our record when collecting patient information. GAPDH was used as endogenous controls. *P < 0.05. (C) Real-time PCR analysis of MAPK8IP1P2 expression in ANT (n = 24), thyroid cancer tissues without lymphatic metastasis (n = 10), and thyroid cancer tissues with lymphatic metastasis (n = 38). GAPDH was used as endogenous controls. n.s. indicates no significance. (D) Real-time PCR analysis of MAPK8IP1P2 expression in ANT (n = 24), thyroid cancer tissues of T1-T2 grade without lymphatic metastasis (n = 10), and thyroid cancer tissues of T1-T2 grade with lymphatic metastasis (n = 27). GAPDH was used as endogenous controls. n.s. indicates no significance. (E) Real-time PCR analysis of MAPK8IP1P2 expression in ANT (n = 24), thyroid cancer tissues with T1-T2 grade (n = 37), and thyroid cancer tissues with T3-T4 grade (n = 11). GAPDH was used as endogenous controls. n.s. indicates no significance. (F) MAPK8IP1P2 expression in ANT (n = 59), thyroid cancer tissues without lymphatic metastasis (n = 230), and thyroid cancer tissues with lymphatic metastasis (n = 225) in the thyroid cancer dataset from TCGA. (G) MAPK8IP1P2 expression in ANT (n = 59), thyroid cancer tissues with T1-T2 grade (n = 309), and thyroid cancer tissues with T3-T4 grade (n = 194) in the thyroid cancer dataset from TCGA. (H) MAPK8IP1P2 expression in ANT (n = 59), thyroid cancer tissues of T1-T2 grade without lymphatic metastasis (n = 164), and thyroid cancer tissues of T1-T2 grade with lymphatic metastasis (n = 110) in the thyroid cancer dataset from TCGA.

Figure 2

Upregulating MAPK8IP1P2 inhibits cancer stem cell characteristics in thyroid cancer cells. (A) Real-time PCR analysis of MAPK8IP1P2 expression in 7 thyroid cancer cells, including 4 PTC cell lines, B-CPAP, BHT101, KTC-1, and K1, and 2 ATC cell lines, CAL-62 and 8305C, and 1 thyroid duct cell carcinoma cells, TT, and a normal thyroid follicular epithelial cell line PTFE. GAPDH was used as endogenous controls. *P < 0.05. (B) MAPK8IP1P2 expression in the vector, MAPK8IP1P2 overexpression scramble, MAPK8IP1P2 shRNA#1, and MAPK8IP1P2 shRNA#2 thyroid cancer cells using real-time PCR. Transcript levels were normalized by GAPDH expression. *P < 0.05. (C) Schematic model of lymphatic metastasis model in vivo. (D) H & E staining analysis of tumors in lymph node from the indicated mice group. (E) The count of tumor cells in the tumor areas of lymph node from the indicated mice group. *P < 0.05.

Figure 3

Upregulating MAPK8IP1P2 does not affect proliferation of thyroid cancer cells. (A–D) The effect of overexpression or silencing MAPK8IP1P2 on the cell growth in the indicated thyroid cancer cells by CCK-8 assay. (E) The effect of overexpression or silencing MAPK8IP1P2 on colony-formation ability of the indicated thyroid cancer cells by colony-formation assay. (F) The effect of overexpression or silencing MAPK8IP1P2 on cell cycle progression of the indicated thyroid cancer cells by flow cytometry. (G) The effect of overexpression or silencing MAPK8IP1P2 on survival ability in the indicated thyroid cancer cells by anchorage-independent growth assay. *P < 0.05.

Figure 4

Upregulating MAPK8IP1P2 inhibits anoikis resistance in thyroid cancer cells. (A) The effect of overexpression or silencing MAPK8IP1P2 on the apoptotic ratio in the indicated thyroid cancer cells by Annexin V-FITC/PI staining. *P < 0.05. (B) The effect of overexpression or silencing MAPK8IP1P2 on mitochondrial potential in the indicated thyroid cancer cells by JC-1 staining. *P < 0.05. (C, D) The effect of overexpression or silencing MAPK8IP1P2 on the activities of caspase-3 (C) and caspase-9 (D) in the indicated thyroid cancer cells. *P < 0.05. (E) Western blotting analysis of the effect of overexpression or silencing MAPK8IP1P2 on anti-apoptotic proteins, BCL2 and BCL2L1, and pro-apoptotic proteins, BAD and BAX, in the indicated thyroid cancer cells. α-Tubulin served as the loading control.

