Transfer RNA Derived Glu^TTC-tRF-3 Facilitates Hypoxia-induced Pulmonary Arterial Hypertension Through Suppressing TDP43 Expression In Vascular Smooth Muscle Cells

OBJECTIVEVascular smooth muscle cell（VSMC）proliferation and hypertrophy are the pathological hallmarkers of pulmonary arterial hypertension（PAH）,but its underlying mechanisms are not fully understood.Transfer RNA（t RNA）is a class of adotor RNA mediting protein translation,and mature t RNA can be cleavaged into t RNA halves（tiRNAs）and t RNA-derived fragments（tRFs）.Accumulated evidence showed these t RNA-derived nc RNAs play an important role in many physiological and pathophysiological processes.However,whether or how the t RNA-derived nc RNAs are involved in pathogenesis of PAH remains unclear.This study is aimed to investigate the impact of upregualted t RNA-derived nc RNAs on pulmonary arterial（PA）SMC proliferation and hypoxia-induced PAH in mice.METHODSHigh-throghput sequencing and q PCR assays were used to explore expression of tRFs and tiRNAs in pulmonary arteries from hypoxia-exposed mice;Synthetic mimics of tRFs and tiRNAs were used to analyze their regulatory functions on the biological behaviors of human and mouse PASMCs.Primary PASMCs were separated in mice and verified by SM22 immunofluorescence.Cells were cultured under either normoxic or hypoxic conditions.Glu^TTC-tRF-3 mimics and inhibitors（LNA Oligos）were obtained by chemical synthesis.Expression plasmid of Glu^TTC-tRF-3 were cloned.After treatment of these mimics,inhibitors or plasmid transfection,biological behavor changes in human and mouse PASMCs were mornitored.Cell proliferation was detected by CCK8 assay;Cell prolferative cycles were seperated by PI chromosomal staining;Cell apoptosis was analyzed by PI and FITC-labeled Annexin-V double-dyed staining followed flow cytometry;Real-time quantitative PCR and western blot analysis were used to detect the expression of targeted genes and proteins;and transwell assays were used to examine cell migration.CRISPR/Cas9 approach was used to construct Glu^TTC-tRF-3 transgenic mice.Hypoxia（10%O2）was used to induce PAH in mice.Right chamber systolic pressure(RVSP＿,ventriculus dexter gravity（RV/LV-S）were measured in mice.The H&E and immunofluorescence staining were performed to evaluate the median thickness（media/CSA）of pulmonary arteries in mice.The collagen expression in lung tissues was stained with mascurome tricolor dyeing.The proliferation of PASMCs in mice was analyzed by immunofluorescent staining of PCNA.RNA-Seq and TMT protein spectrometra were used to analyze the potential downstream targets of Glu^TTC-tRF-3.Potential targeted proteins of Glu^TTC-tRF-3 were cross-analyzed of database mi RDB,RNA-Seq and TMT protein spectrometra.Western blot assay and DNA transfection were used to confirm targeted gene of Glu^TTC-tRF-3.Lentivirus-mediated Glu^TTC-tRF-3 Sponge was intratracheally infused to hypoxia-exposed mice by to examine its therapeutic potentials.Measuement of RVSP and RV/LV-S in mice,and HE,PCNA immunofluorescence stainings in lung tissues were routinely performed to assess the therapeutic effect of Glu^TTC-tRF-3 Sponge on PAH.RESULTS1,Marked expression changes of tRFs and tiRNAs were observed in mouse PAs in response to hypoxia,including 18 upregulated and 1 down-regulated tRFs and tiRNAs.Six tRFs and tiRNAs were highly conservative between mouse and human,and their upregulation was further confirm in hypoxia-treated human and mouse PASMCs.Cell proliferative assay demonstrated that,the upregulated Glu^TTC-tRF-3 dramatically promoted PASMC growth.2,Glu^TTC-tRF-3 mimics or overexpression promoted hypoxia-induced PASMC proliferation,while inhibition of Glu^TTC-tRF-3 suppressed cell proliferation.Overexpression of Glu^TTC-tRF-3 exaggerated hypoxia-indued PAH in mice with elevated RVSP and RV/LV-S,and increased PA remodeling.3,Genome-wide RNA and TMT protein sequencing demonstrated treatment of Glu^TTC-tRF-3 mimics altered gene and protein expression profiles in PASMCs.Combined with the downregulated genes and proteins caused by Glu^TTC-tRF-3 mimics,we found TDP43 was the targeted candidate gene of Glu^TTC-tRF-3,as predicted using mi Target in mi RDB database.Forced expression of TDP43 reversed Glu^TTC-tRF-3-mediated proliferation of PASMCs in response to hypoxia,and infusion of TDP43-expressing lentinvirus mitigated hypoxia-induced PAH in Glu^TTC-tRF-3 transgenic mice.4.Intratracheal infusion of Lentivirus-mediated Glu^TTC-tRF-3 sponge effectively attenuated hypoxia-induced PASMC proliferation and suppressed the progression of hypoxia-induced PAH in mice.CONCLUSION:We found hypoxia increased the expression of t RNA-derived Glu^TTC-tRF-3 in mouse PA and PASMCs,Glu^TTC-tRF-3 facilitated hypoxia-induced PASMC proliferation and PAH in mice by targeting TDP43,and a Lentivirus-mediated Glu^TTC-tRF-3 sponge mitigated hypoxia-induced PAH in mice.Therefore,Glu^TTC-tRF-3 maybe serve as a potential therapeutic target for PAH.