Clin Exp Otorhinolaryngol.  2023 May;16(2):184-197. 10.21053/ceo.2022.01760.

Mitochondrial Ribosomal Protein L14 Promotes Cell Growth and Invasion by Modulating Reactive Oxygen Species in Thyroid Cancer

• Kim HJ
• Nguyen QK
• Jung SN
• Lim MA
• Oh C
• Piao Y
• Jin Y
• Kim JH
• Kim YI
• Kang YE
• Chang JW
• Won HR
• Koo BS

• KMID: 2542360
• DOI: http://doi.org/10.21053/ceo.2022.01760

Abstract

Objectives
. The mitochondrial ribosomal protein L14 (MRPL14) is encoded by a nuclear gene and participates in mitochondrial protein translation. In this study, we aimed to investigate the role of MRPL14 in thyroid cancer.
Methods
. We investigated the association between MRPL14 expression and clinicopathological features using The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) databases. Functional studies of MRPL14, including proliferation, migration, invasion, mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production, were performed in papillary thyroid cancer (PTC) cell lines (B-CPAP and KTC-1).
Results
. Based on the TCGA dataset, PTC tissues lost mitochondrial integrity and showed dysregulated expression of overall mitoribosomal proteins (MRPs) compared with normal thyroid tissues. Of 78 MRPs, MRPL14 was highly expressed in thyroid cancer tissues. MRPL14 overexpression was significantly associated with advanced tumor stage, extrathyroidal extension, and lymph node metastasis. MRPL14 increased cell proliferation of thyroid cancer and promoted cell migration via epithelial-mesenchymal transition-related proteins. Moreover, MRPL14 knockdown reduced the expression of oxidative phosphorylation complex IV (MTCO1) and increased the accumulation of ROS. Cotreatment with a ROS scavenger restored cell proliferation and migration, which had been reduced by MRPL14 knockdown, implying that ROS functions as a key regulator of the oncogenic effects of MRPL14 in thyroid cancer cells.
Conclusion
. Our findings indicate that MRPL14 may promote cell growth, migration, and invasion by modulating ROS in thyroid cancer cells.

Keyword

Papillary Thyroid Cancer; Mitoribosomal Protein 14; Reactive Oxygen Species; Cancer Progression

Figure

• 

  Fig. 1. Abnormal expression of mitoribosomal proteins (MRPs) in The Cancer Genome Atlas (TCGA) patients with thyroid cancer (THCA). (A) Schematic diagram of the analysis of MRPs to define three distinctive signatures (up-MRPs, dn-MRPs, and other-MRPs). (B) Comparison of MRP expression between tumor and normal samples. (C) Comparison of the maximum absolute deviation (MAD) among MRPs’ expression between the thyroid cancer and normal group. (D) Heatmap displays the expression of 78 MRPs in 500 tumor samples and 57 normal samples from the TCGA thyroid cancer cohort. (E) Enrichment of three distinctive signatures (up-MRPs, dn-MRPs, and other-MRPs) in thyroid cancer patients (E1). Heatmap displays the expression of various MRPs (n=32) in thyroid cancer patients (E2). Bar graph shows the fold change in the expression of variable MRPs (n=32) between the tumor and normal samples (E3). FDR, false discovery rate; FC, fold change. ***P<0.001.

• 

  Fig. 2. MRPL14 is highly overexpressed in The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) thyroid cancer (THCA) cohort. (A) Volcano plot showing mitoribosomal protein (MRP) genes in the TCGA THCA cohort. (B) MRPL14 expression levels in 500 tumor and 57 normal samples in the TCGA THCA dataset. (C) Graph showing MRP genes in 364 tumor samples and 244 normal samples from the CNUH_THCA cohort. (D) The protein levels of mitochondrial ribosomal protein L14 (MRPL14) in five tumor–normal pairs were detected by immunoblot analysis. Values are presented as mean±standard error of the mean. **P<0.01, ***P<0.001.

• 

  Fig. 3. MRPL14 knockdown affects the progression of papillary thyroid cancer cells. (A) The expression of MRPL14 mRNA in thyroid cancer cell lines was examined by real-time polymerase chain reaction. (B) The expression of mitochondrial ribosomal protein L14 (MRPL14) protein in thyroid cancer cell lines was examined by Western blot analysis. B-CPAP (C) and KTC-1 (D) cells were transfected with small interfering RNA (siRNA) targeting MRPL14 #1, #2 or negative control siRNA for 48 hours. (E, F) After siRNA against MRPL14 (siMRPL14) transfection, cell proliferation was analyzed using the CCK-8 proliferation assay. (G, H) The expression of Caspase3, Bax and Bcl-xl were examined by Western blot analysis. (I-L) B-CPAP and KTC-1 cells were permitted to migrate for 24 hours in transwell chambers (migration) or in chambers with Matrigel (invasion). 1% Crystal violet staining. The migrated cells were counted under an optical microscope. (M, N) The expression of Slug, Snail, Ecadherin, N-cadherin, and vimentin was studied by immunoblot analysis. Values are presented as the mean±standard deviation of three inde­pendent experiments. NS, not significant.*P<0.05, **P<0.01.

• 

  Fig. 4. MRPL14 knockdown inhibits the expression of oxidative phosphorylation-related protein expression and increases intracellular reactive oxygen species (ROS) in papillary thyroid cancer cell lines. B-CPAP and KTC-1 cells were transfected with small interfering RNA (siRNA) of mitochondrial ribosomal protein L14 (MRPL14) #2 or negative control siRNA. The expression of ATP5A, UQCRC2, MTCO1, SDHA, and NDUFA9 was examined by Western blot analysis in B-CPAP (A) and KTC-1 (B) cells. The production of intracellular ROS was detected using H2DCFDA by flow cytometric analysis in B-CPAP (C) and KTC-1 (D) cells. (E, F) Gene set enrichment analysis results for negative regulation of response to oxidative stress and ROS are shown with normalized enrichment scores (NES) and P-values. The false discovery rate (FDR) for each gene set is noted. (G, H) Scatter plot showing the correlations between MRPL14 expression and that of RACK1 and PINK1. Values are presented as the mean±standard deviation of three independent experiments. **P<0.01, ***P<0.001.

• 

  Fig. 5. N-acetylcysteine (NAC) restores cell progression reduced by MRPL14 knockdown in papillary thyroid cancer cell lines. B-CPAP and KTC-1 cells were transfected with small interfering RNA (siRNA) of mitochondrial ribosomal protein L14 (MRPL14) #2 or negative control siRNA and co-treated with NAC for 48 hours. (A, B) Flow cytometric histogram shows intracellular reactive oxygen species in siMRPL14 transfection and NAC treated cell lines. (C, D) Cell proliferation was analyzed using a CCK-8 proliferation assay. (E, F) The expression of Bax and Bcl-xl were examined by immunoblot analysis. (G, H) The cells were permitted to migrate for 24 hours in transwell chambers. The migrated cells were counted under an optical microscope. 1% Crystal violet staining. (I, J) The expression of vimentin, Slug, and Snail was examined by immunoblot analysis. Values are presented as the mean±standard deviation of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

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