Cell culture
C33A cell [American Type Culture Collection (ATCC), Rockville, MD, USA], which is an HPV16 E6/E7-negative cell line, were maintained in 10 ml of 90% Eagle's minimum essential medium ( Gibco, Thermo Fisher Scientific, Inc., MA, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA). The CaSki cell (ATCC, Rockville, MD, USA), which is an HPV16 E6/E7-positive cell line, were maintained in 10 ml of 90% RPMI-1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA). After mixing, 1 ml of the medium was added to each well of the culture plate and placed in a 5% CO2, 37(C incubator.
The differentially expressed gene was selected by sequencing
siRNA sequences are synthesized by Sangon Biotech (Shanghai) Co., Ltd. sh-E6: F:5´-GGAGCGACCCAGAAAGUUATT-3´,R:5´-UAACUUUCUGGGUCGCUCCTT-3´;Then the interfering shRNA was screened and transfected into CaSki cell. Finally, CaSki cell and siRNA cell were sent to the personalbio (Shanghai, China) for sequencing and screening for differential miRNA. Sample processing: After total RNA was extracted, the RNA quality was detected. Then the small RNA library was constructed by IlluminatheTruSeq Small RNA Sample prep Kit. The enriched library was amplified by PCR and purified by gel electrophoresis. The library quality was checked by Agilent High Sensitivity DNA Kit using Agilent 2100 Bioanalyzer. After testing the qualified library, the library was quantified by Quant-iT Picogreen dsDNA Assay Kit. Finally, single strand library was used as template to bridge PCR amplification, and primers were annealed and sequenced.Data analysis: The original data is de-joined and filtered, and the filtered sequence is de-re-processed. The deleted small RNA sequence is annotated on the basis of the reference genome and the corresponding annotation abundance is given. After statistical analysis, the differentially expressed miRNAs were clustered and predicted, and the predicted target genes were enriched and analyzed.
Construction of miR-4454 mimics and miR-4454 inhibitor
The sequence of the hsa-miR-4454 mature body is 5′-GGAUCCGAGUCACGGCA CCA-3′(http://www.mirbase.org/cgi-bin/mature.pl?mature_acc=MIMAT0018976), which was synthesized by GenScript (Nanjing) Co., Ltd. (Nanjing, Jiangsu, China). The miR-4454 mimics/inhibitor (RIBOBIO, Guangzhou, China) was then successfully constructed based on this sequence.
Construction of pre-ABHD2/NUDT21 and sh-ABHD2/NUDT21 plasmids
The template strand sequences were as follows: ABHD2 positive-sense 5′-GATCGCCAATGGGAGCGTAACAAGTTTCAAGAGAACTTGTTACGCTCCCATTGGCTTTTTT-3′,antisense:5′-AATTAAAAAAGCCAATGGGAGCGTAACAAGTTCTCTTGAAACTTGTTACGCTCCCATTGGC-3′;NUDT21positive-sense:5′-GATCGCGCATGAGGGAAGAATTTGATTCAAGAGATCAAATTCTTCCCTCATGCGCTTTTTT-3′,antisense:5′-AATTAAAAAAGCGCATGAGGGAAGAATTTGATCTCTTGAATCAAATTCTTCCCTCATGCGC-3′. The small hairpin RNA (shRNA) sequences were then obtained by polymerase chain reaction (PCR) amplification, and inserted in the vector plasmid pSIREN connected by EcoRI and BamHI enzymes (Thermo Fisher, MA, USA). Finally, the recombinant plasmids pSIREN-shRNA-ABHD2 and pSIREN-shRNA-NUDT21 were amplified and infected into the CaSki and C33A cells.The template strand sequence of ABHD2 was as follows: forward primer 5′-CGCGGATCCATGAATGCCATGCTG-3′, reverse primer 5′-CCGCTCGAGTCACT CCAGGTCGGCCT-3′. The vector plasmid pcDNA3.1 was digested and connected by EcoRI and XhoI enzymes (Thermo Fisher, MA, USA). The recombinant plasmids pcDNA3.1-pre-ABHD2 and pcDNA3.1-pre-NUDT21 were amplified and infected into the CaSki and C33A cells. The template strand sequence of ABHD2 was as follows: forward primer 5′-CGCGGATCCATGTCTGTGGTAC CG-3′, reverse primer 5′-CCGCTCGAGTCAGTTGTAAATAAAATTG-3′.’
Western blot
Total protein were extracted by RIPA buffer(Beyotime, Shanghai, China) involved in protease inhibitors. The protein concentration measured by BCA protein assay kit (Thermo Fisher Scientific). The supernatant soluble lysate was mixed with loading buffer and the solution boiled for 10 min and stored at -20℃. Isolated proteins were electrophoresed on 8% SDS-PAGE electrophoresisand transferred to a 0.22-μm PVDF membrane and blocked with 5% nonfat milk. Afterward, the membranes were then incubated with primary antibodies against GAPDH (1:5000, ab181602, Abcam, Shanghai, China), ABHD2 (1:1000, ab23041, Abcam, Shanghai, China), NUDT21(1:3000, ab183660, Abcam, Shanghai, China) overnight at 4℃. The membranes were then incubated with the HRP-conjugated secondary antibody (1:10,000, Santa Cruz) and the protein level detected by chemiluminescence .
