Ubiquitin-conjugating enzyme E2T promotes tumor stem cell characteristics and migration of cervical cancer cells by regulating the GRP78/FAK pathway

2.1 Clinical specimens

Forty pairs of cervical cancer and adjacent normal tissues were obtained from patients which were pathologically diagnosed as cervical cancer. Patients with written informed consents were recruited at Affiliated Cancer Hospital of Xinjiang Medical University from 2016 to 2019.

1. Informed consent: Informed consent has been obtained from all individuals included in this study.

2. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration and has been approved by the Affiliated Cancer Hospital of Xinjiang Medical University.

2.2 qRT-PCR

RNAs were isolated from cervical cancer and normal tissues via Trizol (Invitrogen, Carlsbad, CA, USA). The RNAs were reverse-transcribed into cDNAs and conducted with qRT-PCR analysis of UBE2T using SYBR Green Master (Roche, Mannheim, Germany). The following primers for UBE2T (forward: 5′-TTGATTCTGCTGGAAGGATTTG-3′; reverse: 5′-CAGTTGCGATGTTGAGGGAT-3′) and GAPDH (forward: 5′-TGACCACAGTGGATGCCAT-3′; reverse: 5′-TTACTCCTTGGAGGCCATGT-3′) were used in this study. GAPDH was used as endogenous control.

2.3 Cell culture and transfection

Human cervical cancer cell lines (HeLa, Caski, Siha) and normal human endocervical epithelial cell line (Endl/E6E7) were obtained from Cell Resource Center of Shanghai Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (GE Healthcare Life Sciences, Little Chalfont, UK) containing 10% fetal bovine serum (GE Healthcare Life Sciences) at a 37°C incubator. For cell transfection, siRNA-mediated knockdown (si-UBE2T), pcDNA-mediated overexpression of UBE2T (pcDNA–UBE2T), and pcDNA-GRP78 were provided by GenePharma (Suzhou, China). HeLa and Caski were seeded into a 6-well plate and transfected with siRNAs and pcDNAs by Lipofectamine 2000 (Invitrogen).

2.4 Western blot

RIPA buffer (Ding Guo Chang Sheng Biotech, Beijing, China) was used to lyse the tumor tissues and cells on ice. The protein concentration of the supernatants collected from the lysates was calculated using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Samples (30 µg) were separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was probed with anti-UBE2T and anti-GAPDH (1:2,000; Abcam, Cambridge, MA, USA), anti-FAK and anti-p-FAK (1:2,500; Abcam), anti-SOX2 and anti-Oct4 (1:3,000; Abcam), anti-Nanog, or anti-GRP78 (1:4,000; Abcam). Following incubation with horseradish peroxidase-conjugated immunoglobulin G (1:5,000; Abcam), the blots were detected by enhanced chemiluminescence (KeyGen, Nanjin, China).

2.5 Cell viability and proliferation assays

HeLa and Caski under siRNAs or pcDNAs transfection were seeded into a 96-well plate for 24, 48, or 72 hours. MTT solution (5 mg/mL; Beyotime, Beijing, China) was added into each well and incubated for 4 h. Following dissolving of the formazan products by DMSO, absorbance at 490 nm was detected by a microplate reader (Bio-Rad, Hercules, CA, USA). For cell proliferation assay, HeLa and Caski under siRNAs or pcDNAs transfection were seeded in a 6-well plate and cultured for 14 days. Paraformaldehyde-fixed and crystal violet-stained cells were observed under a light microscope (Olympus, Tokyo, Japan).

2.6 Wound healing and invasion assays

For wound healing, HeLa and Caski cells under siRNAs or pcDNAs transfection were seeded into a six-well plate until 90% confluence. A pipette tip was used to induce a scratching wound in the well. Twenty-four hours later, the wound width was calculated under an inverted Olympus IX50 microscope. For invasion assay, HeLa and Caski cells in serum-free medium were placed into the upper chamber of Matrigel-coated Transwell chamber (Biosciences, San Jose, CA, USA). The lower chamber was supplied with medium containing 20% fetal bovine serum. After 24 h of incubation, cells in the lower chamber were fixed in 4% paraformaldehyde and stained with hematoxylin. Cells were counted under the light microscope.

2.7 Tumorsphere culture

HeLa and Caski under siRNAs or pcDNAs transfection were cultured in medium (DMEM/F12 basal medium containing 20 ng/mL basic fibroblastic growth factor, 20 ng/mL human recombinant epidermal growth factor, N2 and B27 supplements) (PeproTech Inc., Rocky Hill, NJ, USA) and seeded into a 24-well ultralow attachment plates at a density of 200 cells per well. Two weeks later, the tumorspheres were aroused and counted under the light microscope.

2.8 Statistical analysis

Results from three independent experiments were presented as mean ± SD. Statistical analyses between different groups were performed with one-way analysis of variance or Student’s t test under SPSS19.0 software. Values were considered significant at p < 0.05.

