Knockdown of TMED3 inhibits cell viability and migration and increases apoptosis in human chordoma cells

Abstract

Chordoma is a rare low‑grade tumor of the axial skeleton. Over previous decades, a range of targeted drugs have been used for treating chordoma, with more specific and effective therapies under investigation. Transmembrane Emp24 protein transport domain containing 3 (TMED3) is a novel gene reported to be a regulator of oncogenesis, cancer development and metastasis; however, its role in chordoma remains unclear. In the present study, the expression of TMED3 was investigated in chordoma cells, and the effect of TMED3 knockdown on chordoma development was examined in vitro and in vivo, followed by exploration of differentially expressed proteins in TMED3‑silenced chordoma cells via an apoptosis antibody array. Reverse transcription‑quantitative PCR and western blot assays were performed to determine the expression levels. It was revealed that TMED3 was highly expressed in chordoma, and that knockdown of TMED3 inhibited cell viability and migration, and enhanced the apoptosis of chordoma cells. Additionally, knockdown of TMED3 inhibited the expression of Bcl‑2, heat shock protein 27, insulin‑like growth factor (IGF)‑I, IGF‑II, IGF binding protein‑2, Livin, Akt, CDK6 and cyclin D1 proteins, whereas MAPK9 was upregulated. Furthermore, a xenograft nude mice model demonstrated that TMED3 expression promoted tumor growth. Collectively, the present findings suggested that knockdown of TMED3 inhibited cell viability and migration, and enhanced apoptosis in chordoma cells, and that TMED3 may be a novel target for chordoma therapy.

Keywords: chordoma; transmembrane Emp24 protein transport domain containing 3; apoptosis; cell viability,; target therapy.

Figure 1

TMED3 is highly expressed in chordoma and knocked down following lentiviral transduction. (A) Expression of TMED3 was detected in MUG-Chor1 and U-CH1 cells via RT-qPCR analysis according to the Cq value (ΔCq=CqTMED3-CqGAPDH), as well as via western blot analysis. Expression was normalized to GAPDH. (B) Efficacy of lentiviral infection of chordoma cells using different shTMED3 sequences was detected via western blot analysis. Cells in the CON group were not exposed to any lentivirus. (C) Efficiency of lentiviral transfection was detected according to the fluorescence intensity after transfection for 72 h, as well as via western blot and RT-qPCR analyses. ^***P<0.001. Expression was normalized to GAPDH. Scale bar, 200 µm. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA; RT-qPCR, reverse transcription-quantitative PCR; Cq, quantification cycle.

Figure 2

Knockdown of TMED3 inhibits chordoma cell proliferation. (A) Cell proliferation ability of MUG-Chor1 and U-CH1 cells was detected using an MTT array. The OD value at 490 nm of each well was measured after cell culture for 1-5 days. (B) Fluorescence-activated cell sorting analysis was performed to determine the effects of TMED3 on the cell cycle progression of MUG-Chor1 and U-CH1 cells; the proportions of cells in G1, S and G2 phases were detected. ^***P<0.001 vs. shCtrl. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA; OD, optical density.

Figure 3

Knockdown of TMED3 inhibits chordoma cell migration. (A) Cell migration ability of MUG-Chor1 and U-CH1 cells was determined via a wound healing assay. The widths of scratches were detected following culture for 0, 24 and 48 h, and the migration rate of each group was calculated. ^**P<0.01, ^***P<0.001. (B) Transwell assays were performed to further evaluate the migration abilities of MUG-Chor1 and U-CH1 cells. After culture for 24 h, migrated cells were counted under a high-power light microscope (magnification, x200) and the migration rate was calculated. ^***P<0.001. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA.

Figure 4

Knockdown of TMED3 increases chordoma cell apoptosis. Fluorescence-activated cell sorting analysis was performed to identify the effects of TMED3 in the apoptosis of MUG-Chor1 and U-CH1 cells; the proportion of apoptotic cells was detected when cells were at 85% confluence following lentiviral transfection. ^***P<0.001. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA.

Figure 5

TMED3 promotes chordoma growth in vivo. (A) At 33 days after inoculation, the tumors in 10 nude mice were detected using an in vivo imaging system and the (B) total and (C) average radiant efficiency were measured, as well as the (D) fluorescence intensity. (E) Tumors in 10 nude mice were harvested and measured after euthanasia. (F) After inoculation, the volumes of tumors in mice were calculated. (G) Xenograft tissues from nude mice were harvested and subsequently stained with hematoxylin and eosin or reacted with anti-Ki67 antibody. ^**P<0.01, ^***P<0.001. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA.

Figure 6

TMED3 regulates the expression of genes involved in the cell cycle, apoptosis and proliferation. (A) Human apoptosis antibody array was performed to identify the effect of TMED3 on downstream genes associated with apoptosis. Proteins encoded by 43 different genes were detected. Dark blue denotes high relative expression and light green denotes low relative expression. FC, red denotes significant upregulation and dark blue denotes downregulation (P<0.05, FC >20% or <-20%). (B) Proteins associated with apoptosis, including Bcl-2, HSP27, IGF-I, IGF-II, IGFBP-2 and Livin, were detected in the human apoptosis antibody array and showed significant differential expression. ^*P<0.05. (C) Western blot analysis was performed to determine the effect of TMED3 on the expression of proteins related to cancer progression. TMED3, transmembrane Emp24 protein transport domain containing 3; sh, short hairpin (RNA); shCtrl, negative control shRNA; HSP27, heat shock protein 27; IGF, insulin-like growth factor, IGFBP2, IGF binding protein 2; p, phosphorylated; FC, fold change.