CC tissue specimens (n =104) and cervicitis tissue (NC, n =31) were collected from patients who underwent surgical resection at the First Affiliated Hospital of Xinjiang Medical University (Urumqi, Xinjiang, China) after obtaining written informed consent in accordance with the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University.
Human CC cell lines (MS751, SiHa, HeLa, and Caski), immortalized cervical epithelial cell line H8, and human lymphatic endothelial cells (HLECs)were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). SiHa, HeLa, and H8 cells were cultured in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA). MS751 cells were cultured in MEM (containing NEAA) medium with 10% FBS, and the Caski cells were cultured in RPMI-1640 medium supplemented with 10% FBS. All cell lines were authenticated by STR profiling test to confirm their identities and in an incubator with 5% CO2 at 37 °C. Human lymphatic endothelial cells (HLECs) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in the ECM (ScienCell, CA, USA).
Lentiviral or plasmid transduction
To knockdown the expression of RACK1, a lentivirus containing short hairpin RNA (shRNA) targeting RACK1 or a non-target oligonucleotide was synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China). The shRNA target sequences were 5′- AGCTGAAGACCAACCACAT-3′ (shRACK1-1) and 5′- TGTGGTTATCTCCTCAGAT -3′ (shRACK1-2). Stable cell lines were selected using 5 μg/mL puromycin (Sigma-Aldrich, USA), and knockdown efficiency was confirmed by western blot assays. The POU2F2 overexpression plasmid and control plasmid were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). Plasmid transfections were performed using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. The OE target sequences are listed in Supplementary Table 1.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was extracted from CC cells using TRIzol reagent (Invitrogen, CA, USA). Reverse transcription of the extracted RNA was performed using a RevertAid First Strand cDNA Sythesis Kit (Thermo, MA, USA) following the manufacturer’s instructions. PCR amplification was performed using QuantiNova SYBR Green PCR Kit (QIAGEN, Frankfurt, Germany). The relative expression of target genes was calculated using the 2-△△CT method, and β-actin was used as a reference gene for normalization. Primer sequences used are listed in Supplementary Table 2.
Western blot analysis
Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease/phosphatase inhibitor cocktail (Solarbio, Beijing, China). Proteins (30 μg) were separated in SDS-PAGE gels and transferred onto PVDF membrane (Millipore, USA). The membranes were then blocked with 5% milk and probed with primary and secondary horseradish-peroxidase- labeled antibodies. After washing thrice, the signaling was detected using an enhanced chemiluminescence detection system (ECL, Biosharp, Anhui, China). The primary antibodies used in this study are listed in Supplementary Table 3.
Immunohistochemistry (IHC) analysis
IHC analysis was performed on formalin-fixed, paraffin-embedded sections of clinical CC and mouse xenograft tissues. Paraffin-embedded tissue sections (4 μM) were rehydrated, blocked with 3% hydrogen peroxide, and treated with hot EDTA-mediated buffer. Next, primary antibodies were added and incubated overnight at 4°C. The results were determined using an IHC detection kit (ZSJQ-Bio, Beijing, China), according to the manufacturer’s protocol. Staining intensity was scored independently by two observers. Briefly, the standard for the proportion of positive staining (1, < 5%, 2, 5–30%, 3, 30–70%, 4, > 70%) and staining intensity (0, no staining, 1, weak, 2, moderate, 3, strong) were multiplied for each observer and then averaged. The antibodies used and their corresponding experimental conditions are listed in Supplementary Table 3.
1H NMR examination
The 1H NMR experiment was used for metabolomics analysis as described previously. Briefly, cell supernatant samples were defrosted at room temperature. Each sample (200 μL) was mixed with 400 μl of saline (0.9% w/v NaCl in 20% v/v D2O and 80% v/v H2O). The mixture at room temperature, and vortex-mixed followed by centrifugation (4°C, 10,000 rpm, 10 minutes). Then, 550 uL supernatant was transferred into a 5-mm NMR tube for 1H NMR (Varian 600 spectrometer with 599.93 MHz resonance frequency of 1H NMR). Transverse relaxation weighting experiments were performed as per the Carr–Purcell–Meiboom–Gill sequence with water peak suppression. Parameters were set as follows: relaxation delay 2.0 seconds, acquisition time 1.64 seconds, spectral width 10,000 Hz, which resulted in an acquisition time of 1.64 sec and a relaxation delay of 2 sec. Raw data were processed according to previous studies. Briefly, 1H NMR spectra were processed and corrected for phase and baseline with Topspin 2.0 software (Brokers Biospin, Rheinstetten, Germany).we manually phased and baseline-corrected the 1H NMR spectra, which were referenced to the anomeric proton of α-glucose at δ 5.233 and the spectra were put into 2,834 integrated regions of 0.003 ppm. The regions of water resonance (δ 4.66–5.20) were excluded for eliminating the baseline effects. Then, we calculated the peak area of each bin. Normalization were performed with the software package SIMCA-P+11 (Umetrics Inc., Umea, Sweden) to compensate for the concentration differences among the samples.
