MicroRNA-144 inhibits proliferation by targeting WW domain-containing transcription regulator protein 1 in papillary thyroid cancer

Abstract

Papillary thyroid carcinoma (PTC), the most common histological subtype of thyroid cancer, accounts for between 80 and 90% of all thyroid cancer cases. Previous studies have suggested that microRNAs (miRNAs/miRs) are involved in the development of PTC. The aim of the present study was to investigate whether miR-144 inhibits cellular proliferation in PTC. The expression of miR-144 was detected in PTC and corresponding adjacent non-cancerous tissues, and in the PTC cell line IHH4, using reverse transcription-quantitative polymerase chain reaction. Associations between miR-144 expression levels and the clinicopathological characteristics were analyzed. Receiver operating characteristic (ROC) curves were used to assess the diagnostic value of miR-144 expression, and the potential function of miR-144 was investigated in IHH4 cells using a Cell Counting Kit-8 and colony formation assays. Western blotting was applied to analyze the expression level of WW domain-containing transcription regulator 1 (WWTR1) in PTC tissues. miR-144 was significantly downregulated in PTC tissues and the PTC cell line. Low expression of miR-144 was associated with larger tumor sizes (P<0.001). The ROC curves demonstrated that miR-144 may be a potential biomarker for identifying PTC and non-cancerous diseases (sensitivity, 58.7%; specificity, 87.3%) as well as to differentiate PTC with tumor sizes ≥2 cm (sensitivity, 79.2%; specificity, 69.2%). Upregulation of miR-144 significantly suppressed proliferation in IHH4 cells. WWTR1 was overexpressed in PTC tissues compared with in adjacent non-cancerous tissues, and the ectopic expression of miR-144 downregulated WWTR1 in IHH4 cells. Co-transfection with pcDNA-WWTR1 and miR-144 'rescued' the proliferation inhibition. The results of the present study collectively demonstrated that miR-144 is downregulated in PTC, that low expression levels of miR-144 are associated with larger tumor sizes and that miR-144 inhibits cellular proliferation in PTC by targeting WWTR1.

Keywords: WW domain-containing transcription regulator protein 1; cell proliferation; miR-144; papillary thyroid cancer; tumor sizes.

Figure 1.

The expression of miR-144 in PTC and adjacent non-cancerous tissues from 63 patients, as well as the PTC cell line IHH4, were analyzed using reverse transcription-quantitative polymerase chain reaction. (A) The expression levels of miR-144 were demonstrated to be significantly downregulated in PTC compared with in paired adjacent non-cancerous tissue (P<0.01). (B) The expression levels of miR-144 were significantly downregulated in the IHH4 cells compared with in the normal human thyroid follicular epithelial cell line Nthy-ori 3-1. **P<0.01. PTC, papillary thyroid carcinoma.

Figure 2.

ROC curves demonstrating the diagnostic value of miR-144. (A) Diagnostic value for differentiating between PTC and non-cancerous tissues (AUC, 0.743; 95% CI, 0.657–0.829; P<0.001). The sensitivity was 58.7%, and the specificity was 87.3%. (B) Diagnostic value of miR-144 for whether the tumor sizes ≥2 cm (AUC; 0.779; 95% CI, 0.661–0.896; P<0.001). The sensitivity was 79.2%, and specificity was 69.2%. ROC, receiver operating characteristic; PTC, papillary thyroid carcinoma; AUC, area under the curve.

Figure 3.

Ectopic overexpression of miR-144 inhibits cellular proliferation in the IHH4 PTC cell line. (A) A CCK-8 cell counting assay was applied to analyze proliferative function in IHH4 cells. Cellular proliferation was significantly inhibited in the miR-144-overexpressing group compared with in the NC group. (B) Colony formation assays indicated that the ectopic overexpression of miR-144 markedly inhibited colony formation. **P<0.01 vs. NC. PTC, papillary thyroid carcinoma; NC, negative control.

Figure 4.

WWTR1 is a target of miR-144. (A) WWTR1 was detected using western blot analysis in PTC, denoted by T, and adjacent non-cancerous tissues, denoted by N, from 15 (–15) patients. WWTR1 was significantly overexpressed in PTC tissues compared with the adjacent non-cancerous tissue. (B) Ectopic overexpression of miR-144 resulted in the downregulation of WWTR1 in the PTC cell line IHH4. The protein expression of WWTR1 was significantly downregulated in the overexpressed miR-144 group compared with in the NC group. *P<0.05, **P<0.01. WWTR1, WW domain containing transcription regulator 1; PTC, papillary thyroid carcinoma; NC, negative control.

Figure 5.

Overexpression of WWTR1 impaired miR-144-induced inhibition of cellular proliferation. (A) Western blot analysis of WWTR1 expression in the miR-144 mimics+pcDNA-WWTR1 group, miR-144 mimics group and NC group in IHH4 cells. (B) A CCK-8 assay demonstrated that cellular proliferation in the miR-144 mimics+pcDNA-WWTR1 group was significantly increased compared with the miR-144 mimic group. *P<0.05, **P<0.01. WWTR1, WW domain containing transcription regulator 1; NC, negative control.