RNA-Seq data processing
RNA-Seq had been performed for hDPSCs under normoxia and hypoxia in our previous study, which can be obtained from NCBI GEO: GSE118046. Fastqc (version 0.11.9) was used to access the sequencing quality and Trimmomatic was used to filter low-quality sequencing reads. After the quality control, reads were aligned to reference genome (Hg19) using HISAT2 (version 2.1.0) and the sam files for each sample were obtained. Next, sam files were converted to sorted bam files using Samtools (version 1.13). Assembly of bam files and gene quantification (FPKM values) for each sample was using StringTie (version 2.1.5) and the '-merge' function was performed to merge the transcript.
Identification of novel lncRNA
The pipeline for identifying novel lncRNA from assembled transcript are as follow: (1) filter transcripts with class code ‘i, j, u, x’ from Gffcompare of StringTie; (2) remove the transcripts that matched annotations of known lncRNA; (3) filter transcripts with length ≥ 200bp, exon numbers ≥ 1; (4) filter transcripts without coding potential using the protein-coding gene prediction tools including Coding-Non-Coding-Index (CNCI, version 2), Coding Potential Calculator (CPC, version 2), Coding Potential Assessment Tool (CPAT) and Pfam-scan (version 14.3). Finally, retaining transcripts of which FPKM > 0 in at least one sample were novel lncRNAs.
Differential expression analysis
Differential expression analysis was used to identify the key novel lncRNAs under hypoxia. Expression matrixs of FPKM values for mRNA and novel lncRNA were imported into R (version 3.63) and analyzed using Linear Models for Microarray data (limma, https://bioconductor.org/ packages/limma/) package in Bioconductor. Up- or downregulated differentially expressed mRNA/novel lncRNA between samples under hypoxia and normoxia were identified with the cut-off criteria of P < 0.05 and |fold change (FC)| > 2.
LncRNA target prediction and functional annotation analysis
In order to understand the function of key lncRNA, LncTar (version) was used to explore the potential interaction between key lncRNA and differentially expressed genes. LncTar is a bioinformatics tool that can calculate the free energy between lncRNA and target genes and a threshold of -0.1 ndG (default parameter) was set. Next, the target genes were imported into Database for Annotation Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) to perform GO enrichment and KEGG pathway analysis. To further explore the function of lncRNA HRL-SC, GSEA (version 4.1.0) was performed using c2.all.v7.4.symbols.gmt (http://software.broadinstitute.org/gsea/downloads.jsp) as a reference gene set. P < 0.05 and FDR < 0.25 were considered statistically significant.
Cell culture and identification
The hDPSCs were obtained from the third molar of patients aged 18-25 years indicated for extraction at the Department of Stomatology in Nanfang Hospital, Southern Medical University,Guangzhou, Guangdong, China. All experimental protocols were approved by the Ethical Committee of Southern Medical University. The isolated hDPSCs were cultured in Dulbecco modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma, St. Louis, Mo, USA) at 37 °C in humidified atmosphere containing 5% CO2 with different oxygen concentrations. Induced conditioned medium was used for cell induction and differentiation. The culture media were renewed every 3 days.
After 21 days of differentiation, Alizarin Red and ALP activity staining were performed to verify the formation of mineral nodes and purple colored precipitate. Besides, the cells were incubated for surface markers including CD45, CD34, CD90, CD44 and CD29 and then were detected by flow cytometry to identify the cell phenotypes of hDPSCs.
The siRNAs against lncRNA and control siRNA were obtained from GeneChem (Shanghai, China). The details of sequences could be seen in Additional file 1 Table S1. The siRNAs and control siRNA were transfected using riboFECT™ CP (Ribobio, GuangZhou, China) according to the manufacturer's protocol. When SC79
Wound healing assay
Migration capability of hDPSCs was characterized by wound healing assay. Cells were cultured with DMEM supplement with 10% FBS in 6-well plates until they reached confluence. After starving for 24h with serum-free DMEM, scratches were generated with a 1-mL pipette tip in each well. After scratching for 0 h (right after scratch), 12h and 24h, cell migration of cells was observed and photographed under inverted microscope. Scratch healing area across each scratch was analyzed calculated by using ImageJ software.
Cell proliferation assay
Proliferation capability of hDPSCs was measured using the EdU (meilunbio, Dalian, China) assay. The hDPSCs were labeling with EdU for 2h and then fixed, washed, permeabilized and stained according to the manufacturer's protocol. After cells washed, the DNA was stained with Hoechst 33342, then each well was observed and photographed under an inverted fluorescent microscope.
RNA extraction and RT-PCR
Total RNA was extracted from each group using EZ-press RNA Purification Kit (EZBiosicence, USA) according to the manufacturer’s protocol and quantified by NanoDrop ND-1000 (Thermo Scientific, USA).
Reverse transcription was performed using Color Reverse Transcription Kit (EZBiosicence, USA) with gDNA remover. A volume of 13µL RNA solution of each sample (containing 1 µg RNA) was treated with 2µL provided gDNA remover to remover genomic DNA. Each reaction was set for 20µL, including 13µL processed RNA solution and 5µL 4× RT Master Mix.
Real time PCR was performed with Color SYBR Green qPCR Master Mix ROX2 plus (EZBiosicence, USA) on Roche LightCycler®480. Each reaction was set for 10 µL: 5µL 2× Color Green qPCR Master Mix, 0.2µL Forward Primer, 0.2µL Reverse Primer, 3.6µL RNase Free dH2O and 1µL Product from RT reaction. Ten differentially expressed novel lncRNAs, primer sequences of which can be seen in Table. All experiments were repeated three times, and relative gene expression was calculated using the 2-ΔΔCt method. The sequences of gene-specific primers are listed in Additional file 1 Table S2.
Western blot analysis
Cells of each group were lysed using the RIPA lysis buffer with protease inhibitors and the total proteins contents were quantitated by the BCA assay (Beyotime, Shanghai, China). The samples were separated by 8% SDS-PAGE, and then were transferred onto polyvinylidene diflfluoride (PVDF) membranes. The PVDF membranes were blocked in protein free rapid blocking buffer (Epizyme Biomedical Technology, Shanghai, China) for 15 min and then incubated overnight at 4 °C with primary antibodies against β-actin, AKT, p-AKT, PI3K, p-PI3K (Bioss, BeiJing, China), which diluted at 1: 1000. Then, the PVDF membranes were incubated with corresponding secondary antibodies (Proteintech, China). The bands were detected using ECL reagents (Biosharp, China). The densities of protein bands were quantified by image J software.