Transwell migration assay of CC cells or HLECs
For the Transwell migration assay of CC cells, 2 × 104 cells were placed in the upper chamber of a Transwell (Corning, NY, USA). Basic medium containing 10% FBS was added to the lower chamber as chemoattractant and the cells were incubated at 37°C for 24 h. For HLECs migration, HLECs (1 × 105) were placed in the upper chamber with serum-free EGM, whereas the lower chamber was filled with conditioned medium derived from 1 × 105 CC cells with EGM medium at 1:1 ratio as chemo attractants and incubated at 37°C for 24 h. The cells attached to the reverse side of the membrane were stained with 0.1% crystal violet and counted under inverted microscope (Nikon E400, Tokyo, Japan) in randomly selected five fields.
Matrigel invasion assay
Matrigel-coated invasion chamber (BD Biosciences, NY, USA) was used to assess cell invasion. Briefly, 2 × 104 CC cells were seeded in the upper chamber of the Transwell insert in serum-free culture medium and cultured for 24-48 h. Penetrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of penetrating cells in each group was counted under the inverted microscope (Nikon E400, Tokyo, Japan).
Determination of glucose consumption and lactate production
Glucose and lactate detection kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) were used to determine glucose and lactate concentrations before and after 24 h of CC cell culture, following the manufacturer’s protocols.
Co-IP assays were conducted to detect the interaction between RACK1 and IGF1R using protein A/G magnetic beads (Bimake, Shanghai, China). MS751 and SiHa cells were collected, lysed, and centrifuged at 4°C and 12,000 rpm for 20 min to obtain the supernatant, which was then mixed with primary antibody and incubated overnight at 4°C. Protein A beads were added and incubated for an additional 4 h. The beads were washed three times with PBST buffer for 5 min each time, 30 μL of loading buffer was then added and boiled for 5 min at 100°C. The supernatant was then used for western blot analysis.
Cells were seeded on coverslips and incubated with antibodies specific for IGF1R and RACK1 at room temperature for 2 h. Next, the coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG for 2 h and stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma, MO, USA) for 10 min. Randomized fields were captured using a microscope.
HLEC tube formation assay
Serum-free conditioned medium was prepared by culturing CC cells (1 × 107 cells per 10 cm dish) in 10 mL serum-free medium for 24 h. The media were collected and centrifuged at 1000 rpm for 5 min to remove cell debris and stored at 4°C to concentrate the conditioned medium. HLECs were seeded into 24-well plates (precoated with 100 μL Matrigel) containing cell conditioned medium and incubated for 10-16 h. Tube formation was quantified by measuring the total length of tube structures or the number of branch sites/nodes in three random fields.
Dual luciferase reporter assay
A plasmid with the RACK1 promoter was designed and constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). The wild-type and mutant sequences of the RACK1 promoter, named GV712-RACK1-WT and GV712-RACK1-mut, were amplified and cloned into the GV712-basic vector. The cells were then collected and lysed 48 h after transfection, and luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, WI, USA) and a luciferase assay kit (Bio-Vision Technologies, PA, USA).
Chromatin immunoprecipitation-PCR (ChIP-PCR) analysis
The chromatin immunoprecipitation assay was performed according to the manufacturer’s instructions and protocols. Cells were (1× 107) fixed with 1% formaldehyde for 10 min at room temperature, and the fixation was stopped with 0.125 M glycine, the cell lysis buffer was then added, and the samples were sonicated to generate 200–1000 bp fragments. The resulting cell lysates were immunoprecipitated using a POU2F2 antibody (Sanying, Wuhan, China) and analyzed using RT-PCR. Anti-POU2F2 antibody and primers used for the PCR assay of ChIP samples are listed in Supplementary Table 1.
All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. Xenograft models were established as previously described. Female BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. For the xenograft LNM model, 5 × 105 cells were inoculated into the footpads of the mice. The mice were sacrificed by cervical dislocation four weeks after the first injection of tumor cells. Footpad tumors and popliteal lymph nodes were removed by hematoxylin and eosin (HE) staining.
The results are presented as the mean ± standard error of the mean (SEM). IBM SPSS Statistics for Windows, version 23.0(IBM Corp., Armonk, NY, USA) and GraphPad (5.0 version) were used for the statistical analyses. Two-tailed Student’s t-test and one-way ANOVA with Tukey’s post-hoc test were used for comparisons between two or multiple groups, respectively. The relationships between RACK1 expression and clinicopatho-logical characteristics were evaluated using Pearson’s χ2 test or Fisher’s exact test. Correlations between measured variables were analyzed using Spearman’s rank correlation analysis. P < 0.05 was considered statistically significant (*, p < 0.05, *, p < 0.01, **, p < 0.001, ***).