Obtain and procession of public pan-cancer and OC data
We obtained lncRNA, miRNA, gene expression and methylation (level 3) data, as well as clinical data of all cancer types including OC, from The Cancer Genome Atlas (TCGA, Release: 2017-09-08, https://portal.gdc.cancer.gov/). The gene expression data of normal tissues for ovarian was obtained from GTEX portal ( https://www.gtexportal.org/home/index.html). Patients in all the cancer types were integrated as pan-cancer patients. The sample numbers in each cancer type were shown in Table S1. To filter gene, miRNA, and lncRNA not expressed across all samples, the items with expression values of 0 in all of the samples were excluded. Any remaining expression values of 0 were set to the minimum value of all samples, and all values were log2-transformed. Expression of FAM83H-AS1 was dichotomized using median expression as the cutoff to define “high value” at or above the median versus “low value” below the median.
Construction of lncRNA-miRNA, lncRNA-protein and lncRNA-mRNA ceRNA networks for FAM83H-AS1
In order to describe the functions of FAM83H-AS1, some interacted regulatory networks were constructed. lncRNA-miRNA network was constructed based on experimentally verified associations between miRNAs and lncRNAs were identified in starBase v3.0 (http://starbase.sysu.edu.cn/) [35] and DIANA-LncBase 3.0 (www.microrna.gr/LncBase) [36]. lncRNA-protein data was download from starBase v3.0 and NPInter v2.0 [37] supported by AGO CLIP-seq data. In order to build the lncRNA–mRNA ceRNA network, a hypergeometric test was used to evaluate whether the two lncRNAs have a potential ceRNA relationship by considering their shared interactive miRNAs. All the networks were constructed by Cytoscape 3.3.0 (http://www.cytoscape.org/).
Differential expressed and co-expressed analyses
T-test was used to calculate the differential expression of all genes and lncRNAs between cancer and normal control samples. Pearson’s correlation coefficients (PCCs) were calculated between FAM83H-AS1 and its interacted miRNAs in lncRNA-miRNA network. In addition, co-expression of methylation level and FAM-83H-AS1 expression was also calculated.
Survival analysis for the FAM83H-AS1 in pan-cancer and OC
The patients were divided into two groups based on median value of FAM83H-AS1 expression. Kaplan–Meier method and log-rank test were used to evaluate the survival difference in patients with high and low expression of FER1L4. P < .05 was regarded as statistically significant.
Tumor-immune infiltrating cells associated with FAM83H-AS1 in OC
The associations between all tumor-immune infiltrating cells and the FAM83H-AS1 were analyzed via the Tumor Immune Estimation Resource (TIMER) platform (https://cistrome. shinyapps.io/timer/), a web tool for studying tumor-infiltrating immune cells and their interactions with cancer cells [38]. B-cells, CD4+T-cells, CD8+T-cells, dendritic cells, macrophages and neutrophils were included in the correlated analyses.
Patients and tissue samples
OC patients with a histological diagnosis who had undergone surgical resection and had not received chemotherapy or radiotherapy were extracted in our study. Lastly, eight OC tissues and corresponding adjacent normal ovarian tissues were obtained from The Third Affiliated Hospital of Harbin Medical University. The tissue samples were frozen in liquid nitrogen and stored at −80°C until experiment. The pathological diagnosis were confirmed by three independent senior pathologists. The study was approved by the Research Ethics Committee of The Third Affiliated Hospital of Harbin Medical University. All patients received written informed consent and disposed of specimens in accordance with accepted ethical standards. The clinicopathological features of all patients are indicated in Table 1.
Quantitative real-time reverse transcription PCR (qRT-PCR)
Total RNA was extracted from fresh frozen samples and cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (2 μg) was reverse-transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Vilvoord, Brussel, Belgium). The relative levels of EGOT to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control transcripts were determined by qPCR using the ABI 7500 Fast Real-Time PCR System (Invitrogen). The primer sequences were as follows. FAM83H-AS1: forward 5’-ACTACAGGCACCCACCACCAC-3’, reverse 5’-TGAGACGGGCGGGATCACAAGG-3’; GAPDH: forward 5’-ACCACAGTCCATGCCATCAC-3’, reverse 5’-TCCACCCTGTTGCTGTA-3’. The qRT-PCR amplification was performed in triplicate reactions starting at 95℃ for 10 min, followed by 40 cycles at 95℃ for 10 s, and 60℃ for 60 s. Quantitative normalization of EGOT cDNA was performed in each sample using GAPDH expression as an internal control. The relative level of EGOT transcripts to control GAPDH was determined by the 2−ΔΔCT method. Each sample was examined in triplicate.