Figure 5

MAPK8IP1P2 activates Hippo signaling pathway in thyroid cancer cells. (A) Gene set enrichment analysis (GSEA) revealed that MAPK8IP1P2 expression positively correlated with Hippo signaling. (B) The effect of overexpression or silencing MAPK8IP1P2 on TEAD transcriptional activity was assessed by HOP-Flash luciferase reporter in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (C) Western blotting analysis of the effect of overexpression or silencing MAPK8IP1P2 on phophorylated MST1/2 (p-MST1/2), phophorylated LATS1 (p-LATS1), phophorylated YAP1 (p-YAP1), total levels of MST1 and LATS1 and nuclear translocation of YAP1 and TAZ in the indicated thyroid cancer cells. α-Tubulin and p84 were served as the cytoplasmic and nuclear loading control respectively. (D) Real-time PCR analysis of the effect of overexpression or silencing MAPK8IP1P2 on CTGF, CYR61, HOXA1, PPIA, RPL13A, and SOX9 in the indicated cells. Transcript levels were normalized by GAPDH expression. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05.

Figure 6

MAPK8IP1P2 activates Hippo signaling by sponging miR-146b-3p. (A) Volcano plot analyzed the clinical correlation of MAPK8IP1P2 with all reported miRNAs in thyroid cancer dataset from TCGA. The orange colors represent significantly and negatively correlated miRNAs with fold change > 2 and r value < -0.2. (B) Predicted recognition sites of miR-146b-3p on MAPK8IP1P2, and predicted miR-146b-3p targeting sequence and mutant sequences in 3’UTR s of NF2, RASSF1, and RASSF5. (C, D) The effect of miR-146b-3p on the luciferase activity of wild-type or mutant MAPK8IP1P2, NF2, RASSF1 and RASSF5 in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (E, F) Real-time PCR analysis of the effect of overexpression or silencing MAPK8IP1P2 on NF2, RASSF1, and RASSF5 expression in the indicated cells. Transcript levels were normalized by GAPDH expression. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (G) Western blot analysis of the effect of overexpression or silencing MAPK8IP1P2 on NF2, RASSF1 and RASSF5 expression in the indicated cells. α-Tubulin served as the loading control. (H, I) Real-time PCR (H) and Western blot (I) analysis of the effect of miR-146b-3p mimics on NF2, RASSF1, and RASSF5 expression in MAPK8IP1P2-overexpressing thyroid cancer cells. Transcript levels were normalized by GAPDH expression. α-Tubulin served as the loading control. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (J) The effect of miR-146b-3p mimics on TEAD transcriptional activity was assessed by HOP-Flash luciferase reporter in MAPK8IP1P2-overexpressing thyroid cancer cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05.

Figure 7

Upregulating MAPK8IP1P2 inhibits anoikis resistance by sponging miR-146b-3p. (A) The effect of miR-146b-3p mimics on colony-formation ability in MAPK8IP1P2-overexpressing thyroid cancer cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (B) The effect of miR-146b-3p mimics on mitochondrial potential in MAPK8IP1P2-overexpressing thyroid cancer cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (C) The effect of miR-146b-3p mimics on apoptotic ratio in MAPK8IP1P2-overexpressing thyroid cancer cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (D, E) The effect of miR-146b-3p mimics on caspase-3 (D) and caspase-9 (E) in MAPK8IP1P2-overexpressing thyroid cancer cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (F) Hypothetical model illustrates the role and underlying mechanism of MAPK8IP1P2 in lymphatic metastasis of thyroid cancer by miR-146b-3p/Hippo signaling axis.