Differential Expressed Exosomal MicroRNAs Identification And Let-7f-5p Biological Function Research In Hepatacellular Carcinoma

Liver cancer is a common malignant tumor with a high morbidity and mortality.50%of liver cancer incidence and death occur in China,and 70~90% of primary liver cancer is hepatocellular carcinoma(HCC).Exosomes are membranous vesicles that can be secreted by a variety of cells including tumor cells,and are approximately 30 to 150 nm in diameter and contain abundant contents,including proteins,lipids,and microRNAs(miRNAs),involved in the exchange of intercellular substances and information.At the time of tumorigenesis,tumor cells secrete more exosomes than normal cells,and the expression profile of exosomal miRNAs is quite different from that under normal physiological conditions.Cells can transmit information of substances through exosomes to regulate the microenvironment of tumors,and then affect the occurrence and development of cancer.Therefore,exosomal miRNAs is not only related to the mechanism of disease development,but also may be a better biomarker for early diagnosis of tumors.This study aimed to screen differentially expressed miRNAs in exosomes of hepatocellular carcinoma cell lines,and explore the effects of differentially expressed miRNAs in exosomes on the function of hepatocellular carcinoma cells,and their clinicalapplication in the diagnosis of hepatocellular carcinoma.This study carried out the following four parts of the experiment.Part 1: Screening differentially expressed miRNAs of HCC cell lines exosomes.Objective: Screening differentially expressed miRNAs of hepatocellular carcinoma exosomes by high-throughput sequencing to lay the foundation for exploring the physiological and pathological functions of exosomal miRNAs in the development of liver cancer and possible biological markers.Methods: Collecting cell supernants of human nromal liver cell line HL-7702,hepatitis B negative HCC cell line Hep G2 and hepatitis B positive HCC cell line Hep 3B,and extracting their exosome.The exosomal markers of CD63 and CD81 were analyzed with flow cytometry,their size and morphology were identified by transmission electron microscopy,The differentially expressed miRNAs were screened by Next Generation Sequencing.The expression of specific miRNAs of cell lines and exosomes were analyzed by quantitative real-time PCR(qRT-PCR).Results:Exosomes we extracted under the transmission electron microscope had complete membrane structure with diameters of around 30~150 nm,Flow cytometry showed that CD63 and CD81 were positively expressed in exosomes.28 miRNAs including 26down-regulated were differentially expressed in HCC cell line exosomes.Six differentially expressed exosomal miRNAs were selected for qRT-PCR verification,the results showed that the expression trends of the six miRNA in the cell supernatant exosomes,cell lines were consistent with the sequencing results.Conclusion: Through high-throughput sequencing of miRNAs of exosomes of cultured cells,a set of miRNAs differentially expressed in exosomes of liver cancer cells was obtained.Part 2: miRNA affects biology behaviors of HCC cells.Objective: This study aimed to value the effects overexpression of miR-98-5p,let-7f-5p and miR-196a-5p with low expression in HCC cell exosomes on HCC cell proliferation,migration,invasion,and apoptosis.Methods: CCK8 was used to assess the effects of overexpresson of miRNA on cell proliferation,wound healing assay used to assess the effects of overexpresson of miRNA on cell migration,transwell assay was used to assess the effects of overexpresson of miRNA on cell migration and invasion,flowcytometry technique to assess the effects of overexpresson of miRNA on apoptosis.Results: After transfection,we found that overexpression of let-7f-5p could inhibit HCC cell proliferation,invasion and migration,but promote its apoptosis compared with the control group.Conclusion: Overexpression of let-7f-5p could inhibit HCC cell proliferation,invasion and metastasis,but promote the apoptosis of them.Part 3: The target gene of let-7f-5p and biological functions of its target gene.Objective: To predict the target gene of let-7f-5p and validate it,and observe the biological behavior of HCC cells after changing the expression of the target gene.Methods: Bioinformatics techniques were used to predict the target gene let-7f-5p.The effects of let-7f-5p on target gene expression were verified by qRT-PCR and western blot(WB)at mRNA and protein levels.The target gene was verified by a dual luciferase gene detection system.The effects of knockdown of target gene on HCC cells was detected by CCK8 assay and transwell assay for their proliferation,migration and invasion.It was verified whether knockdown of the target gene resulted in a similar effect to that of let-7f-5p.To observe whether the co-transfection of let-7f-5p and target gene recombinant expression vector plasmids can restore the influence of let-7f-5p on proliferation,migration and invasion ability of HCC cells.The effects of cotransfection of let-7f-5p and DVL3 recombinant expression vector plasmids on HCC cells was detected by CCK8 assay and transwell assay for their proliferation,migration and invasion.Results:Bioinformatics technology was used to predict the let-7f-5p target gene and dishevelled segment polarity protein 3(DVL3)was selected as the target gene.After overexpression of let-7f-5p in HCC cells,qRT-PCR and Western blot(WB)resuls showed that let-7f-5p inhibited DVL3 gene expression at mRNA and protein levels.The dual-luciferase gene detection system results show that let-7f-5p inhibits the luciferase activity of the wild-type DVL3 3'-UTR reporter vector,but had no effects on the luciferase activity of the reporter vector containing 3'-UTR of DVL3 mutations.CCK8 assay showed that konckdown of DVL3 significantly reduced proliferation of HCC cells,and the co-transfection of let-7f-5p and target gene recombinant expression vector plasmids restored the proliferation of HCC cells.Transewell assay results showed that knockdown of DVL3 significantlydecreased migration and invasion,and the co-transfection of let-7f-5p and DVL3 recombinant expression vector plasmids restored migration and invasion of HCC cells.Conclusion: DVL3 is a direct target gene of let-7f-5p.Altering the expression of DVL3 can change the biological behaviors of cells.Part4 : Exploration of the biological markers of differentially expressed exosomal miRNAsObjective: To investigate the clinical application value of differentially expressed miRNAs in HCC cell exosomes in the diagnosis of HCC.Methods: The expression of differentially expressed miRNA of Hep 3B cell exosomes in serum exosomes of healthy controls(Health),hepatitis B patients(CHB),liver cirrhosis patients(LC)and hepatitis B positive HCC patients were analyzed by qRT-PCR.Results: The expression levels of miR-221-3p and miR-224-5p in exosomes of HCC group were significantly higher than those of Health group,CHB group and LC group(P<0.01).The expression level of serum exosomal miR-124-3p?let-7a-5p and let-7f-5p in HCC group was significantly lower than that of Health group,CHB group and LC group(P<0.05).Among four groups,there was no significant difference in the expression levels of miR-21-5p,miR-191-5p,miR-34a-5p,and miR-122-5p(P>0.05).Conclusion: Serum exosomal miR-221-3p,miR-224-5p,miR-124-3p,let-7a-5p and let-7f-5p may be candidate markers for hepatocellular carcinoma.