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Gene & Protein in Disease A pyroptosis-related gene signature in myeloma

Table 1. (Continued) Table 2. Sequence of primers used in quantitative reverse-
transcription polymerase chain reaction
Gene symbol Definition
Caspase 3 (CASP3)
NLRP3 NLR family pyrin domain containing 3
Forward 5’-CATGGAAGCGAATCAATGGACT-3’
NLRP6 NLR family pyrin domain containing 6
Reverse 5’-CTGTACCAGACCGAGATGTCA-3’
NLRP7 NLR family pyrin domain containing 7
Caspase 8 (CASP8)
NLRP9 NLR family pyrin domain containing 9
Forward 5’-AAGAGCCAGGGTGGTTATTGAAAGT-3’
NOD1 Nucleotide binding oligomerization domain
containing 1 Reverse 5’-ATCTCCTCCTTTCTAGTGTTTAGGT-3’
NOD2 Nucleotide binding oligomerization domain Charged multivesicular body protein 2A (CHMP2A)
containing 2 Forward 5’-CTACTGCGGCAGAACCAG-3’
PJVK Pejvakin/deafness, autosomal recessive 59 Reverse 5’-CATCATTCATCATCTCCTCCT-3’
PLCG1 Phospholipase C gamma 1 Charged multivesicular body protein 3 (CHMP3)
PRKACA Protein kinase cAMP-activated catalytic Forward 5’-ATGGGGCTGTTTGGAAAGACC-3’
subunit alpha
Reverse 5’-TTTGCCTGTCAACAACTCTCAT-3’
PYCARD PYD and CARD domain containing
Charged multivesicular body protein 6 (CHMP6)
SCAF11 SR-related CTD associated factor 11 Forward 5’-AAGGCCATCCTGCAACTGAAG-3’
TIRAP TIR domain containing adaptor protein
Reverse 5’-GCTGCTCCTGGTATCGCTT-3’
TNF Tumor necrosis factor
Forkhead box O3 (FOXO3)
TP53 Tumor Protein P53
Forward 5’-CGGACAAACGGCTCACTCT-3’
TP63 Tumor Protein P63
Reverse 5’-GGACCCGCATGAATCGACTAT-3’
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
to assess the immune environment between different risk Forward 5’-GGAGCGAGATCCCTCCAAAAT-3’
groups. TIDE aims to predict the effectiveness of initial Reverse 5’-GGCTGTTGTCATACTTCTCATGG-3’
37
treatments with immune checkpoint inhibitors, such as Nucleotide-binding oligomerization domain containing 2 (NOD2)
anti-PD1 or anti-CTLA4, in MM patients. Compared to
other markers, including PD-L1 expression and mutation Forward 5’-TGGTTCAGCCTCTCACGATGA-3’
load, TIDE offers higher predictive accuracy. 38,39 Reverse 5’-CAGGACACTCTCGAAGCCTT-3’
Phospholipase C gamma 1 (PLCG1)
2.6. Analyzing the gene expression of the PRG Forward 5’-GCTTCTATGTAGAGGCAAACC-3’
signature in vitro
Reverse 5’-GCCACTTCACGGATCTTTT-3’
The OPM-2 MM cell line was treated with etoposide with
a concentration of 0, 5, or 20 µg/mL for 48 h, and the cells 2.7. Statistical analysis
were then collected to extract total RNA by using the
RNAeasy™ Plus Isolation Kit (Beyotime Biotechnology Co., All statistical analyses were conducted using R software
Ltd., Shanghai, China). The total RNAs were subjected to version 4.0.1, with statistical significance defined as
synthesize cDNA using TransScript Reverse Transcriptase *P < 0.05, **P < 0.01, and ***P < 0.001.
Kit (TransGen Biotech, Beijing, China). Reverse- 3. Results
transcription quantitative polymerase chain reaction
(qRT-PCR) was conducted in accordance to the assay 3.1. The expression profile of PRGs in MM patients
manufacturer’s instructions (TaKaRa Biotech, Dalian, and healthy individuals
China). The primers used for each gene are shown in After a comprehensive literature review, we selected 57
Table 2. The specific steps for qRT-PCR are as follows: (1) PRGs for analysis. The expression landscape of these 57
the reaction mixture was loaded into a qPCR instrument PRGs was examined, and the results are illustrated in the
(TaKaRa Biotech, Dalian, China), and thermal cycling heatmap shown in Figure 1A. Among these PRGs, 53
conditions, typically involving denaturation, annealing, were identified as DEGs, of which 22 genes were markedly
and extension phases, were set. (2) During amplification, downregulated in MM patients, including BAX, CHMP2A,
the fluorescent dye binds to the DNA, allowing real-time CHMP3, CHMP4A, CYCS, GSDME, HMGB1, IL18, IL1A,
quantification of DNA levels. (3) The changes in transcript TP63, CASP-6, -9, GPX4, GSDMA, GSDMC, NLRP2,
levels were quantified using the comparative 2 −∆∆CT method. NOD1, PLCG1, PRKACA, SCAF11, NAIP, and APIP.

Volume 3 Issue 4 (2024) 4 doi: 10.36922/gpd.4534