Biological background of immortalized HFDPCs and AR-expressing immortalized cells

In our previous study, we exogenously introduced an AR expression cassette through retrovirus gene transfer. Although the expression level of AR was undetectable in parent cells, the AR-expressing immortalized HFDPCs showed an intense signal with the expected molecular weight in western blotting (see Fig. 1 of our previous publication, Fukuda et al., 2020). Furthermore, we detected the nuclear localization of AR even without ligand stimulation, which may explain auto-dimerization based on the force expression system (see Fig. 2 of our previous publication, in Fukuda et al., 2020). Furthermore, we detected activation of DKK1 (a major downstream gene of AR) expression in AR-expressing immortalized HFDPCs, which indicated that the AR signaling pathway was reconstituted by exogenous AR introduction.

Fig. 1

Workflow of RNA-Seq analysis and PCA of immortalized human dermal papilla cells (HFDPCs) with K4DT cells and AR-expressing cells. A Workflow of the analysis. B Correlation matrix of all samples. Triplicate samples formed unique clusters. C Mapping ratio of parent immortalized HFDPC-K4DT cells and AR-expressing HFDPC-K4DT cells. The mapping results of parent K4DT were reproduced from our previous publication (Fukuda T., et al., 24: 101929, iScience, 2012). D PCA of expression profiling of HFDPC-K4DT cells with and without AR expression

Fig. 2

Heatmap analysis of differentially expressed genes listed in focal adhesion pathway and Proteoglycans in cancer pathway, which are listed in the KEGG database. Red indicates high expression and blue indicates low expression of genes

Whole-gene transcriptome analysis of parent HFDPC-K4DT and AR-expressing HFDPC-K4DT

To comprehensively compare the expression patterns of whole genes, we carried out RNA-Seq analysis using an Illumina Hiseq X sequencing machine (Illumina, San Diego, CA, USA) and a 150-bp paired end. The sequencing workflow is shown in Fig. 1A. After the removal of the adaptor, we initiated the mapping process. The number of obtained sequence reads was at least 22 M, indicating that the read number was sufficient for quantitation [9]. To evaluate the reproducibility of the data, we carried out RNA-Seq reactions with three biological replicates. We evaluated the quality of the read data using the FASTQC program (Fig. S1 and S2). The results of FASTQC indicated that the average of almost all sequencing data was mapped within the green area, suggesting that the read data was reliable. We next mapped sequencing reads using the STAR program and human reference genome (GRCh38). The mapping ratio and read number are shown in Fig. 1C. The mapping ratio of the samples was more than 95%, indicating that our mapping method was suitable for detecting gene expression. The mapping ratios were 95.9% (HFDPC_K4DT1), 96.2% (HFDPC_K4DT2), 96.3% (HFDPC_K4DT3), 96.4% (HFDPC_K4DT_AR1), 96.3% (HFDPC_K4DT_AR2), and 95.1% (HFDPC_K4DT_AR3).

Fig. 3

Expression level of caveolin-1 and EGF receptors detected by qRT-PCR in identical RNAs, which used for RNA-Seq. Relative RNA quantality which detected with internal control (GAPDH) were shown in the graph. Average value and standard errors are shown with biological triplicated samples

The complete list of expression counts of parent HFDPC-K4DT and AR-expressing HFDPC-K4DT is provided in Figshare ( The sequencing data were submitted to the DDBJ database under Bioproject Submission ID PRJDB10909. We first filtered genes at least 3000 counts on any sample. The number of genes remained after the filtration was 1537 genes. Next, we input the expression counts of the whole genome into TCC-GUI. First, we analyzed the correlation plots of expression profiles, as shown in Fig. 1B. The biological replicates of parent HFDPC-K4DT and AR-expressing HFDPC-K4DT formed unique clusters, indicating that the sequencing results were reproducible. The triplicated data also formed unique clusters in three-dimensional PCA (Fig. 1).

Pathway analysis results

We further analyzed the DEGs with a FDR based Q value of less than 0.01. In total, we narrowed down the DEGs to 1196 as candidate genes. The list of DEGs was submitted to the pathway analysis tool DAVID. The most significant pathway determined in DAVID was the focal adhesion pathway (61 hits in the annotation list), followed by Proteoglycans in cancer (51 hits in the annotation list). Based on the pathway analysis results, we compared 61 genes listed in focal adhesion using heatmap analysis (Fig. 2). Furthermore, the expression levels of Proteoglycans in cancer related genes (51 genes) are shown in Fig. 6. We also mapped the DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG), which showed more than a 2-fold increase or 0.5-fold decrease (at least 3000 counts in any sample) based on bar plots of expression counts and decision criteria [10,11,12]. Mapping of the focal adhesion pathway indicated that collagen-related molecules (FN1, COL1A1, COL27A1, COL4A1, COL4A2, and COL5A3) were either downregulated or upregulated in AR-expressing cells. Furthermore, the expression level of Caveolin 1 was downregulated and EGFR was upregulated in AR expressing HFDPC-K4DT cell (Fig. 5).

Fig. 4

Expression level of caveolin-1 and EGF receptors in HFDPC-K4DT parental cell, and AR expressing HFDPC-K4DT cell, and after the treatment of 50 nM of DHT treatment. A Expression level of caveolin-1, before treatment of DHT (Left side), and after the 8 h of DHT treatment (Right side). B Expression level of EGFR, before before treatment of DHT (Left side), and after the 8 h of DHT treatment (Right side). Average value and standard error are shown with biological triplicated samples

In Proteoglycans in cancer related pathway, the expression level of Twist was elevated which is one of the transcriptional factor in AR expressing cell.

Validation of RNA-Seq results with qPCR analysis

We furthermore detected the expression levels of Caveolin 1 downregulated and EGFR with qPCR analysis. As the first evidence, we detected expression of these two genes in identical RNAs which used for RNA-Seq. The downregulation of Caveolin 1 and upregulation of EGFR in AR expressing cell were reproduced with qPCR analysis (Fig. 3). Furthermore, we carried out treatment of 50 nM of dihydrotestosterone (DHT) with and without AR expressing cells. Under the intact condition, downregulation of Caveolin 1 and upregulation of EGFR in AR expressing cell were also reproduced (Fig. 4A and B, Left side). However, expression of Caveolin 1 was strongly suppressed after the treatment of DHT even in parent K4DT cell. The expression levek of Caveolin 1 was elevated in AR expressing cells. These data indicate that 50 nM DHT treatment causes various types of expression change, which independent to AR signaling.

Fig. 5

Location of upregulated or downregulated genes in AR-expressing HFDPC-K4DT cells in focal adhesion pathway from KEGG

Fig. 6

Location of upregulated or downregulated genes in AR-expressing HFDPCK4DT cell in Proteoglycans in cancer pathway from KEGG

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