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Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells.

ABSTRACT: MicroRNAs are key post-transcriptional gene regulators often displaying aberrant expression patterns in cancer. As microRNAs are promising disease-associated biomarkers and modulators of responsiveness to anti-cancer therapies, a solid understanding of their targetome is crucial. Despite enormous research efforts, the success rates of available tools to reliably predict microRNAs (miRNA)-target interactions remains limited. To investigate the disease-associated miRNA targetome, we have applied modified cross-linking ligation and sequencing of hybrids (qCLASH) to BRAF-mutant melanoma cells. The resulting RNA-RNA hybrid molecules provide a comprehensive and unbiased snapshot of direct miRNA-target interactions. The regulatory effects on selected miRNA target genes in predicted vs. non-predicted binding regions was validated by miRNA mimic experiments. Most miRNA-target interactions deviate from the central dogma of miRNA targeting up to 60% interactions occur via non-canonical seed pairing with a strong contribution of the 3' miRNA sequence, and over 50% display a clear bias towards the coding sequence of mRNAs. miRNAs targeting the coding sequence can directly reduce gene expression (miR-34a/CD68), while the majority of non-canonical miRNA interactions appear to have roles beyond target gene suppression (miR-100/AXL). Additionally, non-mRNA targets of miRNAs (lncRNAs) whose interactions mainly occur via non-canonical binding were identified in melanoma. This first application of CLASH sequencing to cancer cells identified over 8 K distinct miRNA-target interactions in melanoma cells. Our data highlight the importance non-canonical interactions, revealing further layers of complexity of post-transcriptional gene regulation in melanoma, thus expanding the pool of miRNA-target interactions, which have so far been omitted in the cancer field.

SUBMITTER: Kozar I

PROVIDER: S-EPMC7961530 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis.IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will help us understand their role in viral oncogenesis. The cross-linking and sequencing of hybrids (CLASH) protocol is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than for its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with wild-type (WT) KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of the cellular oncogenic miRNA (oncomiR) miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, apoptosis, and cell cycle control.

Project description:In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.

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Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells.

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