Cell culture and transfection
The papillary thyroid carcinoma cell line BCPAP was obtained from the Stem Cell Bank, Chinese Academy of Sciences. The follicular thyroid cancer cell line WRO was obtained from the Endocrinology Department of the Affiliated Second Xiangya Hospital of Central South University. The BCPAP cells were grown in RPMI-1640 medium (HyClone, America) supplemented with 10% fetal bovine serum (Gibco, Biological Industries, Israel), 1% sodium pyruvate (Gibco, Biological Industries, Israel), 1% NEAA (Gibco, Biological Industries, Israel) and 1% glutamax (Gibco, Biological Industries, Israel) in a 37°C humidified incubator with 5% CO2. WRO cells was grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. BCPAP and WRO cell were transfected with SREBP1 small interfering RNA (siSREBF1#1 and siSREBF1#2) and a negative control (control siRNA), respectively, which were purchased from RIBOBIO, Guangzhou, China. BCPAP and WRO cells were plated in 6-well culture plates for 24 h before transfection, and then the medium was discarded and replaced with fresh complete RPMI-1640 medium containing siRNA and iMAX transfection reagent for 24 h.
Sample preparation and small RNA sequencing
Novogene (Beijing, China) performed the RNA extraction, library preparation, and sequencing analyses. After that, the complete database sequencing process is carried out (Supplemental Fig. 1). At 24 h posttransfection, the cells were harvested, and total RNA was extracted using TRIzol reagent (Takara Bio Inc, Japan). RNA degradation and contamination were monitored on 1% agarose gels, and then a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit associated with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) were used to detect the RNA purity and contamination, concentration and integrity of the samples, respectively.
A total amount of 3 µg total RNA per sample was used for the small RNA library. Then, the miRNA libraries were generated using the NEBNext® Multiplex Small RNA Library Prep Kit from Illumina® (NEB, USA) according to the manufacturer’s instructions. By using the particular structure at the 3’ and 5’ end of the small RNA, the adapter was added at the 3’ and 5’ ends and reverse-transcribed into cDNA, followed by PCR amplification. PCR products were purified on an 8% polyacrylamide gel, and size selection was used to generate cDNA libraries. After construction of the libraries, Qubit 2.0 was used for quantification, and the Agilent 2100 Bioanalyzer was used to test the insert size. Finally, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. The Illumina Hiseq 2500/2000 platform was then carried out after the library test.
Filtering sRNA reads and miRNA identification
Raw reads in FASTQ format were processed using base-calling. Clean reads were obtained by removing low-quality reads (smaller than 18 nt and longer than 35 nt) containing poly-N, with 5’ adapter contaminants and without the 3’ adapter or insert tags, and with poly A/T/G/C. Then, sRNA reads of the specified size with a length of 18–35 nt were screened and mapped to determine the sequence for downstream analyses. Simultaneously, the Q20, Q30, and GC contents of the raw data were calculated.
The small RNA tags were mapped to the reference sequence by Bowtie without mismatches. Mapped sRNA reads were examined for known miRNAs using miRBase20.0 (http://www.mirbase.org/index.shtml) as a reference, and the modified software miRDeep2 and sRNA-tools-cli were used to obtain the potential miRNA. Detailed information for the sRNA of each sample, including the miRNA secondary structure, sequence and length, were obtained by contrast with the specific reads. Subsequently, the unannotated sRNA reads were further filtered by rRNA, tRNA, snRNA, and snoRNA from Rfam, and they were compared with the exons and introns of mRNAs to identify the degradation fragment of the mRNA and dislodge them. Finally, The remaining sRNAs reads were further analyzed by exploring the secondary structure, the Dicer cleavage site and the minimum free energy using miRDeep2 and miREvo methods to predict novel miRNAs. Subsequently, summarizing all alignments and annotations obtained, base edit analysis of the miRNAs was performed by aligning all the sRNA tags to mature miRNA and miRNA families.
Differential expression analysis of miRNAs
miRNA expression levels were normalized using the TPM (transcript per million) method: normalized expression = mapped read count × 106/total reads. Then, the obtained read counts were used to conduct the miRNA differential expression analysis. The DESeq R package (Novogene, Beijing, China) was used to analyze differentially expressed miRNA. The P-values were adjusted using the q-value. The adjusted P < 0.05 was considered significantly differentially expressed. A Volcano Plot was used to deduce the distribution of differential miRNAs. Cluster analysis was applied to determine the clustering pattern of miRNA expression under different experimental conditions. Moreover, a Venn chart was used to compare the miRNA numbers that were common and unique to each group.
Target gene prediction and GO and KEGG enrichment analysis
For differentially expressed miRNA target gene prediction, miRanda (http://www.mirandaim.org), PITA and RNAhybrid were used. Furthermore, Gene Ontology (GO) enrichment analysis (http://www.feneontology.org/) was used for target gene candidates of differentially expressed miRNAs with terms of biological process (BP), cellular component (CC) and molecular function (MF). The GOseq-based Wallenius noncentral hypergeometric distribution, which can adjust for gene length bias, was implemented for GO enrichment analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) enrichment analysis was applied for predicting the biochemical metabolic pathways and signal transduction pathways of candidate target genes. KOBAS (Mao et al, 2005) software was used to test the statistical enrichment of the target gene candidates in the KEGG pathways.
Real-time qPCR validation
Quantitative real time polymerase chain reaction (qRT-PCR) was performed to further validate selected differentially expressed miRNAs that target SREBP1. The cells were collected from one 6-well culture plate, and total RNA was isolated from thyroid cancer BCPAP and WRO cell lines following the method described previously. Then, total RNA was reverse-transcribed into cDNA using random primers (for SREBP1) and specific stem-loop RT primers (for miRNA) with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc, Japan). Moreover, differentially expressed miRNAs targeting SREBP1 were also validated in thyroid cancer tissue and adjacent normal tissues. Total RNA was reverse-transcribed by polyadenylation of Oligo dT adaptor primer with the Mir-X™ miRNA Frist-Strand Synthesis Kit (Takara Bio Inc, Japan). Real-time qPCR was performed using SYBR® Premix DimerEraser™ (Perfect Real Time) (Takara Bio Inc, Japan) following the manufacturer’s instructions. The process was performed on the LightCycle 480 II (Roche) as follows: 95°C for 30 sec followed by 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds. Each sample was analyzed in triplicate. β-actin was used as an endogenous control to normalize the data, and miRNAs were normalized to nuclear RNA U6. The expression level was calculated using the 2 − ΔΔCT method. Bulge-Loop miRNA qRT-PCR primer sets (one reverse transcription primer and a pair of qPCR primers for each set) specific for the corresponding miRNAs (hsa-miR-100-3p, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-21-5p and hsa-miR-941) and RNU6B (U6) were designed by and purchased from RiboBio.
All statistical analyses were performed using SPSS 18.0 software (IBM, Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Detailed information for the sRNA of each sample, including the miRNA secondary structure, sequence and length, were obtained by contrast with the specific reads. The experimental results are displayed as the mean ± SD. P < 0.05 was considered statistically significant in the comparisons.