MiR-516a-3p Is A Novel Mediator Of Hepatocellularcarcinoma Oncogenic Activity And Cellular Metabolism

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors with poor prognosis.The incidence of HCC is increasing more rapidly than any other cancers,which deserves great attention.The mortality of HCC increases both in women and men during the last decades.Surgery is a crucial treatment for HCC.Despite the combination of targeted drug therapy,local ablation,transcatheter arterial chemoembolization,and even liver transplantation,the prognosis of HCC remains grave.Thus,to better understand the pathogenesis and explore possible therapeutic targets of HCC is mandatory.Mi RNA cluster,defined as a set of precursor miRNAs transcribed from the same orientation or adjacent genome,are particularly attractive.Their regulation and expression are characterized by a high degree of consistency.Evidence proved that they could target the same genes or pathways and achieve common effects on the regulation of tumorigenesis.Chromosome 19 micro RNA cluster(C19MC),as the largest miRNA cluster,contains 46 precursor miRNAs(pre-miRNAs).By mining The Cancer Genome Atlas TCGA(TCGA)pre-miRNA profiles,we demonstrated that the members of C19 MC showed consistent high expression levels in HCC.We also found that four members of C19MC(mir-516a-1,mir-516a-2,mir-516b-1 and mir-516b-2)were correlated with poor prognosis of HCC,and were also the risk factors of HCC.And the four oncogenic pre-miRNAs of C19 MC co-spliced an identical mature miRNA named as miR-516a-3p.The results of HCC clinical samples showed that miR-516a-3p was upregulated in HCC sample,compared with the adjacent non-tumor tissues.By assessing the clinical value of miR-516a-3p,we confirmed that higher expression of miR-516a-3p positively correlated with HCC features(tumor size > 8 cm;multiple tumors;AJCC stage III-IV).Patients with high tumor miR-516a-3p levels showed a significant lower tumor-free survival and lower overall survival as compared to those with low miR-516a-3p levels.In multivariate Cox analysis,tumor miR-516a-3p expression was proved to be an independent influencing factor of HCC recurrence and HCC mortality.The high tumor miR-516a-3p expression also improved the prediction of HCC recurrence and mortality.Meanwhile,miR-516a-3p increased the proliferation,invasiveness,metastasis of HCC cells in vitro and in vivo.Mi RNA could be transported among different cell types including cancer cells and adjacent cells,and affects the biology of recipient cells,with the carriers of exosomes or extracellular vesicles.Some reports have suggested that the functions of C19 MC were transferred via exosomes in human placental trophoblasts.In this study,we proved that,among cancer cells,miR-516a-3p could be delivered via exosomes or extracellular vesicles and increased the oncogenic activity of recipient cells.Overwhelming evidence proved that multiomics analysis could comprehensively explain the potential cancer biology mechanism.We first performed comprehensive transcriptomics,proteomics,and metabolomics analysis on the potential mechanism underlying miR-516a-3p-promoted oncogenicity.We further identified 6 proteins(LMBR1,CHST9,RBM3,SLC7A6,PTGFRN,and NOL12)as the direct targets of miR-516a-3p and central players in the miR-516a-3p-mediated metabolism regulation.The integrated multi-omics and co-enriched pathway analysis showed that miR-516a-3p regulated the metabolic pathways of HCC cells particularly the purine and pyrimidine metabolism.Mix Omic DIABLO analysis showed close correlation and strong cluster consistency between proteomics and metabolomics datasets,while the variables in transcriptomics were relatively discriminated against with the other two datasets.In conclusion,our results provide evidence that the analysis of co-spliced mature miRNA products might be a new strategy to investigate the functions of miRNA cluster.This study reveals the oncogenicity of four key members of C19 MC by exploring one mature miRNA,which may be called “killing four birds with one stone”.Mi R-516a-3p can enhance tumor malignant behaviors and affect neighboring HCC cells via the exosome delivery system.The finding is expected to provide a novel molecular target for HCC therapy and prognostic prediction.Furthermore,to the best of our knowledge,this is the first study to explore the function of miRNA using a multiomics approach,which has many advantages.The integrative analysis sheds new light on the miRNA regulation of cancer metabolism and malignant activity.