Hypoxia-induced TRF-3^Thr-CGT Promotes The Migration And Invasion By Inhibiting The Expression Of MtDNA-encoded Protein In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is the second most deadly malignancy in China in recent years.Tumor recurrence and migration are the underlying causes of the dramatic increase in mortality in HCC patients.Therefore,this study investigates the molecular mechanisms of HCC migration and invasion,and searches for the main therapeutic targets for HCC,in order to provide an effective way to solve the clinical practice problems such as low survival rate and poor prognosis of HCC patients.Hypoxia is one of the key features of the tumor cell microenvironment and plays an important role in tumor angiogenesis,invasion,migration and drug resistance.Despite the results achieved through liver transplantation,intra-arterial chemoembolization and interferon therapy,the practical clinical problems of postoperative recurrence and ischemia and hypoxia in HCC,especially the high invasiveness and metastatic nature of HCC cells due to hypoxia,have become bottlenecks limiting the treatment of HCC.Adaptive shifts in mitochondrial energy metabolism in tumor cells under hypoxic conditions are key to maintaining tumor cell invasion and metastasis and are one of the main mechanisms contributing to poor prognosis in HCC patients.In contrast,mitochondria are the most direct receptors of oxygen,and their oxidative phosphorylation system is regulated by a dual nuclear-mitochondrial genome;therefore,the role of hypoxia in the regulation of mitochondrial energy cannot be fully explained from the perspective of previous single nuclear transcription studies alone.Hypoxia primarily inhibits the translation of mitochondrial deoxyribonucleic acid(mtDNA)encoded proteins and affects mitochondrial function to promote reorganization of cellular energy metabolism.It is suggested that affecting the translation of the mtDNAencoded protein may be a breakthrough in elucidating the ability of hypoxia to coordinate mitochondrial energy metabolism for invasive migration in HCC,with a view to providing an effective way to improve the survival and prognosis of HCC patients.In many biological processes,including tumor progression,non-coding RNAs are involved in important pathways related to cellular invasion and metastasis under various stress conditions by regulating the expression of target genes at the transcriptional and post-transcriptional levels,and are considered to be one of the core molecules of translational signaling.Hypoxia-induced tRNA-derived fragments(tRF s)have become a new hot topic in biomedical research as novel splice molecules for decoding transcription and translation.tRNAs,which are 60-100 times more abundant than mRNAs in the nucleus and cytoplasm,can interfere with the transcription and translation of mRNAs and play an important role.Disturbances in the expression of tRFs can lead to the development of disease and can contribute to the invasion and migration of cancer cells by inhibiting the synthesis of respiratory chain proteins and causing energy metabolic conversion,among other things.Although there is growing evidence that tRFs are closely associated with tumor invasion and metastasis,identifying HCC-associated tRFs and elucidating their mechanisms of action remain key questions that we need to address.In this study,based on transcriptome sequencing analysis,we enriched the differential expression of tRFs and related pathways in HCC cells under hypoxic conditions and found that hypoxia led to high expression of tRF-3Thr-CGT.By examining the effect of tRF-3Thr-CGT on human mitochondrial peptide deformylase(HsPDF)and By examining the effect of tRF-3Thr-CGT on the expression of human mitochondrial peptide deformylase(HsPDF)and mtDNA-encoded proteins,we investigated whether tRF-3Thr-CGT could promote HCC cell invasion and migration through the inhibition of mtDNA-encoded protein expression by HsPDF,and provided new ideas for the development of tRF-3Thr-CGT-based treatment protocols.Objective:Based on in vivo and ex vivo experiments,we investigated the effect of hypoxiainduced tRF-3Thr-CGT on invasive metastasis of HCC cells,elucidated the mechanism by which tRF-3 Thr-CGT finely regulates the translation of mtDNA-encoded proteins,and revealed the role of hypoxia-mediated tRF-3 Thr-CGT in regulating the reorganization of mitochondrial energy metabolism in the invasive migration of HCC cells.Method:1.Transwell and scratch assays under hypoxia(5%CO2,1%O2,94%N2)to assay the invasion and migration ability of SNU-387 and SNU-449 cells.SNU-387 and SNU449 cells were treated with 1%O2 and the expression levels of ATPSA,UQCRC2,SDHB,COX2,ATP6,CYTB and Peptide demethylase were detected in SNU-387 and SNU-449 cells using Western Blot.2.HsPDF expression levels were detected under hypoxia(5%CO2,1%O2,94%N2)using Western Blot;ATPSA,UQCRC2,SDHB,ATP6,CYTB protein levels were detected in SNU-387 and SNU-449 cells using Western Blot;ATP6,ND1,CYTB and COX2 transcript levels were detected in SNU-387 and SNU-449 cells using q-PCR.3.Western Blot assay to detect the changes of HsPDF inhibitor actinonin on the expression levels of ATPSA,UQCRC2,SDHB,ATP6,CYTB and HsPDF in