CX-5461, a novel selective RNA polymerase I inhibitor, shows potential anti-inflammatory and immunosuppressive activities. However, the molecular mechanisms underlying the inhibitory effects of CX-5461 on macrophage-mediated inflammation remain to be clarified. In the present study, we attempted to identify the systemic biological processes which were modulated by CX-5461 in inflammatory macrophages. Primary peritoneal macrophages were isolated from normal Sprague Dawley rats, and primed with lipopolysaccharide or interferon-gamma. Genome-wide RNA sequencing was performed. The study suggests that limiting cell proliferation predominates in the inhibitory effects of CX-5461 on macrophage-mediated inflammation. 

The RNA processing and sequencing services were provided by LC-Bio Technology (Hangzhou, Zhejiang Province, China). Total RNA was extracted using Trizol reagent (Thermo Fisher) following the manufacturer's instructions. The RNA quantity and purity were analyzed using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (from Agilent, Santa Clara, CA, USA). RNA samples with RIN number > 7.0 were used for library construction. Purified mRNA was obtained from 5 mg of total RNA using Dynabeads Oligo (dT) (Thermo Fisher) with two rounds of purification; the mRNA was then fragmented using divalent cations (Magnesium RNA Fragmentation Module from New England Biolabs, Ipswich, MA, USA) under 94oC for 5 min. The cleaved mRNAs were reverse-transcribed to cDNAs using SuperScript II Reverse Transcriptase (Thermo Fisher), which were used as templates to synthesize U-labeled second-strand DNAs with E. coli DNA polymerase I, RNase H (all from New England Biolabs) and dUTP (Thermo Fisher). After ligation with Dual-index adapters, the DNA library was treated with heat-labile UDG enzyme (New England Biolabs) to remove the U-labeled second strands, and amplified by PCR. The average insert size of the final cDNA library was 300 ± 50 bp. Paired-end sequencing (2 × 150 bp) was performed using a NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). High quality clean reads were obtained by filtering with Cutadapt (version 1.9) (https://cutadapt.readthedocs.io/en/stable/) and verified with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Read alignment to the Rattus norvegicus reference genome was performed using HISAT2 package (version 2.0.4) (https://daehwankimlab.github.io/hisat2). Read assembly was performed using StringTie (version 1.3.4d) (http://ccb.jhu.edu/software/stringtie). StringTie and Ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the relative expression levels (expressed as fragment per kilobase per million reads/FPKM values).

In each experimental group, 3 independent samples were included.

Sample identity:

control1-control3: Control group

IFN1-IFN3: Interferon-gamma-treated group

IFN_CX1- IFN_CX3: Interferon-gamma + CX-5461 co-treated group

LPS1-LPS3: Lipopolysaccharide-treated group

LPS_CX1- LPS_CX3: Lipopolysaccharide + CX-5461 co-treated group

Concentrations used:

CX-5461 (1 uM)

IFN-gamma (150 U/mL)

LPS (1 ug/mL)