2.1 Tissue specimens
Total of twenty-one CSCC and eighteen normal cervical epithelial (NCE) tissues used in this study were collected at Nanfang Hospital from September 2019 to September 2020. Informed consent was obtained from all patients. No patients had been treated with radiotherapy or chemotherapy before surgery and all specimens preserved in sample protector for RNA/DNA (Takara, Japan) at −80°C. This study was approved by the Ethics Committee of Southern Medical University.
2.2 Cell culture
The human cervical squamous cancer cell lines CaSki, SiHa and HEK293T cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All the cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L D-glucose) supplemented with 10% (v/v) fetal bovine serum (FBS) and incubated at 37°C in the humidified incubator with 5% CO2.
2.3 Cell transfection
The small interfering RNAs (siRNAs) separately targeting HPV16 E6 (si-E6), HPV16 E7 (si-E7), MIR210HG (si-MIR210HG), HIF-1α (si-HIF-1α), PGK1 (si-PGK1) and a scrambled oligonucleotide control were purchased from GenePharma (Shanghai, China). The sequences of siRNA used for RNA interference are listed in Table S2. The short hairpin RNA (shRNA) vector specific to MIR210HG (sh-MIR210HG) and a scrambled negative control vector were obtained from Genechem (Shanghai, China). To construct an overexpression plasmid, the full-length of MIR210HG (NR_038262) was synthesized and cloned into the pcDNA3.1 vector by Genechem (Shanghai, China).
Cell transfection was performed using Lipofectamine™ 2000 Transfection Reagent (Invitrogen). The stably shRNA transfected cells were screened under G418 (Genview) pressure. Cells were harvested for further analyses 24–72 h after transfection.
2.4 RNA isolation, cDNA synthesis and qRT-PCR
Total RNA was extracted from the cells or tissues using RNAiso Plus reagent (Takara) following the manufacture’s protocol. The concentration and purity of extracted RNA were assessed by NanoDrop 2000 (Thermo). The reverse transcription reactions were performed by using a PrimeScript RT reagent kit (RR047A, TaKaRa). qRT-PCRs were performed with a SYBR Premix Ex Taq kit (RR420A, TaKaRa) on a ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression levels relative to β-actin were calculated by 2−ΔΔCT method. All the primer sequences used in this study are listed in Table S1.
2.5 CCK-8 proliferation assay
After transfection, CaSki and SiHa cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3 × 103 cells per well and incubated for 1.5 h at 37 °C and 5% CO2 before the optical density (OD) at 450 nm was detected. Proliferation was measured by the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) every 24 h after transfection for 4 days.
2.6 Transwell assay
For migration and invasion assays, transwell assay was conducted using 24-well transwell chamber pre-coated with or without Matrigel (BD). Treated CaSki and SiHa cells (4 × 104/well) in serum-free DMEM medium were added to the upper chamber and 20% fetal bovine serum medium was added in the lower chamber. Cells were cultured at 37 °C with 5% CO2 for 24 h and then those on the lower surface were fixed with methanol and stained with 0.1% crystal violet. Cells were counted in five randomly selected areas under microscope field.
2.7 Western blot
Total cell lysates were prepared using immunoprecipitation assay lysis buffer containing protease and phosphorylase inhibitors and clarified by centrifugation (12 000 g for 15 min at 4°C). The protein concentration was detected by a BCA Protein Assay Kit (Beyotime). Proteins were mixed with loading buffer, then cell lysates were separated by sodium dodecyl sulphate SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were subsequently blocked in 5% skimmed milk for 2 h. Afterwards, primary antibodies against E6 (ab70, Abcam), E7 (ab20191, Abcam), HIF-1α (36169S, CST), PGK1 (17811-1-AP, Proteintech) were incubated overnight at 4°C. The next day, the membrane was incubated with second antibody at room temperature for 2 h, washed with TBST and then developed with the ECL system and normalized to the gray value of β-actin.
2.8 Isolation of nuclear and cytoplasmic RNA
Cells were partitioned into nuclear and cytoplasmic fractions using the PARIS™ Kit (Invitrogen) according to the manufacturer’s instructions. RNA isolated from each of the fractions was analyzed by qRT-PCR to determine the expression levels of MIR210HG, nuclear control transcript (MALAT1) and cytoplasmic control transcript (GAPDH).
2.9 Luciferase reporter assay
The length of MIR210HG promoter region containing two kilobase (kb) was constructed into pGL3-based vectors. To determine the effect of HIF-1α on MIR210HG promoter, pGL3-based construct containing MIR210HG promoter sequences and renilla luciferase reporter plasmid were cotransfected into HEK293T cells with or without HIF-1α knockdown. Cultured under normoxia or 250 µM CoCl2 24 h after transfection, firefly and renilla luciferase activity were measured by dual-luciferase reporter assay kits (Beyotime). The ratio of firefly luciferase to renilla activity was calculated for each sample.
2.10 Xenografted tumor and pulmonary metastasis model in vivo
All animal experiments were approved by the Institutional Committee on Animal Care and Use of Southern Medical University. The female nude mice between 4 and 5 weeks were purchased from Beijing HFK Bioscience Co., Ltd. SiHa cells transfected with the negative control vector or the sh-MIR210HG vector were paired, 1 × 107 cells were inoculated subcutaneously into either side of flank of the same female nude mouse and 1 × 106 cells were injected intravenously into tail. The tumor volumes were measured every week and calculated as Length × Width2 × 0.5. After 5 weeks, the mice were euthanized after injection and the tumors were fixed for hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining.
2.11 Statistical analysis
For the cell functional analyses, results are presented as mean ± standard deviation (SD). The comparison of means between two groups was conducted using Student’s t test, while one-way analysis of variance was used for multiple comparisons. Correlation was calculated according to Pearson correlation. P < 0.05 is considered significant. All the experiments were repeated at least three times.