MicroRNA-mRNA Co-sequencing Identifies Transcriptional and Post-transcriptional Regulatory Networks Underlying Muscle Wasting in Cancer Cachexia

Abstract

Cachexia is a complex metabolic syndrome characterized by loss of skeletal muscle, leading to a significant weight loss that impacts patient morbidity and mortality. Given the complexity of gene regulatory networks that control gene expression, our objective was to perform an integrative mRNA and miRNA profiling to identify genetic programs that capture essential mechanistic details that promote muscle atrophy in cancer cachexia. Here, we used RNA sequencing to analyze miRNAs and mRNAs expression profiles in tibialis anterior (TA) muscles of the Lewis lung carcinoma model of cancer cachexia. In addition, we compared these findings with RNA-Seq data from C2C12 myotubes treated in vitro with the cachectic factors tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Extracellular matrix (ECM) alterations were validated by picrosirius staining, western blot, and fractal dimension analyses. We found 1,008 mRNAs and 18 miRNAs differentially expressed in cachectic mice. This set of genes was associated with the ECM, proteolysis, and inflammatory response. Enrichment analysis of transcriptional factor binding sites revealed activation of the atrophy-related transcriptional factors: NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and miRNA expression profiles identified posttranscriptional regulation by miRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and FoxO signaling pathway. C2C12 myotubes treated with TNF-α and IFN-γ similarly down-regulate subsets of ECM genes, including collagens. Our integrative analysis of miRNA-mRNA co-profiles comprehensive characterized regulatory relationships of molecular pathways and revealed miRNAs targeting ECM-associated genes in cancer cachexia. We also confirmed in C2C12 myotubes that changes in ECM-associated genes are dependent on inflammatory signaling of the cytokines TNF-α and IFN-γ.

Non-coding RNAs as Putative Biomarkers of Cancer-Associated Cachexia

“…The integrative analysis allowed the authors to generate a miRNA-mRNA network composed of 171 interactions between 18 miRNAs and 131 target mRNAs. This analysis revealed enrichment for genes involved in extracellular matrix organization, highlighting their contribution to cancerassociated cachexia (Fernandez et al, 2019).…”

“…This suggests the possibility for myomiR to be novel mediators and powerful biomarkers for cancer-associated cachexia. In the last years, different studies on muscle tissue from mice models of cachexia or from cachectic cancer patients revealed a correlation between myomiRs deregulation and cachexia occurrence and maintenance (Soares et al, 2014;Lee et al, 2017;Fernandez et al, 2019).…”

“…More recently, Fernandez and collaborators, performed an integrated genome wide analysis by combining miRNA/mRNA sequencing from the same set of skeletal muscles derived from mice models of cancer-associated cachexia (Fernandez et al, 2019). In particular, they identified 18 miRNAs differentially expressed in skeletal muscle of cachectic mice compared to the controls.…”

“…In particular, several studies have reported that low concentrations of myomiRs in blood correlate with poor prognosis in non-small cell lung cancer, melanoma, astrocytoma, or osteosarcoma patients (Hu et al, 2010;Li et al, 2014; Soares et al, 2014Soares et al, miR-147, miR-205-3p, miR-229a3p, miR-233 3p, miR-431-5p, miR-511-3p, miR-665-3p, miR-1933 Mice muscles Lee et al, 2017Lee et al, miR-10b-5p, 1249Lee et al, -3p, miR-144-3p, miR-144-5p, miR-146a-5p, miR-146b-5p, miR-181c-3p, miR-183-5p, miR-1843a Mice muscles Fernandez et al, 2019Fernandez et al, miR-3184-3p, miR-423-5p, let-7d-3p, miR-1296 Human skeletal muscle Zhang C. et al, 2014;Tian et al, 2015). This because skeletal muscle, under physiological conditions, continuously releases exosomes contained myomiRs in the blood (Guescini et al, 2015), but in advanced cancers, when a substantial reduction of muscle mass occurs, there is a consequent decrease in circulating myomiRs.…”

MicroRNA-mRNA Co-sequencing Identifies Transcriptional and Post-transcriptional Regulatory Networks Underlying Muscle Wasting in Cancer Cachexia

“…This manuscript has been released as a pre-print at https://www. preprints.org/ (Fernandez et al, 2019).…”