Knockdown of TFRC suppressed the progression of nasopharyngeal carcinoma by downregulating the PI3K/Akt/mTOR pathway

Abstract

Background: The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer. TfR is highly expressed in many cancers and is expected to be a promising new target for cancer therapy; however, its role in nasopharyngeal carcinoma (NPC) remains unknown.

Methods: The TfR levels were investigated in NPC tissues and cell lines using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. Knockdown of TFRC using two siRNA to investigate the effects on intracellular iron level and biological functions, including proliferation by CKK-8 assay, colony formation, cell apoptosis and cell cycle by flow cytometry, migration and invasion, and tumor growth in vivo by nude mouse xenografts. RNA sequencing was performed to find possible mechanism after TFRC knockdown on NPC cells and further verified by western blotting.

Results: TfR was overexpressed in NPC cell lines and tissues. Knockdown of TFRC inhibited cell proliferation concomitant with increased apoptosis and cell cycle arrest, and it decreased intracellular iron, colony formation, migration, invasion, and epithelial-mesenchymal transition in HK1-EBV cells. Western blotting showed that TFRC knockdown suppressed the levels of the iron storage protein FTH1, anti-apoptotic marker BCL-xL, and epithelial-mesenchymal transition markers. We confirmed in vivo that TFRC knockdown also inhibited NPC tumor growth and decreased Ki67 expression in tumor tissues of nude mouse xenografts. RNA sequencing and western blotting revealed that TFRC silencing inhibited the PI3K/Akt/mTOR signaling pathway.

Conclusions: These results indicated that TfR was overexpressed in NPC, and TFRC knockdown inhibited NPC progression by suppressing the PI3K/Akt/mTOR signaling pathway. Thus, TfR may serve as a novel biomarker and therapeutic target for NPC.

Keywords: Nasopharyngeal carcinoma; PI3K/Akt/mTOR signaling pathway; RNA-seq; TFRC; siRNA.

Fig. 1

TfR was upregulated in cancers, including nasopharyngeal carcinoma (NPC). (A) GEPIA database showed TFRC was widely upregulated in most cancer types, including head and neck squamous carcinoma (HNSC). (B) High TFRC expression in HNSC patients is associated with shorter overall survival. (C) TFRC was upregulated in NPC tissues than control tissues in four NPC GEO datasets. (D) TFRC was upregulated in 9 NPC cell lines than normal nasopharyngeal epithelial cell line NP69, as assessed by RT-qPCR. (E) TfR was upregulated in NPC tissues by IHC. Asterisks indicate *p < 0.05, ***p < 0.001 compared to control

Fig. 2

TFRC knockdown inhibited cell growth in vitro. (A) Successful knockdown of TFRC levels using two different siRNAs in HK1-EBV cells. (B) The intracellular Fe²⁺ visualized using FerroOrange was decreased after TFRC knockdown compared to the level in control cells. (C) Knockdown of TFRC inhibited the iron storage protein FTH1. (D) The CCK-8 assays showed knockdown of TFRC significantly suppressed cell proliferation from day 2 compared to that in control cells. (E) Representative images from colony formation cells, and the quantitative data indicate that knockdown of TFRC inhibited the colony formation ability in NPC cell line. (F) Representative distribution of the apoptotic cells using the Muse cell analyzer, and the quantitative data indicating total apoptotic cells significantly increased in siTFRC#1(23.9%) and siTFRC#2(22.7%) than that in the control (17.1%). (G) Knockdown of TFRC inhibited anti-apoptosis protein BCL-xL. (H) Representative cell cycle plots for the cells using Muse™ Cell analyzer, and the quantitative data indicating a significant arrest of siTFRC#1 group (54.4%) and siTFRC#2(58.4%) cells in the G0/G1 cell cycle phases compared to that in the control (43.5%). The results are given for three independent experiments. Asterisks indicate *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig. 3

Knockdown of TFRC inhibited cell migration, invasion, and epithelial–mesenchymal transition in vitro. (A) Representative images from wound healing assays showing knockdown of TFRC suppressed cell migration. (B) Summary bar graph showing percentage wound closure in TFRC knockdown group is lower than that in the control group at the 6 and 24 h time points. (C) Representative images of the invasion assay. (D) Summary bar graph showing the invaded cells in the TFRC knockdown cells as less than that in the control cells. (E-G) Knockdown of TFRC inhibits Vimentin and N-cadherin (mesenchymal phenotype), and increases E-cadherin (epithelial phenotype). The results are given for three independent experiments. Asterisks indicate *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig. 4

Xenograft tumor formation of HK1-EBV cell with knockdown of TFRC in nude mice. (A) There was no difference on mouse body weight. (B) Growth curves showed that knockdown of TFRC significantly decreased tumor volume from day 9 compared that in control mouse. (C) Nude mice were sacrificed 19 days after initial implantation and (D) surgically excised tumor tissues. (E) Average weight of implanted tumors in siTFRC#2-HK1-EBV and siCtrl-HK1-EBV xenograft-bearing mice. (F, G) The xenograft tumors were stained and show that TfR level was significantly downregulated, and the Ki67 staining was significantly decreased in TFRC knockdown group compared with control group, as assessed by IHC. Asterisks indicate **p < 0.01, ***p < 0.001 compared to control

Fig. 5

RNA sequencing analysis after TFRC knockdown in HK1-EBV cells. (A) mRNA level of TFRC  was significantly decreased in siTFRC#2-HK1-EBV samples, as assessed by RT-qPCR. (B) Proportion pie chart showing GO analysis of altered biological process after knockdown of TFRC, using DAVID database. (C) Proportion pie chart showed altering pathways after knockdown of TFRC, using KEGG data from DAVID database. (D) Knockdown of TFRC inhibited the phospho-Akt (p-Akt) level, which is the PI3K-Akt signaling pathway marker. (E) Knockdown of TFRC inhibited the mTOR level. The results are given for three independent experiments. Asterisks indicate *p < 0.05 compared to control

Fig. 6

Schematic diagram of the proposed mechanisms. TfR was overexpressed in NPC tissues and cells. Knockdown of TFRC led to iron deprivation and inhibited cell proliferation, migration, invasion, and EMT, which may function by inhibiting the activity of the PI3K/Akt/mTOR signaling pathway. TFRC participates in NPC development and may serve as a novel biomarker and therapeutic target for NPC.