SNU-387 and SNU-449 cells.4.ATP production was detected by chemiluminescence and oxygen consumption levels of cells were measured using oxygen-sensitive probes to investigate the regulation of mitochondrial oxidative phosphorylation levels by HsPDF in SNU-387 and SNU-449 cells.5.Genome-wide microarray data analysis based on clinical hepatocellular carcinoma patients using the GEO database and the bioinformatics prediction website miRanda;transcriptome sequencing analysis to enrich the differential expression of tRFs and associated pathways in HCC cells under hypoxic conditions and predict high expression of tRF-3 Thr-CGT and its target gene HsPDF in hypoxia.6.The tRF-3 Thr-CGT overexpression vector(tRF-3 Thr-CGT mimic)was constructed.After overexpression of tRF-3 Thr-CGT in SNU-387 and SNU-449 cells,the transcript levels of tRF-3 Thr-CGT were detected by q-PCR;the expression levels of HsPDF in SNU387 and SNU-449 cells were detected by q-PCR and Western Blot.7.Overexpression of tRF-3Thr-CGT in SNU-387 and SNU-449 cells.q-PCR and Western Blot assays were used to detect changes in ATPSA,UQCRC2,SDHB,ATP6 and CYTB protein levels and ATP6,ND1,CYTB and COX2 transcript levels in SNU387 and SNU-449 cells.8.Overexpression of tRF-3 Thr-CGT in SNU-387 and SNU-449 cells and detection of ATP production by chemiluminescence and oxygen consumption levels in cells using an oxygen-sensitive probe.9.The tRF-3 Thr-CGT overexpression vector and an inhibitor(tRF-3 Thr-CGT inhibitor)were constructed to overexpress and underexpress tRF-3 Thr-CGTin SNU-387 and SNU449 cells.Western Blot assays were performed to detect the expression levels of EMTrelated proteins E-cadherin,N-cadherin and Vimentin.10.Transwell and scratch assays examined the effects of overexpression and inhibition of tRF-3 Thr-CGT on the invasion and migration ability of SNU-387 and SNU449 cells.11.Construction of a mouse primary displacement colonoma model using a mouse hepatocellular carcinoma cell-luciferase labeled cell line,Hep-1-6-LUC and quantitative validation of the effect of tRF-3 Thr-CGT on the migration of tumor nodules in the mouse liver using small animal biopsy techniques for metastasis throughout the liver.Result:1.Hypoxia(5%CO2,1%O2,94%N2)promote the ability of HCC cells to invade and migrate compared to normoxia(5%CO2,1%O2).2.The expression levels of HsPDF decreased under hypoxic(5%CO2,1%O2,94%N2)compared to normoxia(5%CO2,1%O2)conditions;the expression levels of the nuclear gene-encoded proteins ATPSA,UQCRC2 and SDHB remained unchanged,and the transcript levels of the mtDNA-encoded proteins ND1,COX2,ATP6 and CYTB did not decrease,while the expression levels of ATP6 and CYTB decreased.3.Compared to the NC group,protein expression of HsPDF,ATP6 and CYTB was reduced in SNU-387 and SNU-449 cells after inhibition of HsPDF expression using actinonin,and the expression levels of the nuclear gene-encoded proteins ATPSA,UQCRC2 and SDHB were largely unchanged.4.ATP production was reduced and oxygen consumption was significantly reduced in SNU-387 and SNU-449 cells after inhibition of HsPDF expression using actinonin compared to the NC group.5.Compared to the NC group,transfection with the tRF-3Thr-CGT overexpression vector resulted in increased tRF-3Thr-CGT transcript levels in SNU-387 and SNU-449 cells;transcript levels and protein expression in HsPDF were decreased.6.Compared to the NC group,transfection with the tRF-3Thr-CGT overexpression vector resulted in essentially unchanged protein expression of the SNU-387 and SNU449 cell nuclear gene-encoded proteins ATPSA,UQCRC2 and SDHB,and no decrease in transcript levels of ATP6,ND1,CYTB and COX2,and decreased protein expression levels of ATP6 and CYTB.7.ATP production was reduced and oxygen consumption was significantly reduced in SNU-387 and SNU-449 cells after transfection with the tRF-3Thr-CGT overexpression vector compared to the NC group.8.Compared with the NC group,the expression levels of N-cadherin and Vimentin protein increased and E-cadherin protein decreased after transfection with tRF-3Thr-CGT overexpression vector;E-cadherin protein expression levels increased and N-cadherin and Vimentin protein expression levels decreased after transfection with tRF-3Thr-CGT repression vector.9.Transfection of tRF-3Thr-CGT overexpression vector promoted SNU-387 and SNU-449 cell invasion and migration compared to NC group;transfection of tRF-3ThrCGT suppression vector inhibited SNU-387 and SNU-449 cell invasion and migration.10.Compared with the number of nodules metastasizing from hepatocellular carcinoma in the NC group,the number of nodules metastasizing from hepatocellular carcinoma in the tRF-3Thr-CGT(mimic)group increased and the number of nodules metastasizing from hepatocellular carcinoma in the tRF-3Thr-CGT(inhibitor)group decreased.Conclusion:tRF-3Thr-CGT has the ability to promote HCC cell invasion and migration,while hypoxia,an important factor,significantly induces tRF-3Thr-CGT expression and affects HCC cell mtDNA-encoded protein translation through regulation of HsPDF,leading to reorganization of energy metabolism affecting the EMT pathway to promote HCC cell invasion and migration,which could be a novel diagnostic biomarker and therapeutic target for HCC.