3.1 Upregulation of UBE2T in cervical cancer

Microarray analysis based on GEPIA (Gene Expression Profiling Interactive Analysis) database showed that UBE2T was upregulated in the cervical cancer tissues (n = 306) compared to the normal tissues (n = 13) (Figure 1a). Western blotting assay also identified the upregulation of UBE2T in the tumor tissues (Figure 1b) and cervical cancer cell lines (Figure 1c). Moreover, mRNA expression of UBE2T was also upregulated in the cervical cancer tissues compared with normal tissues (Figure 2b). These results suggested that UBE2T might be related to cervical cancer progression.

Figure 1

Upregulation of UBE2T in cervical cancer. (a) UBE2T was upregulated in the cervical cancer tissues (n = 306) compared to the normal tissues (n = 13) based on GEPIA database shown. (b) Protein and mRNA expression of UBE2T were upregulated in the cervical cancer tissues compared to the normal tissues. (c) Protein expression of UBE2T was upregulated in the cervical cancer cells (HeLa, Caski, and Siha) compared to the normal cells (End1/E6E7).

Figure 2

UBE2T contributed to cervical cancer cell growth. (a) Transfection with si-UBE2T in HeLa cells decreased the protein expression of UBE2T, while pcDNA–UBE2T increased the expression. (b) Cell viability of HeLa and Caski was reduced by si-UBE2T, while enhanced by pcDNA–UBE2T. (c) Knockdown of UBE2T repressed the cell proliferation of HeLa and Caski, while overexpression of UBE2T promoted the proliferation. (d) Knockdown of UBE2T repressed the cell invasion of HeLa and Caski, while overexpression of UBE2T promoted the invasion. (e) Knockdown of UBE2T repressed the cell migration of HeLa and Caski, while overexpression of UBE2T promoted the migration. *, ** vs siNC, p < 0.05, p < 0.01. ## vs pcDNA, p < 0.01.

3.2 UBE2T contributed to cervical cancer cell growth

HeLa and Caski were conducted with gain- and loss-of-functional assays through transfection with siRNA and pcDNA, respectively. Transfection with si-UBE2T decreased the protein expression of UBE2T, while pcDNA–UBE2T increased its expression (Figure 2a). Cell viability of HeLa and Caski was reduced by si-UBE2T, while enhanced by pcDNA–UBE2T (Figure 2b). Moreover, knockdown of UBE2T repressed the cell proliferation (Figure 2c), invasion (Figure 2d), and migration (Figure 2e) of HeLa and Caski. However, the cell proliferation (Figure 2c), invasion (Figure 2d), and migration (Figure 2e) of HeLa and Caski were promoted by overexpression of UBE2T. These results indicated the proliferative and pro-invasive effects of UBE2T on cervical cancer.

3.3 UBE2T contributed to stemness of cervical cancer cell

The effect of UBE2T on self-renewal capacity of cervical cancer cell was assessed through tumorsphere formation assay. UBE2T-silenced HeLa and Caski cells showed reduced typical tumorspheres compared with the control (Figure 3a), while overexpression of UBE2T enhanced the tumorspheres (Figure 3a). The stem cell-related transcription factors, including SOX2, Oct4, and Nanog, were decreased in UBE2T-silenced cells (Figure 3b) and increased in UBE2T-overexpressed cells (Figure 3b), revealing that cervical cancer stem cell traits were promoted by UBE2T.

Figure 3

UBE2T contributed to stemness of cervical cancer cell. (a) UBE2T-silenced HeLa and Caski cells showed reduced typical tumorspheres compared to the control, while overexpression of UBE2T enhanced the tumorspheres. (b) Knockdown of UBE2T decreased protein expression of SOX2, Oct4, and Nanog in HeLa cells, while overexpression of UBE2T increased the protein expression. ** vs siNC, p < 0.01. ## vs pcDNA, p < 0.01.

3.4 UBE2T contributed to GRP78/FAK activation

Protein expression of FAK was not affected by UBE2T-silencing or UBE2T-overexpressing in HeLa cells (Figure 4). However, FAK phosphorylation was reduced in UBE2T-silenced HeLa cells and enhanced in UBE2T-overexpressing HeLa cells (Figure 4). Moreover, GRP78 was also downregulated by knockdown of UBE2T, while upregulated by overexpression of UBE2T (Figure 4). HeLa was cotransfected with si-UBE2T and pcDNA-GRP78. Overexpression of GRP78 attenuated UBE2T silencing-induced decrease in p-FAK (Figure A1). Decreased cell viability in HeLa induced by silencing of UBE2T was also restored by overexpression of GRP78 (Figure A1), demonstrating that UBE2T contributed to cervical cancer cell growth and metastasis through GRP78/FAK activation.

Figure 4

UBE2T contributed to GRP78/FAK activation. Knockdown of UBE2T decreased protein expression of FAK phosphorylation and GRP78 in HeLa cells, while overexpression of UBE2T increased the protein expression. ** vs siNC, p < 0.01. ## vs pcDNA, p < 0.01.