The Study On The Function And Molecular Mechanism Of Exosomal MiR-92a-3p In Hepatocelluar Carcinoma

Background:Primary liver cancer is one of the most common malignant tumors,and about 90%of them are hepatocellular carcinoma(HCC).Although the treatment methods and diagnosis technology of liver cancer have been continuously improved in recent years,the prognosis of patients with metastatic liver cancer has always been poor,and the mechanism of occurrence and metastasis of liver cancer is still blurred.Exosomes are extracellular vesicles secreted by cells with a diameter of about 30-150 nm.Exosomes carry kinds of biomolecules,such as non-coding RNAs(including miRNAs),messenger RNAs and proteins,which play important roles in cellular signalings communication.miRNA is the most abundant component in exosomes.In recent years,various miRNAs have been shown to play an indispensable role in the development of cancer.However,the specific role of exosome miRNA in hepatocellular carcinoma metastasis,tumor progression,and its underlying mechanism still need to be further explored.Objectives:This study aims to analyze the differential exosomal miRNAs in liver cancer cells with different metastatic capabilities through exosomal miRNA sequencing,and to find miRNAs that play an important role in the process of hepatocellular carcinoma metastasis,and to explore the development and metastasis process of HCC and further explore the downstream regulatory mechanism of the target miRNA.And analyzing the upstream regulatory factors that regulate miRNA expression is one of our objectives as well.All of these may help to propose new ideas for the diagnosis and treatment of metastatic HCC.Methods:HCC cell lines with high metastatic capacity(97hm and Huhm)were constructed by continuously applying selective pressure on HCC cell lines.Then,the effects of highly metastatic liver cancer cells on low metastatic liver cancer cells were verified through co-culture experiments and in vivo lung metastasis experiments.Exosomes were obtained by enriching starved cell supernatant and high-speed centrifugation,and its contents were analyzed by sequencing the exosomal miRNA sequencing.miR-92a-3p is the most abundant miRNA in the potential differential miRNAs,and RT-PCR was performed in exosomes of liver cancer cell,plasma of mouse metastasis models,and plasma samples of clinical HCC patients to verify the potential miRNA levels.In the following experiments,miR-92a-3p mimics were used to overexpress miR-92a-3p,and inhibitor(in vitro)and antigomiR(in vivo)were used to knock down miR-92a-3p levels.CCK8,clonal formation and subendothelial tumorigenesis experiments were used to verify miR-92a-3p affect proliferation ability of HCC.The transwells assay,wound healing assay,and in vivo lung and liver metastasis assay were used to verify the effect of miR-92a-3p on the metastasis ability of liver cancer cells.As for mechanism,the potential target genes of miR-92a-3p are predicted through the bioinformatics website,and the target genes and binding sites of miR-92a-3p are analyzed by RT-PCR and luciferase dual reporter genes.For upstream regulatory genes,the potential regulatory transcription factors of miR-92a-3p were predicted by sequencing transcriptomes of hepatocellular carcinoma cells with different metastatic abilities and the bioinformatics website.RT-PCR,chromatin immunoprecipitation(Chip)and luciferase double-report experiments were used to analyze the transcriptional activation of miR-92a-3p by E2F1 and C-myc.Results:1.Exosomes of high-metastatic HCC cells can transfer metastatic ability to low-metastatic HCC cells.Exosomes derived from high-metastatic HCC cells can improve the migration ability of the recipient liver cancer cells in vitro and promote lung metastasis ability of HCC cells in vivo.2.Through analysing of exosome miRNA sequencing results,miR-92a-3p is the highest miRNA among the differential miRNAs(p value is less than 0.05)in the high-metastatic HCC cells.The results of RT-PCR experiments reveal that miR-92a-3p is highly expressed in both the exosomes of high-metastatic HCC cells and the plasma exosomes of lung metastatic mice compared with the control group.In the mice model,the level of exosome miR-92a-3p increases with the development of HCC.In clinical samples,exosomal miR-92a-3p is highly expressed in plasma of HCC patients with metastasis,and high levels of plasma exosomal miR-92a-3p are associated with poor prognosis in HCC patients.3.In terms of biological functions,in vivo and in vitro experimental data show that after overexpression of miR-92a-3p,the proliferation ability and invasion and migration ability of liver cancer cells are improved.In contrast,inhibition of miR-92a-3p significantly inhibits the proliferation ability of liver cancer cells.The ability of tumor and metastasis in vivo and in vitro.And western blot experiments indicate that miR-92a-3p promotes epithelial-mesenchymal transition by reducing E-cadherin and promoting expression of β-catenin and snail.4.The miRNA database were used to predict the potential target genes of miR-92a-3p.RT-PCR results indicate that miR-92a-3p significantly reduces the m RNA level of PTEN.Luciferase report assay indicate that miR-92a-3p can directly bind to the3’-UTR region of PTEN,inhibiting the expression of PTEN.Furthermore,western blot results indicate that miR-92a-3p can inhibit PTEN,following promote the p-akt / snail pathway and promote metastasis of HCC.Moreover,overexpression of PTEN can counteract the increased metastatic ability of HCC cells caused by overexpression of miR-92a-3p.5.Combining the transcriptome sequencing results of HCC cells with different metastatic ability and bioinformatic analysis,E2F1 and C-myc are potential regulatory transcription factors for miR-92a-3p.RT-PCR results indicated that highly metastatic HCC cells contained higher levels of E2F1 and C-myc,and overexpression of E2F1 and C-myc promoted the expression of miR-92a-3p.Chromatin immunoprecipitation experiments proved that E2F1 and C-myc can directly combine with the upstream of the miR17 HG promoter.Fluorescent dual-report assay showed that E2F1 and C-myc can transcribe and activate miR-92a-3p to promote its expression.Conclusions:1.Compared with low-metastatic HCC cells,high-metastatic HCC cell exosomes contain high levels of miR-92a-3p,and metastatic HCC patients have high expression of exosomal miR-92a-3p in plasma,and high levels of plasma exosomal miR-92a-3p are associated with poor prognosis of HCC patients.2.Ectopic overexpression of miR-92a-3p promotes the proliferation,invasion,migration and metastasis of HCC cells both in vitro and in vivo.Conversely,knockdown of miR-92a-3p can inhibit the proliferation,tumorigenic ability and metastatic ability of HCC cells.3.Mi R-92a-3p inhibits the expression of PTEN by targeting 3’-UTR of PTEN,promotes the phosphorylation of akt and the expression of snail,thereby promoting EMT and metastasis of HCC cells.4.The transcription factors E2F1 and C-myc can directly bind to the upstream region of the promoter of miR-92a-3p host gene miR17 HG,transcriptionally activate miR-92a-3p and promote the development of metastatic HCC.