Background
Dysregulated microRNAs (miRNAs) have been implicated in the pathogenesis and progression of rectal adenocarcinoma. Various non-coding, small RNA segments can act as oncogenes or tumor suppressors, thus influencing the progression of tumor processes. Although several miRNAs have been described as CRC-specific miRNA signatures, their biomarker effects in relation to therapy efficiency have only been partially confirmed.
Purpose/Objective(s)
This study aimed to define miRNAs in rectal cancer patients that change in patients' saliva due to the neoadjuvant concurrent chemoradiotherapy. Furthermore, we investigated the perceivable differences in correlations between microRNAs in healthy and rectal cancer groups. Our further aim was to identify possible miRNAs that may serve as potential markers of the effectiveness in neoadjuvant concurrent chemoradiotherapy in further studies.
Materials/Methods
Eleven patients with locally advanced rectal adenocarcinoma receiving concurrent neoadjuvant chemoradiotherapy were included in the study. Saliva samples were taken from the patients before the first and last treatment fractions. We performed an in-depth analysis of high-throughput small transcriptome sequencing data obtained from the saliva samples of our study cohort of 31 including the 11 rectal cancer patients and 20 healthy volunteers. Total RNA isolation was performed using the MagMaxTM mirVanaTM Total RNA Isolation Kit. RNA quality was measured using Agilent Tapestation 4200. Small RNA libraries were prepared using NebNext Small RNA Library Prep Set kit. Small RNA libraries were sequenced on Illumina NextSeq 550 System with read lengths of 75 bp, single-end reads. After in silico bioinformatics analyses of sequenced data, microRNAs were annotated and differential expression analyses were performed using EdgeR R package. Differentially expressed miRNAs were validated using RT-qPCR reaction.
Results
By integrating in silico bioinformatic analyses of small noncoding RNA data, 147 miRNAs were identified as having a read number above 10 RPM (RPM>10) in both healthy controls and patient study groups (before the first and the last radiation fraction). For further analysis we assessed this set of miRNAs. According to our results, 37 miRNAs showed significant expression differences in rectal cancer patients compared to healthy control groups. Furthermore, 7 miRNAs (hsa-miR-378a-3p, hsa-miR-203a-3p, hsa-miR-200a-3p, hsa-miR-152-3p, hsa-miR-361-5p, hsa-miR-107 and hsa-miR-221-5p) showed significantly altered expression associated with concurrent chemoradiotherapy. The expression levels of these miRNAs were successfully quantified by qRT-PCR.
Conclusion
miRNAs as epigenetic modulators have been extensively researched. In recent years, many miRNAs have been discovered as tumor suppressors or oncogenes and miRNA expression levels determined in blood or feces can provide useful information for diagnosis, prognosis, or therapeutic outcome. The present study provides insightful results on the changes observed in saliva, which can serve as valuable biomarkers for assessing therapeutic effectiveness.