Non-coding RNA profiling by high throughput sequencing
Summary
Background: MicroRNAs (miRs) are small non-coding RNAs that regulate gene (mRNA) expression. Although pathological role of miRs have been studied in muscle wasting conditions such as age, obesity, starvation and muscular dystrophy, their roles in Cancer Cachexia CC are still emerging. Objectives: (i) to profile human skeletal muscle expressed miRs; (ii) to identify differentially expressed (DE) miRs between cachectic and non-cachectic cancer patients; (iii) to identify mRNA targets for the DE miRs to gain mechanistic insights and (iv) to investigate if miRs show potential prognostic and predictive value. Methods: 43 cancer patients were included, of which 19 were cachectic cases exhibiting pre-illness weight loss ≥ 5% and 24 non-cachectic cancer controls who were weight stable in the preceding 6 months. Total RNA isolated from muscle biopsies were subjected to next generation sequencing (NGS). Results: 781 miRs were profiled and 82 miRs with read counts of ≥ 5 in 80% of samples were retained for analysis. We identified 7 DE miRs (up-regulated, fold change of ≥1.4 at p< 0.05). Potential mRNA targets for the DE miRs were identified using previously described human skeletal muscle mRNA expression data (n=90) and identified 212 potential mRNA targets. Ingenuity Pathway Analysis identified 41 pathways (p<0.05) including pathways of myogenesis and inflammation. qRT-PCR analysis of representative miRs, showed similar direction of effect (p<0.05) as in NGS. And lastly, we evaluated the identified miRs for prognostic and predictive value. Conclusions: We identified seven novel miRs associated with CC and pathways that are regulated in CC.
Overall design
Identification of miRs for cancer Cachexia using human skeletal muscle biopsies
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