| Hepatocellular carcinoma(HCC)is one of the most common liver malignancies,in which hepatitis B virus(Hepatitis B Virus,HBV)and hepatitis C virus(Hepatitis C Virus,HCV)infection are the main causes of HCC.At present,HCC accounts for the fifth place among all new cancer cases in my country and the second place among all cancer fatal diseases,which seriously endangers human health.Early diagnosis of HCC,comprehensive and individualized treatment,and monitoring of recurrence and distant metastasis after treatment are important means to improve the diagnosis and cure rate of liver cancer.MicroRNA(miRNA)is a type of endogenous smallRNA with a length of about 20-24 nucleotides.It has a variety of important regulatory effects in cells.It is related to cell growth,metabolism,apoptosis,and tumorigenesis.Tumor cell invasiveness and tumor treatment are also closely related with miRNA.So far,many studies have confirmed the abnormal expression of miRNA in a variety of malignant tumors,such as breast cancer,brain cancer,colorectal cancer,HCC,lung cancer,lymphoma,prostate cancer,and thyroid cancer.In HCC,miRNA can be used as a diagnostic marker miRNA to assist in the diagnosis and treatment of HCC,and it also affects the occurrence and development of HCC through a variety of cell signaling pathways.As a self-protection mechanism of cells,autophagy is closely related to the process of tumorigenesis.Since autophagy is closely related to cell metabolism,protein and organelle turnover,cell survival,etc,the role of autophagy in tumors shows dynamic and diverse changes,with a high degree of complexity.How miRNAs regulate autophagy has not yet been clearly defined.The continuous discovery of new miRNAs regulating autophagy suggests that,as important molecules,they can target the expression of autophagy-related genes,affect the process of autophagy and regulate disease progression.Therefore,it is worthwhile to further explore the relationship between the regulation of autophagy by abnormally expressed miRNAs and disease progression.In this study,we screened miRNAs related to liver cancer autophagy through public databases and analyzed their clinical significance.In HCC cell lines,autophagy was induced by hypoxia.Cells in each group were collected for miRNA and mRNA sequencing,and autophagy-related miRNA and its possible mechanism in different HCC cells were analyzed according to transcriptome sequencing data.Key miRNAs and their target genes involved in autophagy of liver cancer were preliminarily identified.After stable overexpression/inhibition of miRNA,autophagy activity was detected,autophagy phenomenon was observed,and gene changes in the downstream signaling pathway of miRNA were detected,so as to provide theoretical and experimental basis for the diagnosis,treatment and follow-up of miRNA in HCC.Part One: The value of miRNA as biomarker for diagnosis of liver cancer was studied by machine learning method based on TCGA database.Objective: Potential biomarkers for HCC diagnosis were screened to preliminarily identify miRNAs with diagnostic potential.Methods: According to The Cancer Genome Atlas(TCGA)atabase,The differentially expressed miRNAs with The most diagnostic value were preliminarily identified by random forest algorithm.A taxonomic model was established to distinguish between patients with HCC and normal individuals,and then a regulatory network was constructed between differentially expressed miRNAs and mRNAs.The GSE63046 dataset was used to verify the expression of the identified differentially expressed miRNAs,and the diagnosis and prognosis of differentially expressed miRNAs were analyzed.Results:1.A total of 14 differentially expressed miRNAs(all upregulated)and 2,982 differentially expressed mRNAs(1,989 upregulated and 993down-regulated)were found,among which hsa-miR-10b-5p,hsa-miR-10b-3p,hsa-miR-224-5p,hsa-miR-183-5p and hsa-miR-182-5p were considered as possible biomarkers for the diagnosis of hepatocellular carcinoma;2.The five miRNA-targeted mRNAs include SFRP1,EDNRB,NR4A3,FHL2,NKX3-1,IL6 ST,and Fox O1.The "Bile acid biosynthesis and cholesterol metabolism" pathway is the most abundant signaling pathway for these target mRNAs;3.The validation results of the five miRNAs initially identified were consistent with the validation results of the GSE63046 dataset;4.Hsa-miR-10b-5p and hsa-miR-10b-3p may have important prognostic value in patients with hepatocellular carcinoma.Summary:1.Five differentially expressed miRNAs can be regarded as diagnostic biomarkers in patients with HCC.2.Five differential expression levels of miRNA-targeted mRNAs(including SFRP1,EDNRB,NR4A3,FHL2,NKX3-1,IL6 ST and Fox O1)may be associated with the occurrence of hepatocellular carcinoma.Part Two: In Vitro Validation of MiRNA as a Biomarker for Hepatocarcinoma DiagnosisObjective: Clinical and in vitro validation of differential expression of miRNA in HCC.Methods: Hepatocellular carcinoma tissues and adjacent 2cm tissues of 7cases of hepatocellular carcinoma were collected,and the levels of differentially expressed miRNAs(hsa-miR-10b-5p,hsa-miR-10b-3p,hsa-miR-224-5p,hsa-miR-183-5p,and hsa-miR-182-5p)in HCC were quantitatively analyzed by RT-q PCR.Results: The expression levels of 5 differentially expressed miRNAs in 7HCC patients were all upregulated,and hsa-miR-224-5p was the most significantly upregulated,which was consistent with the results of bioinformatics analysis in Part I.Summary:1.Five differentially expressed miRNAs can be regarded as diagnostic biomarkers in patients with HCC.2.The miRNA levels in liver tissue samples were consistent with the results from the public dataset.Part Three: Study on autophagy-related mechanisms of liver cancer based on transcriptome sequencingObjective: To explore new molecules related to autophagy in HCC,and further clarify the mechanism of their role in the regulation of autophagy affecting the progression of HCC.Methods: In this part,the hypoxia-induced autophagy model was first established,and the number of autophagy vesicles was observed by electron microscopy.Western Blot and Transwell assay were used to analyze the expression levels of autophagy-related proteins and cell invasion.Transcriptome sequencing was used to analyze the differentially expressed mRNA and miRNA in HCC cells between the control group and the hypoxia group.Establishment of miRNA-mRNA interaction network and functional enrichment analysis of miRNA-targeted genes to explore the potential mechanism of hypoxia-induced autophagy in HCC.Results: 1.We found that hypoxia may promote the invasion of HCC cells by inducing autophagy;2.Compared with the normal control group,407 shared mRNAs and 57 shared miRNAs were identified in the HCCLM3-induced autophagy group and SMMC-7721 autophagy group.Summary:1.Nineteen highly expressed miRNAs were obtained based on the miRNA-mRNA interaction network.2.Hsa-miR-483-5p,hsa-miR-4739,has-miR-214-3p and has-miR-296-5p may be potential molecular markers associated with autophagy in HCC.Conclusions:1.Hsa-miR-10b-5p,hsa-miR-10b-3p,hsa-miR-224-5p,hsa-miR-183-5p and hsa-miR-182-5p may be diagnostic markers for hepatocellular carcinoma.2.Clinical tissue samples confirmed that these miRNAs may be clinical markers for the diagnosis of HCC.3.At the cellular level,hsa-miR-483-5p,hsa-miR-4739,has-miR-214-3p and has-miR-296-5p may be important molecules related to autophagy in liver cancer.4.Screening of hepatocellular carcinoma and autophagy-related miRNA of hepatocellular carcinoma can help to understand the potential molecular mechanism of autophagy of hepatocellular carcinoma,and provide new therapeutic targets for clinical diagnosis and prognosis monitoring. |
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