Identification of microRNAs in skeletal muscle associated with lung cancer cachexia

Abstract

Background Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets. Methods A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcriptionquantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients. Results We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six topranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival. Conclusions We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.

Sex Differences in Cancer Cachexia

“…Because differentially expressed miRNAs have been associated with metabolic alterations, inflammatory responses, and cancer-induced muscle wasting in humans and rodent models and the molecular targets for these miRNAs are involved in the regulation of catabolism, acute phase response, and muscle degenerative or regenerative capacity [85][86][87][88][89], we wondered whether sex-related factors such as sex chromosomes-linked genes would be the source of differences in the roles of altered miRNAs expression in cancer cachexia. However, no publication has addressed this in cancer cachexia.…”

“…However, no publication has addressed this in cancer cachexia. To begin addressing the possibility, we investigated the validated cachexia-associated miRNAs reported in the literature [86,88] to see whether any of the miRNAs are located on X chromosome by aligning them with the miRNA genes listed in the miRBase [90] (mirbase.org). The alignment indeed identified several on X chromosome, including mir-106a, mir-221, mir-223, mir-362, mir-384, mir-424, mir-450a, and mir-450b, which are shown in Table 1 along with their target genes.…”

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

“…More recently, Van de Worp et al (2019), by performing miRNA profiling in skeletal muscle of cachectic lung cancer patients compared to matched healthy controls, identified a signature of 28 miRNAs differentially expressed. Interestingly, 4 miRNAs our of 5 which resulted to be up-regulated in cachetic patients, belong to the same cluster (miR-450a-5p, miR-450b-5p, miR-424-5p, and miR-424-3) (Van de Worp et al, 2019).…”

Regulation of Skeletal Muscle Atrophy in Cachexia by MicroRNAs and Long Non-coding RNAs

“…Also, miR-424-5p regulates rRNA synthesis by inhibiting Pol I pre-initiation complex formation, which reduces the capacity of the protein synthesis machinery, contributing to inhibition of protein synthesis and loss of muscle mass in patients with ICU-acquired weakness or COPD, patients undergoing aortic surgery, and in individuals from the Hertfordshire Sarcopenia Study (Connolly et al, 2018). miR-424-5p expression is elevated in muscle of cachectic non-small cell lung cancer (NSCLC) patients compared with healthy control subjects; however, the mechanism underlying the altered expression of miR-424-5p in NSCLC muscle atrophy is unknown (Worp et al, 2020). miR-142a-5p can inhibit mitofusin-1 (MFN1) expression by binding to the 3'UTR of its mRNA.…”