RRS1 gene expression involved in the progression of papillary thyroid carcinoma

Abstract

Background: Papillary thyroid carcinoma (PTC) is one of the most frequent malignancies of the endocrine system, whose mechanisms of pathogenesis, progression and prognosis are still far from being clearly elucidated. Despite an increasing body of evidences highlights ribosome biogenesis regulator homolog (RRS1) as a ribosome biogenesis protein in yeast and plants, little is known about human RRS1 function.

Methods: Proliferation, cell cycle and apoptosis of PTC cells were assessed following the knockdown of RRS1 expression though MTT, colony formation assay, and flow cytometry. Then, transcriptome profiling was conducted to explore pathway changes after RRS1 silencing in PTC cells. Receiver operating characteristic curve and Youden's index were performed in twenty-four thyroid carcinoma samples to assess their potential clinical diagnostic value.

Results: Firstly, we found that silencing RRS1 significantly reduced cell proliferation, inhibited cell cycle, and promoted apoptosis in PTC cell line. The result also showed that knock-down of RRS1 could up-regulate genes involving apoptosis and metabolism, while, down-regulate genes relative to cell proliferation and blood vessel development. Notably, the present study confirmed the diagnostic value of RRS1 for thyroid carcinoma in both children and adults.

Conclusions: In conclusion, these data afford a comprehensive view of a novel function of human RRS1 by promoting cell proliferation and could be a potential indicator for papillary thyroid carcinoma.

Keywords: Cancer progression; Papillary thyroid carcinoma; RRS1.

Fig. 1

Knock-down of RRS1 inhibited TPC-1 cells proliferation. a, b Real-time PCR and Western blotting were performed to detect the efficiency of RRS1 knock-down. c Proliferation of TPC-1 cells was expressing shRNA targeted against negative and RRS1. Plotted data were mean ± s.e.m. of three independent experiments. *P < 0.05 from student T-test. d Colony formation assays of TPC-1 cells expressing shRNAs were quantified for relative colony numbers of three independent experiments. *P < 0.05 from student T-test. Three independent experiments were performed

Fig. 2

Knock-down of RRS1 induced G₂/M phase arrest and apoptosis in TPC-1 cells. a Left: one representative flow cytometry analysis of the cell cycle; right: histograms showed the percentage of cell subpopulations in G₁, S, and G₂/M phases. Data represent mean ± SE (n = 3). b Left: one representative flow cytometry analysis of the Annexin-V; right: histograms showed the percentage of cell subpopulations in M1 gate (Annexin-V positive cells). Data represent mean ± SE (n = 3). Three independent experiments were performed. *P value < 0.1, **P value < 0.01, ***P value < 0.001

Fig. 3

Microarray assay of RRS1 knock-down cells revealed dramatic alterations of the cancer cell transcriptome. a Heatmap of the 1127 genes altered in both negative control and RRS1 silencing, showing up- (red) and down- (green) regulated genes. b Pie chart showing 489 up- (red) and 683 down- (blue) regulated genes as calculated by both DESeq2 and edgeR algorithms with cutoffs as indicated. c Dot plots of IPA diseases and functions enriched in both knock-downs were shown in dots scaled by − log (P)

Fig. 4

Bioinformatics analyze of RRS1 knock-down cells revealed dramatic alterations of transcriptome in the thyroid cancer cells. a Dot plots of gene ontology terms enriched on RRS1 knock-down upregulated genes (top panel, red) and downregulated genes (bottom panel, blue) were shown in dots scaled by − log10 (P). b Example gene set enrichment analysis of genes altered in RRS1 knock-downs. Top panel: Ribosome related genes had a significant negative normalized enrichment score (NES) upon RRS1 knock-down. Middle-up panel: HES/HEY pathway related genes had a significant NES upon RRS1 knock-down. Middle-down panel: MEF2D pathway related genes had a significant NES upon RRS1 knock-down. Bottom panel: Glycan degradation gene set had a significant positive NES upon knock-down of RRS1. c Summary of microarray assay by multiple bioinformatics tool

Fig. 5

Real-time PCR detected the difference expression and diagnostic value of RRS1 for thyroid carcinoma in children and adults. a Expression of RRS1 in PTC (mean = 1.51, n = 24) is higher than that in pericarcinous tissues (mean = 0.54, n = 14, unpaired t-test, P < 0.01). b Real-time PCR detects RRS1 expression in 12 paired carcinoma and pericarcinous tissues (paired t-test, P < 0.01). c Pearson correlation coefficient measures the relationship between RRS1 expression and age in PTC tumors (R = 0.2638). d Receiver operating characteristic (ROC) curve of 22 PTC tissues and 12 pericarcinous tissues (AUC is 0.850, P < 0.0001). *P value < 0.1, **P value < 0.01, ***P value < 0.001