Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer.

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination.

The Detection of Cancer Epigenetic Traces in Cell-Free DNA.

Nucleic acid fragments found in blood circulation originate mostly from dying cells and carry signs pointing to specific features of the parental cell types. Deciphering these clues may be transformative for numerous research and clinical applications but strongly depends on the development and implementation of robust analytical methods. Remarkable progress has been achieved in the reliable detection of sequence alterations in cell-free DNA while decoding epigenetic information from methylation and fragmentation patterns requires more sophisticated approaches. This review discusses the currently available strategies for detecting and analyzing the epigenetic marks in the liquid biopsies.

Breast cancer organoid model allowed to reveal potentially beneficial combinations of 3,3'-diindolylmethane and chemotherapy drugs.

Epigenetic alterations represent promising therapeutic targets in cancer treatment. Recently it was revealed that small molecules have the potential to act as microRNA silencers. Capacity to bind the discrete stem-looped structure of pre-miR-21 and prevent its maturation opens opportunities to utilize such compounds for the prevention of initiation, progression, and chemoresistance of cancer. Molecular simulations performed earlier identified 3,3'-diindolylmethane (DIM) as a potent microRNA-21 antagonist. However, data on DIM and microRNA-21 interplay is controversial, which may be caused by the limitations of the cell lines.

TATA-Like Boxes in RNA Polymerase III Promoters: Requirements for Nucleotide Sequences.

tRNA and some other non-coding RNA genes are transcribed by RNA polymerase III (pol III), due to the presence of intragenic promoter, consisting of boxes A and B spaced by 30-40 bp. Such pol III promoters, called type 2, are also intrinsic to Short Interspersed Elements (SINEs). The contribution of 5'-flanking sequences to the transcription efficiency of genes containing type 2 promoters is still studied insufficiently. Here, we studied this issue, focusing on the genes of two small non-coding RNAs (4.5SH and 4.5SI), as well as B1 and B2 SINEs from the mouse genome. We found that the regions from position -31 to -24 may significantly influence the transcription of genes and SINEs. We studied the influence of nucleotide substitutions in these sites, representing TATA-like boxes, on transcription of 4.5SH and 4.5SI RNA genes. As a rule, the substitutions of A and T to G or C reduced the transcription level, although the replacement of C with A also lowered it. In 4.5SH gene, five distal nucleotides of -31/-24 box (TTCAAGTA) appeared to be the most important, while in the box -31/-24 of 4.5SI gene (CTACATGA), all nucleotides, except for the first one, contributed significantly to the transcription efficiency. Random sequences occurring at positions -31/-24 upstream of SINE copies integrated into genome, promoted their transcription with different efficacy. In the 5'-flanking sequences of 4.5SH and 4.5SI RNA genes, the recognition sites of CREB, C/EBP, and Sp1 factors were found, and their deletion decreased the transcription.

A 5'-3' terminal stem in small non-coding RNAs extends their lifetime.

4.5SI and 4.5SH are two non-coding RNAs about 100nt long, synthesized by RNA polymerase III in cells of various rodents including mice, rats, and hamsters. The first RNA is long-lived whereas the half-life of the second is only 20min. We previously found that the 16bp double-stranded structure (stem), formed by 4.5SI RNA termini, contributes essentially to the long lifetime of this RNA (Koval et al., 2012). The rapid decay of 4.5SH RNA seems to be related to the lack of a similar structure in this RNA. The aim of this work was to verify whether the lifetime of any other short-lived non-coding RNA can be prolonged following creation of the double-stranded structure with its terminal regions. Here RNAs transcribed by RNA polymerase III from short interspersed elements (SINEs) B2 and Rhin-1 from the genomes of mouse and horseshoe bat, respectively, were used. Replacement of 16nt at the 3'-terminal region by the sequence complementary to the 5' end region of B2 and Rhin-1 RNA increased their half-life more than 4 fold. In addition, we demonstrated that shortening of the terminal stem from 16 to 8bp decreased only slightly the 4.5SI RNA lifetime. Finally, we showed that the disruption of an internal (non-terminal) stem in 4.5SI RNA did not accelerate its decay in cells. Possible mechanisms of the small non-coding RNA lifetime extension are discussed.

Complementarity of end regions increases the lifetime of small RNAs in mammalian cells.

