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. 2021 Mar;10(3):1444-1456.
doi: 10.21037/tlcr-21-219.

Isochorismatase domain-containing protein 1 (ISOC1) participates in DNA damage repair and inflammation-related pathways to promote lung cancer development

Jinghan Shi  1 , Fujun Yang  1 , Nanfeng Zhou  2 , Yan Jiang  1 , Yanfeng Zhao  1 , Junjie Zhu  1 , Arsela Prelaj  3   4 , Jyoti Malhotra  5 , Nicola Normanno  6 , Elisa Danese  7 , Andrés F Cardona  8 , Xuan Hong  9 , Gening Jiang  1 , Xiao Song  1
Affiliations

Isochorismatase domain-containing protein 1 (ISOC1) participates in DNA damage repair and inflammation-related pathways to promote lung cancer development

Jinghan Shi et al. Transl Lung Cancer Res. 2021 Mar.

Abstract

Background: The advent of novel molecular targets has dramatically changed the treatment landscape of lung cancer in recent years. Isochorismatase domain-containing protein 1 (ISOC1) has been reported as a potential biomarker in gastrointestinal cancer, while its function in lung cancer has not been determined.

Methods: The expression levels and prognostic significance of ISOC1 were assessed using bioinformatic analysis. Overexpression of ISOC1 and miR-4633, and knockdown of ISOC1 in non-small cell lung cancer (NSCLC) cell lines were generated by lentiviral infection with overexpressed or shRNA plasmids. CRISPR/Cas9 system was applied to knockout ISOC1 in A549 cells. The functions of ISOC1 and miR-4633 in lung cancer development were investigated using cell proliferation, migration, and invasion assays. Xenograft tumor growth assays in nude mice were further assessed the effect of ISOC1 in the tumorigenesis of NSCLC in vivo. Cell cycle distribution analysis was performed to uncover the underlying mechanism of ISOC1 and miR-4633 in promoting NSCLC cell proliferation. Co-immunoprecipitation combined with mass spectrometry and RNA sequencing were performed to uncover the potential mechanism of ISOC1 in lung cancer development.

Results: Our results found that ISOC1 expression was upregulated in NSCLC tissues and that increased expression of ISOC1 was significantly associated with worse disease-free survival in NSCLC patients. Overexpression of ISOC1 could increase the proliferation, viability, migration, and invasion of NSCLC cells. Furthermore, miR-4633, located in the first intron of ISOC1, could also promote tumor cell progression and metastasis. Mice xenograft tumor assay showed that knockout of ISOC1 could significantly inhibit tumor growth in vivo. Besides, co-immunoprecipitation combined with mass spectrometry assay revealed that ISOC1 interacted with the proteins of DNA damage repair pathways and that upregulated ISOC1 expression could significantly increase the number of DNA damage lesions. RNA sequencing analysis showed that the downstream signaling pathways mediated by ISOC1 were mainly inflammation-related.

Conclusions: We demonstrated that ISOC1 and its intronic miR-4633, both of them could promote NSCLC cell proliferation, migration, invasion, and cell cycle progression. ISOC1 participates in DNA damage repair and inflammation to promote lung cancer development.

Keywords: DNA damage repair; Isochorismatase domain-containing protein 1 (ISOC1); inflammation; lung cancer; miR-4633.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-21-219). Dr. JM has served on the Advisory Board of Astra Zeneca and Blueprint Medicines. The authors have no other conflicts of interest to declare.

Figures

Figure 1
Figure 1
Effects of ISOC1 on NSCLC cell progression, migration, and invasion. (A) OE, KD (shISOC1), and KO efficiency of ISOC1 was determined by Western blot and qRT-PCR. (B) CCK-8 cell proliferation assays in ISOC1 OE, KD, and KO A549 cells from days 1 to 5. (C) Transwell migration and invasion assays in ISOC1 OE, KD, and KO A549 cells (×200). Cells were stained with crystal violet. (D) Effects of OE, KD and KO of ISOC1 on colony formation in A549 cell line. (E) Tumor xenograft assay performed in the ISOC1 KO group and hematoxylin-eosin staining confirmed tissues as tumorous (×400). The number of mice in each group was 7. Three mice in the ISOC1-KO group died 3 weeks after tumor implantation and no suspicious tumor tissues were found subcutaneously. *, P<0.05; **, P<0.01; ***, P<0.001. OE, overexpression; KD, knockdown; KO, knockout; EV, empty vector; WT, wild-type.
Figure 2
Figure 2
miR-4633 promoted cell viability in NSCLC. (A) Relative luciferase activity of WT ISOC1-3'UTR by miR-4633. (B) Forced expression of miR-4633 in A549 cells was assessed by qRT-PCR. (C) Effects of miR-4633 on A549 cell proliferation was examined by CCK-8 assay. (D) Transwell assay was used to assess effects of miR-4633 on A549 migration and invasion (×200). Cells were stained with crystal violet. (E) Colony formation assays in A549 cells infected with EV or miR-4633 lentivirus. *, P<0.05; **, P<0.01; ***, P<0.001. WT, wild-type; EV, empty vector.
Figure 3
Figure 3
Effects of ISOC1 and miR-4633 on cell cycle distribution. (A) ISOC1 OE in A549 cells caused an increases cell accumulation in S and G2/M phases. (B) ISOC1 knockdown induced G1 arrest in A549 cells. (C) ISOC1 deficiency caused decrease of G2/M phase cells in the A549 cell line. (D) miR-4633 promoted G1/S progression in the A549 cell line. *, P<0.05; **, P<0.01; ***, P<0.001. OE, overexpression; KD, knockdown; KO, knockout; EV, empty vector; WT, wild-type; N.S., not significant.
Figure 4
Figure 4
ISOC1 was involved in DNA damage and inflammation-related pathways. (A) Quantitation of the number of DNA damage sites in A549 cells treated with cisplatin (20 μM) and detected 2 h thereafter. (B) KEGG pathway enrichment analysis results based on the RNA sequencing assay in A549 cells with ISOC1 OE. (C) KEGG pathway enrichment analysis results detected by the RNA sequencing assay in A549 cells with ISOC1 KO. *, P<0.05; **, P<0.01; ***, P<0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes; OE, overexpression; KO, knockout.
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