Supplementary Material for: Inhibition of LncRNA-HRIM Increases Cell Viability by Regulating Autophagy Levels During Hypoxia/Reoxygenation in Myocytes

Backgrund/Aims: Ischemia reperfusion (I/R) promotes the severity of cardiomyocyte injury. Long noncoding RNAs (LncRNAs) are key regulators in cardiovascular diseases. However, the association between LncRNAs and myocardial I/R injury has not been thoroughly characterized to date. We attempted to clarify the potential biological role of a LncRNA (E230034O05Rik), which we named hypoxia/reoxygenation (H/R) injury-related factor in myocytes (HRIM), by investigating the differential expression of LncRNAs between groups of myocytes exposed to either a normal level of oxygen or to H/R. Methods: Microarray analysis was used to determine analyze the global differential expression of LncRNAs in H9c2 myocytes exposed either to a normal level of oxygen or to H/R. Target LncRNA levels were further verified in vitro and ex vivo by real-time polymerase chain reaction (qPCR). Cell viability was analyzed using the Cell Counting Kit-8 assay. Autophagy levels were confirmed by Western blotting, transmission electron microscopy, and autophagic double-labeled (mRFP-GFP-LC3) adenovirus analyses. Results: Gene expression profiling revealed that 797 LncRNAs and 1898 mRNAs were differentially expressed in the H/R group compared with the normal oxygen group. Among these LncRNAs and mRNAs, 6 upregulated LncRNAs and 2 downregulated LncRNAs in the H/R group were selected and further validated by qPCR in vitro and ex vivo. Additionally, LncRNA-HRIM was inhibited by specific siRNAs in H9c2 myocytes exposed to H/R. The inhibition of LncRNA-HRIM by siRNA prevented cell death by suppressing excessive autophagic activity in myocytes, This finding suggests a detrimental role of LncRNA-HRIM in the regulation of I/R injury. Conclusions: LncRNAs are involved in H/R injury of H9c2 myocytes. Inhibition of LncRNA-HRIM increased cell viability by reducing autophagy in myocytes during H/R.