The lncRNA gene copy number variation as a low-invasive marker of rectal tumor cell sensitivity to radiotherapy.
Authors: Kutilin Denis S. , Kosheleva Natalia G , Gusareva Marina A. , Zinkovich Mikhail S. , Solntseva Anna A. , Fatkina Natalya B. , Vasilieva Ekaterina O. , Tolmacheva Ekaterina A. , Martynova Ksenia V. , Gappoeva Madina A. , Udalenkova Irina A. , Shlyakhova Olga V. , Pavlyatenko Inna V. , Gabrichidze Petr N. , Gvaramiya Astanda K. , Rozenko Lyudmila Ya , Legostaev Vladislav M. , Kit Oleg I. 
Published: June 2022

e15536 Background: Radiotherapy (RT) is a key component of rectal cancer (RC) treatment, however, cases of non-response to preoperative RT in patients are very common, which is associated with radioresistance of tumor cells, mediated by their molecular characteristics, including the expression level of long non-coding RNA (lncRNA). lncRNAs are longer than 200 bp, show specific expression to various incentives, regulate transcription by acting as molecular traps, and can serve as biomarkers. The number of genes encoding lncRNA is 2 times greater than the number of protein coding genes. However, the rapid degradation of lncRNA molecules in the extracellular environment makes this indicator unsuitable for low invasive diagnostics. The problem can be solved by switching to a more stable marker - gene copy number variation (CNV) of lncRNA, which can be determined in extracellular DNA (cfDNA) of blood plasma. Therefore, the aim of this study was to identify the relationship between CNV of lncRNA genes in cfDNA of blood plasma and the RT effectiveness for rectal tumors. Methods: We used cfDNA from blood plasma obtained before RT from 200 RC patients and from the blood plasma of 50 apparently healthy donors (without cancer, AHD). RT was carried out on Novalis TX linear accelerator (TFD = 54 Gy). Blood samples were separated into plasma and cell fraction by centrifugation. Isolation of cfDNA from plasma was carried out by the phenol-chloroform extraction method. Determination of lncRNA genes CNV ( XIST, HELLPAR, NEAT1, AC008124.1, AC016717.2, AC093297.2, AC097634.1, BX890604.1, CASC9, IQCH-AS1, LINC00662, LINC00707, LINC01089, LINC01468, LINC0154, MALAT0154, MUC20-OT1, PVT1, VASH1-AS1) was performed by Real-Time qPCR. Differences were assessed using the Mann-Whitney test (Bonferroni correction was used). Results: An analysis of RT results in 200 patients allowed dividing them into 2 groups: complete tumor regression (group 1, n = 120) and minor or no tumor regression (group 2, n = 80). In cfDNA of patients in group 1, a decrease (p < 0.05) in the CNV of lncRNA XIST, HELLPAR, NEAT1, AC008124.1 and AC016717.2 genes was found by 2.5, 3.3, 2.0, 2.0, and 5.0 times, respectively, relative to the AHD. In cfDNA of patients in group 2, an increase (p < 0.05) in the CNV of lncRNA XIST, HELLPAR, NEAT1, AC008124.1, LINC01089, LINC01547 and VASH1-AS1 genes by 1.9, 2.9, 3.5, 3.7, 2.6, 4.7 and 1.9 times was found respectively relative to the AHD. Differences (p < 0.05) by 4.8, 9.7, 7.0, 7.4, 5.0, 3.1, 4.8 and 2.2 times were observed between 2 groups of patients in terms of CNV of XIST, HELLPAR, NEAT1, AC008124.1, AC016717.2, LINC01089, LINC01547 and VASH1-AS1 genes, respectively. Conclusions: Increased CNV of XIST, HELLPAR, NEAT1, AC008124.1, LINC01089, LINC01547 and VASH1-AS1 genes in blood plasma cfDNA is associated with low RT efficiency.