HeLa cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences in 2018. HeLa cell was maintained as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS,Sijiqing Biological Engineering Materials Co., Hangzhou, China) at 37°C in the presence of 5% CO2-balanced air. The other three stable transformants was maintained as a monolayer in DMEM with 10% FBS and 2ug/ ml puromycin (Sigma, German).
To induce hypoxia, HeLa cell was rendered in a chamber with a gas mixture of 1% O2 and 5% CO2-balanced N2 at 37°C. The level of oxygen in the chamber was verified using a gas monitor (SKC, Inc., Eighty Four, PA). To mimic hypoxia using chemicals, cells were cultured under 20% oxygen in the presence or absence of 100 µM CoCl2 for a specified time period.
Plasmid Construct And Transfection
Full-length human HIF3A cDNA was cloned into a Pcmv-ORF-flag-his expression vector and HIF1A was cloned into a pBabe-puro-HA expression vector (TranSheepBio, Shanghai, China). Transfection was performed with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) 24 h after cell seeding. Lentiviral constructs containing up-regulating miR-630 (LV-hsa-mir-630), inhibiting miR-630 (LV-hsa-mir-630-inhibitor) and the negative control lenti-vector (LV-Negative Control) were designed and provided by Genechem lnc. (Shanghai, China). 60–70% confluent HeLa cell was infected with three lenti-virus at multiplicity of infection (MOI) of 10 with enhanced infection solution (ENI.S) according to the manufacturer’s protocol. 10 hours post-infection, viruses were replaced by complete DMEM and 48 hours post-infection, three stably infected cells (LV-hsa-mir-630, LV-hsa-mir-630-inhibitor, LV-Negative Control) were selected by DMEM with 2 µg/ ml puromycin (Sigma, German). The expression level of miR-630 in the stable transformants were identified by qRT-PCR.
Quantitative Real-time Pcr
The expression level of miR-630 in the cells were determined using real-time PCR. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcription was performed according to manufacturer’s instructions (638315, Clontech Laboratories, Inc. A Takara Bio company, USA). qRT-PCR was performed to determine the expression level of miR-630 using the exact sequences (U to T) of miR-630 as the forward primer and the unique q-PCR primer from the cDNA Synthesis Kit as the reverse primer. U6 was used as an internal control, and each plate contains one cDNA sample for each primer as a correct sample. Primers were shown in Table S1.
Transwell Chamber Assay
The invasive ability of cells was performed in 24-well transwell chambers (Corning, NY). The polycarbonate filters containing 8-µm pores were coated on ice with 80 µl of Matrigel (Sigma-Aldrich) at 5 mg/L. After blocking with 1% BSA for 1h at 37°C the cells (5 x 105/mL) were suspended in serum-free culture medium, 100µl were added to the upper compartments of chamber. In each lower chamber, 500µl of medium (10% FBS) was added. After 24h incubation the cells from the upper compartment were removed, and the cells on the lower surface were fixed in ethanol and stained with hematoxylin-eosin. Cells on the lower surface were quantified in 10 random microscopic fields per filter at a magnification of x200 (DMI4000B, LEICA, Germany).
Western Blot Analysis
Western blot analysis
When 80% confluence in 25 cm2 flasks (Nunc, Roskilde, Denmark) was reached, the cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) on ice, and then centrifuged at 12000 rpm for 20 min. Supernatants were collected and protein was determined using Bicinchoninic acid (BCA) kit (Boster, Wuhan, China). The extracts (20 µg per lane) were fractionated by 10% SDS-PAGE and then transferred onto PVDF membranes (0.45 µm; Millipore, Bedford, MA). After blocking with TBST buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% non-fat milk for 1h at 25℃, the membranes were incubated with primary antibodies against E-cadherin、N-cadherin (1:5000,Epitomics, USA), Cytokeratin (1:400, Boster, China), EP300 (1:1000, Abnova, USA) and β-actin (1:5000, Cmctag, USA) overnight at 4°C. Membranes were washed three times x 5 min with TBST before incubation with horseradish peroxidase–conjugated secondary antibody (1:3000, CST, USA) for 60 min at 25℃. The membranes was exposed by chemiluminescence (Millipore, Billerica, MA), and images were acquired by ChemiDoc XRS (Bio-Rad, Hercules, CA). Semi-quantification of scanned films was performed using Quantity One-4.6.2 (Bio-Rad).
