Transcriptome Analysis of the Effects of X-Ray Radiotherapy on Non-small-cell Lung Cancer Using Next-generation Sequencing

Abstract

Background/aim: This study investigated the acute effects of flattening filter (FF) and flattening filter-free (FFF) beams on gene expression in non-small-cell lung cancer (NSCLC).

Materials and methods: Thirty-six adult athymic nude mice were divided into five groups. The control group did not undergo any radiotherapy or treatment procedures, whereas in the lung cancer (LCa) group, a cancer model was created but not irradiated. LCa models received 20 Gy radiotherapy with FF at 400 MU/min, or with FFF at 1,000 or 1,800 MU/min dose rates. The mice were irradiated 20 days after A549 cancer cell-line implantation and sacrificed 48 h after irradiation for genetic analysis.

Results: Twelve genes were identified as being common across all radiotherapy groups. The expression of most of these genes changed as the dose rate increased. Seven of these genes were also common to the LCa and control groups. Three genes down-regulated in the untreated cancer group showed increased expression with higher dose rates in treated groups. Significant differences were observed in glutamatergic synapse, actin cytoskeleton regulation, and steroid synthesis in FF-400 and FFF-1000. The FFF-1800 group exhibited significant changes in RNA transport, actin cytoskeleton regulation, and phagosome-associated pathways.

Conclusion: FFF beams induced more extensive and pronounced gene-expression changes compared to FF beams in NSCLC.

Keywords: Lung cancer; RNA sequencing; flattering filter; flattering filter free; next-generation sequencing; radiotherapy.

Figure 1

Volcano plots of differentially expressed genes in murine data. The genes with the highest or lowest expression among the differentially expressed genes are indicated. Mice were without lung cancer (A1), with lung cancer but untreated (A2), treated with 20 Gy radiotherapy with flattening filter (FF) beam at 400 MU/min (A3), with flattening filter-free (FFF) beams at 1,000 (A4) and 1,800 (A5) MU/min dose rates. The mice were irradiated 20 days after A549 cancer cell-line implantation and sacrificed 48 h after irradiation for genetic analysis. Green dot: |log2FC| <1, Red dot: p<0.05 and |log2FC|<1; NS: Not significant.

Figure 2

Venn diagram analysis of differentially expressed genes in study groups. Mice were without lung cancer (A1), with lung cancer but untreated (A2) or treated with 20 Gy radiotherapy with flattening filter (FF) beam at 400 MU/min (A3), or with flattening filter-free (FFF) beams at 1,000 (A4) or 1,800 (A5) MU/min dose rates murine data. A: Up/down-regulated genes for all groups. B: Up/down-regulated genes for groups receiving radiotherapy at different dose rates compared with the untreated lung cancer group (A2).

Figure 3

Comparison of gene expression profiles between human cancer tissues and key genes differentially expressed in radiotherapy-treated mice with non-small-cell lung cancer. Box plots display the expression levels of selected target genes based on data from The Cancer Genome Atlas (TCGA). *Only genes showing significant expression differences (p<0.01) similar to those observed in the mouse model are included. In the plots, red bars represent expression in primary tumor tissues (n=515), while blue bars represent expression in normal (healthy) tissues (n=59); n: Number of samples. AOAH: Acyloxyacyl hydrolase; ATAD1: ATPase family AAA domain containing 1; DERL1: derlin1; FAM83A: family with sequence similarity 83 member A; GPN1: GPN-loop GTPase 1; ITGAM: integrin subunit alpha M; LSS: lanosterol synthase; MKLN1: muskelin 1; MRM1: mitochondrial rRNA methyltransferase 1; NFYB: nuclear transcription factor Y subunit beta; PODXL: podocalyxin like; POLA1: DNA polymerase alpha 1, catalytic subunit.

Figure 3

Comparison of gene expression profiles between human cancer tissues and key genes differentially expressed in radiotherapy-treated mice with non-small-cell lung cancer. Box plots display the expression levels of selected target genes based on data from The Cancer Genome Atlas (TCGA). *Only genes showing significant expression differences (p<0.01) similar to those observed in the mouse model are included. In the plots, red bars represent expression in primary tumor tissues (n=515), while blue bars represent expression in normal (healthy) tissues (n=59); n: Number of samples. AOAH: Acyloxyacyl hydrolase; ATAD1: ATPase family AAA domain containing 1; DERL1: derlin1; FAM83A: family with sequence similarity 83 member A; GPN1: GPN-loop GTPase 1; ITGAM: integrin subunit alpha M; LSS: lanosterol synthase; MKLN1: muskelin 1; MRM1: mitochondrial rRNA methyltransferase 1; NFYB: nuclear transcription factor Y subunit beta; PODXL: podocalyxin like; POLA1: DNA polymerase alpha 1, catalytic subunit.

Figure 4

Correlation analysis of human genes matching our murine data using TIMER 2.0. AOAH: Acyloxyacyl hydrolase; CNOT4: CCR4-NOT transcription complex, subunit 4; ITGAD: integrin subunit alpha D; ITGAM: integrin subunit alpha M; Log2TPM: log base 2 of transcripts per million; MKLN1: muskelin 1; POLA1: DNA polymerase alpha 1, catalytic subunit.

Figure 5

Survival analysis according to expression of human genes that were found to be significant and matched in our murine data. AOAH: Acyloxyacyl hydrolase; ARMC5: armadillo repeat containing 5; ARMCX3: armadillo repeat containing X-linked 3; ATAD1: ATPase family AAA domain containing 1; DERL1: derlin1; DMAC2: distal membrane arm assembly component 2; FAM83A: family with sequence similarity 83 member A; GPN1: GPN-loop GTPase 1; HR: hazard ratio; ITGAD: integrin subunit alpha D; ITGAM: integrin subunit alpha M; LSS: lanosterol synthase; TBC1D31: TBC1 domain family member 31; TVP23A: trans-Golgi network vesicle protein 23 homolog A.

Figure 5

Survival analysis according to expression of human genes that were found to be significant and matched in our murine data. AOAH: Acyloxyacyl hydrolase; ARMC5: armadillo repeat containing 5; ARMCX3: armadillo repeat containing X-linked 3; ATAD1: ATPase family AAA domain containing 1; DERL1: derlin1; DMAC2: distal membrane arm assembly component 2; FAM83A: family with sequence similarity 83 member A; GPN1: GPN-loop GTPase 1; HR: hazard ratio; ITGAD: integrin subunit alpha D; ITGAM: integrin subunit alpha M; LSS: lanosterol synthase; TBC1D31: TBC1 domain family member 31; TVP23A: trans-Golgi network vesicle protein 23 homolog A.

Figure 5

Survival analysis according to expression of human genes that were found to be significant and matched in our murine data. AOAH: Acyloxyacyl hydrolase; ARMC5: armadillo repeat containing 5; ARMCX3: armadillo repeat containing X-linked 3; ATAD1: ATPase family AAA domain containing 1; DERL1: derlin1; DMAC2: distal membrane arm assembly component 2; FAM83A: family with sequence similarity 83 member A; GPN1: GPN-loop GTPase 1; HR: hazard ratio; ITGAD: integrin subunit alpha D; ITGAM: integrin subunit alpha M; LSS: lanosterol synthase; TBC1D31: TBC1 domain family member 31; TVP23A: trans-Golgi network vesicle protein 23 homolog A.