Association between polymorphism and ceRNA network of differential expressed gene in resistance and sensitive non-small-cell lung cancer to radiotherapy
Association between polymorphism and ceRNA network of differential expressed gene in resistance and sensitive non-small-cell lung cancer to radiotherapy
Introduction: Lung cancer has been counted as one of the most malignant tumors which people in both poor and well-developed countries may face it. Nowadays, radiotherapy is one of the main arms of oncology in the post-surgical process of lung cancer patients. Radioresistancy, an important limiting factor, decreases the efficacy of radiotherapy for lung cancer patients. References determine the microRNAs (miRNAs) as important regulatory agents which can participate in radiation responses such as radioresistance or radiosensitization. On the other hand, studies have shown that resistance or sensitivity of the patients to the treatment can be related to single nucleotide polymorphisms (SNPs). Thus, the aim of this study is to investigate the effect of the two regulatory factors, miRNA and SNP, in the CPS1, which, based on our bioinformatic approaches, plays a role in the radioresistance in lung cancer.
Methods: Gene expression data of lung cancer cell lines were obtained from the National Center for Biotechnology Information (NCBI), Gene Expression Omnibus (GEO), and then analyzed by GEO2R to find differentially expressed genes (DEGs). Furthermore, miRWalk was utilized to find significant miRNA-mRNA interactions in the coding sequence (CDS) region. Additionally, the selected miRNA was searched in LncBase v.3 to find strong interaction with lncRNAs and construct a predictive competing endogenous RNA (ceRNA) network. The Pathway enrichment analysis was carried out using the hub for long non-coding RNAs (lncHUB) and Kyoto Encyclopedia of Genes and Genomes (Kegg) online databases. SNPs of carbamoyl-phosphate synthase 1 (CPS1) were extracted from the Database of Single Nucleotide Polymorphisms (dbSNP), and identification of deleterious SNPs brought out from the Sorting Intolerant From Tolerant (SIFT) database. Biophysical validation of deleterious SNPs was realized from HOPE software. CrisPam was used to identify the suitable clustered regularly interspaced short palindromic repeats (CRISPR) system to target the SNP.
Results: Based on microarray analysis, CPS1 has a significant down-regulation in the radioresistance of non-small-cell lung cancer (NSCLC) cell lines samples compared to radiosensitive NSCLC samples (log fold change; logFC: -7.65, adjp-value < 1.54E-15). Analysis of possible miRNA-mRNA interactions revealed hsa-miR-27b-5p as a significant interactor to CPS1 mRNA. This miRNA was then searched in LncBase v.3 (marrow), showing that CPS1 Intronic Transcript 1 (CPS1-IT1) had the strongest interactions. Kegg reveals that CPS1 has an effect on nitrogen metabolism, arginine biosynthesis, alanine, aspartate, and glutamate metabolism; by using LncHUB, we found that CPS1 and CPS1-IT1 affect arginine biosynthesis. From all of the extracted SNPs on the coding region, SIFT online software revealed that rs28940283 is the most significant deleterious SNP in the protein-coding region CPS1. Based on the biophysical validation of HOPE, the mutated residue is located in a domain crucial for the protein's functionality and in contact with another domain that is also essential for the activity. The mutation can disturb the interaction of these domains, which might affect the protein's function. CrisPam suggests xCas9 and Streptococcus pyogenes Cas9 (SpCas9)-NG to target the SNP rs28940283.
Conclusion: In conclusion, we noticed that radioresistancy in NSCLC is associated with regulatory agents such as miRNA and SNP. rs28940283 can promote resistance in lung cancer cells by changing the correct folding and protein interactions of CPS1 and the misregulation of glutamine amidotransferase type-1 activity which can cause a reduction in the CSP1's function. Moreover, an interaction with hsa-miR-27b-5p forms a possible ceRNA network that can lead to the downregulation of the CPS1.