Determination of Common microRNA Biomarker Candidates in Stage IV Melanoma Patients and a Human Melanoma Cell Line: A Potential Anti-Melanoma Agent Screening Model
Authors: Antonova Elena , Hambikova Anastasia , Shcherbakov Denis , Sukhov Vitaly , Vysochanskaya Sonya , Fadeeva Inna , Gorshenin Denis , Sidorova Ekaterina , Kashutina Maria , Zhdanova Alina , Mitrokhin Oleg , Avvakumova Nadezhda , Zhernov Yury 
Published: May 2023

MicroRNAs (miRNAs) are small, non-coding RNAs that play an important role in regulating gene expression. Dysregulation of miRNA expression is commonly observed in cancer, and it can contribute to malignant cell growth. Melanoma is the most fatal type of skin malignant neoplasia. Some microRNAs can be prospective biomarkers for melanoma in stage IV (advanced) at higher risk of relapses and require validation for diagnostic purposes. This work aimed to (1) determine the most significant microRNA biomarker candidates in melanoma using content analysis of the scientific literature, (2) to show microRNA biomarker candidates’ diagnostic efficacy between melanoma patients and healthy control groups in a small-scale preliminary study by blood plasma PCR analysis, (3) to determine significant microRNA markers of the MelCher human melanoma cell line, which are also detected in patients with melanoma, that can be used as markers of drug anti-melanoma activity, and (4) test anti-melanoma activity of humic substances and chitosan by their ability to reduce level of marker microRNAs. The content analysis of the scientific literature showed that hsa-miR-149-3p, hsa-miR-150-5p, hsa-miR-193a-3p, hsa-miR-21-5p, and hsa-miR-155-5p are promising microRNA biomarker candidates for diagnosing melanoma. Estimating microRNA in plasma samples showed that hsa-miR-150-5p and hsa-miR-155-5p may have a diagnostic value for melanoma in stage IV (advanced). When comparing ΔCt hsa-miR-150-5p and ΔCt hsa-miR-155-5p levels in melanoma patients and healthy donors, statistically significant differences were found (p = 0.001 and p = 0.001 respectively). Rates ΔCt were significantly higher among melanoma patients (medians concerning the reference gene miR-320a were 1.63 (1.435; 2.975) and 6.345 (4.45; 6.98), respectively). Therefore, they persist only in plasma from the melanoma patients group but not in the healthy donors group. In human wild-type stage IV melanoma (MelCher) cell culture, the presence of hsa-miR-150-5p and hsa-miR-155-5p in supernatant was detected. The ability of humic substance fractions and chitosan to reduce levels of hsa-miR-150-5p and hsa-miR-155-5p was tested on MelCher cultures, which is associated with anti-melanoma activity. It was found that the hymatomelanic acid (HMA) fraction and its subfraction UPLC-HMA statistically significantly reduced the expression of miR-150-5p and miR-155-5p (p ≤ 0.05). For the humic acid (HA) fraction, this activity was determined only to reduce miR-155-5p (p ≤ 0.05). Ability to reduce miR-150-5p and miR-155-5p expression on MelCher cultures was not determined for chitosan fractions with a molecular weight of 10 kDa, 120 kDa, or 500 kDa. Anti-melanoma activity was also determined in the MTT test on MelCher cultures for explored substances. The median toxic concentration (TC50) was determined for HA, HMA and UPLC-HMA (39.3, 39.7 and 52.0 μg/mL, respectively). For 10 kDa, 120 kDa, or 500 kDa chitosan fractions TC50 was much higher compared to humic substances (508.9, 6615.9, 11352.3 μg/mL, respectively). Thus, our pilot study identified significant microRNAs for testing the in vitro anti-melanoma activity of promising drugs and melanoma diagnostics in patients. Using human melanoma cell cultures gives opportunities to test new drugs on a culture that has a microRNA profile similar to that of patients with melanoma, unlike, for example, murine melanoma cell cultures. It is necessary to conduct further studies with a large number of volunteers, which will make it possible to correlate the profile of individual microRNAs with specific patient data, including the correlation of the microRNA profile with the stage of melanoma.