Two RNAs (4.5SH and 4.5SI) with unknown functions share a number of features: short length (about 100 nt), transcription by RNA polymerase III, predominately nuclear localization, the presence in various tissues, and relatively narrow taxonomic distribution (4 and 3 rodent families, respectively). It was reported that 4.5SH RNA turns over rapidly, whereas 4.5SI RNA is stable in the cell, but their lifetimes remained unknown. We showed that 4.5SH is indeed short-lived (t(1/2)~18 min) and 4.5SI is long-lived (t(1/2)~22 h) in Krebs ascites carcinoma cells. The RNA structures specifying rapid or slow decay of different small cellular RNAs remain unstudied. We searched for RNA structural features that determine the short lifetime of 4.5SH in comparison with the long lifetime of 4.5SI RNA. The sequences of genes of 4.5SH and 4.5SI RNAs were altered and human cells (HeLa) were transfected with these genes. The decay rate of the original and altered RNAs was measured. The complementarity of 16-nt end regions of 4.5SI RNA proved to contribute to its stability in cells, whereas the lack of such complementarity in 4.5SH RNA caused its rapid decay. Possible mechanisms of the phenomenon are discussed.

Additional box B of RNA polymerase III promoter in SINE B1 can be functional.

Many genes of small RNAs and short interspersed elements (SINEs) are transcribed by RNA polymerase III due to an internal promoter that is composed of two boxes (A and B) spaced by 30-45bp. Rodent SINE B1 originated from 7SL RNA, and a 29-bp tandem duplication took place in B1 at an early stage of its evolution. As a result of this duplication, an additional box B (named B') located at a distance of 79-82bp from box A arose in SINE B1. Here we have shown that despite the unusually large distance between boxes A and B', they can form an active promoter. In chinchillas, guinea pigs, and other rodents belonging to clade Ctenohystrica, structure of the B' box was well preserved and closely resembles the canonical B box. One may suggest therefore, that box B' can functionally replace box B in those copies of B1 where the latter has lost activity due to mutations.

5'-flanking sequences can dramatically influence 4.5SH RNA gene transcription by RNA-polymerase III.

4.5SH RNA is a 94 nt small nuclear RNA with an unknown function. Hundreds of its genes are present in the genomes of rodents of six families including Muridae. 4.5SH RNA genes contain an internal RNA-polymerase III promoter consisting of A and B boxes. Here we studied the influence of 5'-flanking sequences on the transcription of a mouse 4.5SH RNA gene. We found that replacement of the upstream sequence can dramatically change the 4.5SH RNA gene transcription efficiency. Various DNA fragments inserted immediately upstream from 4.5SH RNA gene completely inhibited its in vitro transcription, whereas others promoted it. The shortening of the native mouse 5'-flanking sequence of 4.5SH RNA gene to 42 bp resulted in the activation of an additional illegal transcription start site in upstream region. Transcription of the 4.5SH RNA gene with various upstream sequences in transfected HeLa cells revealed the differences between the tests performed in vivo and in vitro: in whole cells, only the construct with 5'-flanking native sequence could be transcribed. Apparently, at least some regions of the native 5'-flanking sequence of 4.5SH RNA genes have been selected during evolution for high transcription activity.

Evolutionary history of 4.5SH RNA.

4.5SH RNA is a 94-nt small RNA with unknown function. This RNA is known to be present in the mouse, rat, and hamster cells; however, it is not found in human, rabbit, and chicken. In the mouse genome, the 4.5SH RNA gene is a part of a long (4.2 kb) tandem repeat ( approximately 800 copies) unit. Here, we found that 4.5SH RNA genes are present only in rodents of six families that comprise the Myodonta clade: Muridae, Cricetidae, Spalacidae, Rhizomyidae, Zapodidae, and Dipodidae. The analysis of complementary DNA derived from the rodents of these families showed general evolutionary conservation of 4.5SH RNA and some intraspecific heterogeneity of these RNA molecules. 4.5SH RNA genes in the Norway rat, mole rat, hamster and jerboa genomes are included in the repeated sequences. In the jerboa genome these repeats are 4.0-kb long and arranged tandemly, similar to the corresponding arrangements in the mouse and rat genomic DNA. Sequencing of the rat and jerboa DNA repeats containing 4.5SH RNA genes showed fast evolution of the gene-flanking sequences. The repeat sequences of the distantly related rodents (mouse and rat vs. jerboa) have no apparent similarity except for the 4.5SH RNA gene itself. Conservation of the 4.5SH RNA gene nucleotide sequence indicates that this RNA is likely to be under selection pressure and, thus, may have a function. The repeats from the different rodents have similar lengths and contain many simple short repeats. The data obtained suggest that long insertions, deletions, and simple sequence amplifications significantly contribute in the evolution of the repeats containing 4.5SH RNA genes. The 4.5SH RNA gene seems to have originated 50-85 MYA in a Myodonta ancestor from a copy of the B1 short interspersed element. The amplification of the gene with the flanking sequences could result from the supposed cellular requirement of the intensive synthesis of 4.5SH RNA. Further Myodonta evolution led to dramatic changes of the repeat sequences in every lineage with the conservation of the 4.5SH RNA genes only. This gene, like some other relatively recently originated genes, could be a useful model for studying generation and evolution of non-protein-coding genes.