Cells were grown on 24-well m-Slides (NEST Biotechnology Co.LTD. Wuxi, China) and fixed with 4% paraformaldehyde for 30 min at 4°C, followed by treatment with 0.1% Triton for 10 min. The samples were blocked with PBST buffer (0.1% Tween-20) containing 10% goat serum at room temperature for 1 h and incubated with primary antibodies E-cadherin, N-cadherin (1:500, Epitomics, USA), Cytokeratin (1:200, Boster, China), for overnight at 4°C. The cells then washed in PBST and incubated with DyLight 488 and DyLight 594 conjugated secondary antibodies (ZS-Bio) at room temperature in the dark for 1 h. Nuclei were counterstained with DAPI for 5 min. After washing three times, the cells were maintained with 50% glycerin in PBS and observed by laser confocal microsopy (Fluoview FV1000; Olympus, Tokyo, Japan). Photographs were taken through a digital camera (Olympus Fluoview FV1000) attached to the microscope. Ten images (approximately 30 cells per field) were acquired in each group, the quantification of gray value was analyzed with Olympus Fluoview software FV10-ASW 1.7.
Rna-seq Library Construction And Sequencing
5µg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo (dT)-conjugated magnetic beads (invitrogen) before directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95℃ followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. PCR products corresponding to 200–500 bps were purified, quantified and stored at -80℃ until sequencing. The libraries were prepared and applied to illumina NextSeq 500 system with 150x2 paired-end type (ABLife Inc., Wuhan, China).
Mirna-seq Library Construction And Sequencing
Total RNA (3µg) was used for small RNA cDNA library preparation with Balancer NGS Library Preparation Kit for small/microRNA (Genome Gen). Briefly, RNAs were ligated to 3' and 5' adaptor sequentially and reverse transcribed to cDNA and then PCR amplified. Whole library was applied to 10% native PAGE gel electrophoresis and bands corresponding to microRNA insertion were cut and eluted. The purified small RNA libraries were quantified and stored at -80℃. The libraries were prepared following the instructions and applied to illumine NextSeq 500 system with 150x2 paired-end type (ABLife Inc., Wuhan, China).
Chromatin Immunoprecipitation (Chip) Library Construction And Sequencing
Total cell extracts were prepared from 2x107 formaldehyde fixed cells resuspended in 1 mL lysis buffer containing 50 mM Tris 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5% deoxycholate. The suspension was sonicated to generate DNA fragments of 200-500bp, centrifuged for 10 min at 12,000 g, 1000µL cell extracts were incubated with HIF3A antibody (orb101652, Biobyt, China) overnight at 4 ℃. The immunoprecipitates were further incubated with protein A Dynabeads for 3h at 4 ℃. After applying to magnet and removing the supernatants, the beads were sequentially washed with lysis buffer, high-salt buffer (250 mM Tris 7.4, 750 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5 deoxycholate), and PNK buffer (50 mM Tris, 20 mM EGTA and 0.5% NP-40) for two times. The immunoprecipitates were eluted from the beads with elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and reverse cross-linked overnight-incubated at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. Purified DNA fragments were end-repaired, adenylated, ligated to adaptors and PCR amplified for 12 cycles. The PCR products corresponding to 300–500 bps were gel purified, quantified and stored at -80 ℃. The libraries were prepared and applied to illumina X-Ten system with 150x2 paired-end type by Novogene.
Rna-seq Data Processing And Alignment
Raw reads containing more than 2-N bases were first discarded. Then adaptors and low-quality bases were trimmed from raw sequencing reads using FASTX-Toolkit (Version 0.0.13). The short reads less than 16 nt were also dropped. After that, clean reads were aligned to the GRCh38 genome by TopHat2  allowing 4 mismatches. Uniquely mapped reads were used to calculate reads number and FPKM value (fragments per kilobase of transcript per million fragments mapped)  for each gene.
Differentially Expressed Genes Analysis
The R Bioconductor package edgeR  was utilized to screen out the differentially expressed genes (DEGs). A false discovery rate < 0.05 and fold change > 2 or < 0.5 were set as the cut-off criteria.
To predict the gene function and calculate the functional category distribution frequency, Gene Ontology (GO) analyses and enriched KEGG pathway were identified using KOBAS 2.0 server . Hypergeometric test and Benjamini-Hochberg FDR controlling procedure were used to define the enrichment of each pathway (corrected p-value < 0.05).
All the statistics were expressed as mean ± standard deviation (SD) and processed using SPSS 16.0 statistical software (Chicago, IL). All experiments were performed at least three times independently,and P < 0.05 were considered significant.