SUMMARY
Globally, lung cancer has the highest mortality rate compared to all other forms of cancer. Non-small cell lung cancer (NSCLC) is the 2nd major cause of cancer-related deaths in Iraq and all over the world due to its delayed diagnosis. It is still unclear exactly which molecular mechanisms drive the progression and spread of NSCLC. NSCLC is still a problem that requires immediate attention due to its late diagnosis and poor prognosis. Therefore, it is essential to thoroughly examine the mechanisms and factors that contribute to the development of NSCLC in order to create efficient treatments. Due to its multifactorial nature, studies are underway to identify novel molecular targets which can serve as potential biomarkers for diagnosis and treatment to reduce the disease burden in the Kurdistan region of Iraq. According to numerous studies, the initiation and development of NSCLC have been linked to a number of genomic aberrations that are specific to tumors.
The transmembrane protein known as Epidermal Growth Factor Receptor (EGFR), functions as a tyrosine kinase receptor for various ligands that control cellular proliferation, differentiation, and viability. The first distinguishable abnormalities in lung cancer that could be targeted were EGFR mutations, and they are known to be the most common reasons for NSCLC. Prior to the development of targeted treatment, elevated levels of EGFR in NSCLC were known to have some correlation with an increased risk of metastasis, a lack of tumor grade, and accelerated tumor growth. In addition, the microRNAs (miRNAs) are responsible for various biological activities like cell differentiation, cell proliferation, disease initiation, cell migration, disease progression, and eventually apoptosis. Various studies have proposed that miRNAs can act as potential diagnostic biomarkers for NSCLC. Thus, the research study aimed to evaluate the association of EGFR gene mutation with microRNA-146a gene polymorphism in NSCLC development.
For this purpose, total 141 participants (25 control+116 cases) were recruited after their informed consent. a histochemical study was performed on lung cancer biopsy samples. 70 EGFR, which was recently identified as a critical molecule biomarker in the investigation of lung cancer, was the subject of a novel therapeutic approach based on molecular analysis. EGFR and microRNA-146a genes were amplified by the amplification refractory mutation system (ARMS)-PCR and allele-specific polymerase chain reaction (AS-PCR), respectively. Then, the sanger sequencing was done for the five samples of NSCLC patients for microRNA-146a and EGFR gene exons. In order to look into the distribution of EGFR and microRNA-146a gene mutations in NSCLC patients, this study was carried out in Korea (Macrogen Inc. Korea). The forward and reverse primers for both of microRNA-146a and EGFR gene exons were used for sequencing. The purpose of sanger sequencing is to examine the characteristics of the EGFR and microRNA-146a gene’s distribution in patients suffering from NSCLC and then analyzing the results to find any impact on NSCLC. The EGFR exons with the highest percentage of mutations were EGFR20, which was present in 64 (91.42 %) of the patients, or 70 (58 males and 12 females), and EGFR21, which was present in 59 (84.28 %) patients (47 males and 8 females). While only 35 (50%) patients confirmed positive for mutated EGFR19, 41 (58.57%) patients had mutated EGFR18. Males constituted the majority of the mutant EGFRs. The GC genotype of miRNA-146a was found to be slightly higher in cancer patients with odd ratio=4.8 (confidence interval= 0.75-55.6 p-value=0.2532) as compared to 25 healthy controls. More susceptible to get lung cancer between EGFR mutations and miRNA-146a rs2910164 C>G nucleotide polymorphism was observed in this study. Thus, miRNA-146a could act as a potential target for treatment.
In this study, Sanger DNA sequencing revealed various DNA abnormalities in four different exon regions of the EGFR gene. A total of 51 mutations were detected within the EGFR gene in which 43 were substitution mutations and 8 were deletions, leading to alterations in 29 different amino acid types. Exon 18 featuring heterozygous substitutions (C to CA, causing a silent mutation) and a homozygous deletion mutation (155494_155495delTT). Likewise, exon 19 mutations were detected, including homozygous and heterozygous substitutions, resulting in synonymous or missense mutations. Furthermore, heterozygous substitution at position 162886 (162886G>GA:787Q>Q/Q) was detected in exon 20 which led to a synonymous mutation without altering the amino acid glutamine at codon 787 (Q787Q/Q). Moreover, heterozygous substitutions and homozygous deletions were detected in exon 21.
In the present study, Sanger DNA sequencing has been conducted to identify mutations in the miRNA146a gene. These mutations encompassed two homozygous substitutions involving a change from C to G at nucleotide -560C>G80. Additionally, there were eight heterozygous substitutions observed, including A to T (-683A>AT8), C to G (-692C>CG149), G to C (-562G>C81), G to T (-619G>GT138), T to G (-436T>TG7), and C to A (-512C>CA7), which resulted in synonymous, silent mutations. Furthermore, a homozygous deletion mutation (691delG10) was also detected within this specific genomic region of the miRNA146a gene.
Overall, the study underscores the diversity of EGFR and miRNA146a genes mutations in NSCLC patients and their potential implications for treatment response, emphasizing the importance of precise diagnostic testing and subclassification of these mutations for personalized therapy.