Transwell assay
A transwell assay was performed to investigate the migration and invasion of C33A and CaSki cells. The cells were harvested for 24 h before cell suspension, and then the cells were digested to be used as signal cells at a 5 × 105/ml concentration. The cells were suspended using a serum-free medium containing bovine serum albumin, and 100 µl of the cell suspension was added to the transwell chamber. The 24-well plate chamber was filled with 600 µl of medium per well containing 10% FBS, and then the plate was incubated for 24 h at 37(C. After incubation, tweezers were used to carefully remove the chamber. The upper chamber fluid was drained and approximately 800 µl of methanol was added to each well for fixation for 30 min at room temperature. The chamber was taken out and dyed with 1 ml of 0.5% crystal violet solution for 30 min. The cells were observed using a microscope (TS100-F, Nikon, Tokyo, Japan). The experimental protocol for the invasion assay of C33A and CaSki cells was similar to that of the migration assay, except for the addition of Matrigel (BD, NJ, USA) and the serum-free medium diluted at a 1:8 ratio at 4(C. The plate was coated with the upper chamber surface of the bottom membrane in the transwell membrane. The Matrigel was polymerized to a gel at 37(C for 30 min.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay
The logarithmic-phase cells from cell subculture were plated in 96-well plates with 180 µl (5 × 103 cells) per well, and the plates were incubated at 37(C overnight. The effect of miR-4454, ABHD2, and NUDT21 on the proliferation of HPV16-positive CaSki cells and HPV16-negative C33A cells was detected at 24 h, 48 h, and 72 h. After the addition of 20 µl MTT (Beyotime, Shanghai, China) solution (5 mg/ml) to the cell culture plates of different groups, the plates were incubated for 4 h. After the supernatant was removed, 150 μl of dimethyl sulfoxide (Beyotime) solution was added to each well and mixed, and the absorbance value of each well was measured at 490 nm.
Flow cytometry assay
After the C33A and CaSki cells of the different treatment groups were collected, they were centrifuged at 1000 ×g for 5 min, and then suspended by phosphate-buffered saline (PBS) and counted. Approximately 2 × 106 cells/ml were suspended with binding buffer, and then centrifuged at 300 ×g for 10 min.14 Subsequently, 5 µl Annexin V-FITC (Solarbio, Beijing, China) was added to 100 µl of the cell suspension and mixed at room temperature for 10 min, followed by the addition of 5 µl propidium iodide and mixed at room temperature for 5 min. Finally, the PBS was added to reach a constant volume (500 µl), and cell apoptosis was detected by flow cytometry within 1 h.
Reverse transcription-quantitative PCR (RT-qPCR) assay
CaSki and C33A cells were harvested and the total RNA was extracted using Trizol reagent (Ambion, TX, USA). The total RNA was reverse-transcribed to cDNA by PrimeScript™ RT Master Mix (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The mRNA was quantified by SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian, China). The following primers were designed by Nanjing Genscript Biological Technology Co., Ltd.: miR-4454F:5′-GGGGGATCCGAGT CA-3′,R:5′-AACTGGTGTCGTGGAGTCGGC-3′;ABHD2F:5′-CGTTGACTACGCCCAGAA-3′,R:5′-AAGCCCACGACGACCAG-3′;NUDT21F:5′-AAACTACCTGGTGGTGAACT-3′, R:5′-CTTAGGCTTTGTAATATGTGC-3′;U6 F:5′-CTCGCTTCGGC AGCACA-3′, R:5′-AACGCTTCACGAATTTGCGT-3′; β-actin F: 5′-ACACTGTG CCCATCTACG-3′, R:5′-TGTCACGCACGATTTCC-3′. The experiment was executed independently in three biological replicates. General thermal cycling conditions for PCR: 95 °C, 3 min; 95 °C, 5 sec; 56 °C, 10 sec; 72 °C, 25 sec;(39 cycles) 65 °C, 5 sec; 95 °C, 50 sec.
Dual luciferase reporter gene assay
The 3′ untranslated regions (UTRs) of the ABHD2 and NUDT21 genes were cloned and amplified in 293 cells. Mutations of the 3′ UTR of the ABHD2 and NUDT21 genes were generated by the QuickChange Site-Directed Mutagenesis kit (Stratagene, CA, USA) at the putative binding site of miR-4454. The wild and mutant ABHD2/NUDT21 genes were cloned into the pmirGLO-vector (Promega, WI, USA) and then co-transfected with pmirGLO-3’ UTR-ABHD2 (50 ng) or pmirGlO-3′ UTR-NUDT21 (50 ng) and miR-4454 (50 nM) using Lipofectamine® 2000 (Invitrogen, MA, USA). The reporter gene plasmid and transcription factor expression plasmid-co-transfected 293 cells were seeded in 24-well plates and incubated for 12 h. After transfection, cell lysates were prepared for 48 h using Passive Lysis Buffer (Promega, WI, USA). The dual luciferase activity was then examined using the Luciferase Assay System (Promega, WI, USA).
Statistical analysis
All data were analyzed by the data analysis software SPSS19.0 (SPSS, IL, USA), and are presented as the mean ± standard deviation. Data were analyzed by one-way analysis of variance followed by Dunnett's or Duncan's test. A P-value less than 0.05 was considered statistically significant.