| ObjectivePrimary liver cancer(PLC)is one of the most common malignant tumors in the world,including three different pathological types: hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICCA),and combined hepatocellular and cholangiocarcinoma(cHCC-CC).Due to the early non-specific symptoms of PLC,most of the diagnosis is in the advanced stage,and the disease progresses rapidly,so the treatment is difficult and the prognosis is poor.At present,the treatment methods of PLC include surgical treatment,radiotherapy,chemical medicine treatment,targeted therapy,etc.,but most PLCs still have poor prognosis.Therefore,it is an urgent problem to be solved in the field of PLC research to explore the mechanism of PLC development and explore effective early diagnostic markers and molecular therapeutic targets.cHCC-CC is a rare primary liver cancer,which means that HCC and ICCA exist in the same tumor body for mixed growth.cHCC-CC has the differentiation characteristics of HCC and ICCA double phenotype,and is similar to HCC or ICCA in clinical symptoms.From this we hypothesized that the presence of mixed liver cancer indicates that there may be common mechanisms of occurrence or regulation in HCC and ICCA,and the effects on this mechanism or pathway may include HCC,ICCA,and cHCC-CC,thereby contributing to treatment of PLC.Because cHCC-CC is very rare in the clinic,it is reported to account for only 0.4% to 14.2% of primary liver cancer.Therefore,we choose HCC and ICCA as the research objects,and combine the two into the same research,respectively conduct bioinformatics research and comprehensive analysis to explore the possible common molecular targets and regulatory pathways between the two,thus providing new treatment strategies for comprehensive treatment for PLC.In this study,two different pathological types of PLC,HCC and ICCA,were selected as research objects.The differentially expressed mRNA and miRNA were obtained by screening and analyzing the database of GEO database,and the miRNA-transcription factor co-regulation network was constructed,and the network was combined with TCGA database.Functional and enrichment analysis of key genes in the study revealed that key genes in the co-regulatory network are mainly enriched in signaling pathways involved in cell proliferation and apoptosis,and there may be a common miR-181a-5p→EGR1/FOS in HCC and ICCA.Subsequently,we performed real-time PCR and immunohistochemical verification of HCC and ICCA tissues,confirming the differential expression of miR-181a-5p and EGR1 and FOS between cancer tissues and adjacent tissues.Finally,we performed cytological validation on the HCC cell line SMMC-7721 and the ICCA cell line RBE,and explored that each molecule in the miR-181a-5p→EGR1/FOS regulatory pathway may play a role in the proliferation and apoptosis of PLC.The role of further improving the activity of PLC cells by targeting key molecules and providing new ideas for the treatment of PLC.Method(1)Combining the GEO database and the TCGA database for bioinformatics analysis of HCC and ICCA,and constructing a joint regulation network of miRNA-transcription factors of primary liver cancer through joint analysis of multiple databases such as TTRUST Version 2,TRED and miRTarbase,and regulating key genes in the network were analyzed for GO and KEGG enrichment and functional analysis to explore the relationship between differentially expressed mRNA and miRNA and PLC progression.(2)Quantitative RT-PCR and immunohistochemical techniques were used to verify the differential expression of miR-181a-5p,EGR1 and FOS in PLC tissues,and to detect the expression levels of their mRNAs and proteins.(3)To construct a model of PLC with over-expression and low-expression of miR-181a-5p,and to verify the expression of mi-181a-5p and the expression of downstream target genes by qRT-PCR and Western Blot.(4)To explore the role of miR-181a-5p in proliferation and anti-apoptosis of PLC including HCC and ICCA by MTS,clone formation,flow cytometry.(5)To explore the role of miR-181a-5p in HCC and ICCA apoptosis was investigated by MTS assay,flow cytometry and Western blot.Result(1)By screening and analyzing expression profiles in GEO and TCGA databases and integrating the information of transcription factors and target genes of miRNA predicted based on TRED,TTRUST and miRTarbase databases,miRNA-TF co-regulatory network of in HCC and ICCA were constructed,and the key genes in the regulatory network were analyzed and annotated.The enrichment results showed that the key genes in the two regulatory networks were all enriched in p53 and cell cycle signaling pathways,suggesting that these genes were related to the proliferation and apoptosis of tumors.(2)Through comprehensive bioinformatics analysis and verification,it was found that there is a common miR-181a-5p→EGR1/FOS regulatory pathway in HCC and ICCA.The results of qRT-PCR showed that the expression levels of miR-181a-5p in HCC and ICCA tissues were both significantly higher than those in adjacent tissues.The expression levels of EGR1 and FOS mRNA were significantly lower than those in adjacent tissues.The results of immunohistochemistry showed that the expression of EGR1 and FOS proteins in HCC and ICCA tissues was significantly lower than those in adjacent tissues.(3)The miR-181a-5p overexpression and low expression of HCC and ICCA cell models were constructed by lipofection,and confirmed by qRT-PCR and Western blot.The expression level of EGR1 and FOS in miR-181a-5p overexpressing cell model significantly decreased,and the expression level of EGR1 and FOS was significantly increased in the miR-181a-5p low expression cell model.(4)Through MTS and clone formation experiments,it was found that the proliferation rate and colony formation rate of miR-181a-5p overexpressing cells were significantly higher than that of the control group,while proliferation rate and colony formation rate of the miR-181a-5p low expression cell were significantly lower that of the control group.(5)By flow cytometry,the percentage of cells in the S phase of miR-181a-5p overexpressing cells was significantly increased compared with the control cells(P<0.05),and the percentage of cells in G0/G1 phase was significantly decreased(P<0.05).As for miR-181a-5p low-expressing cells,the percentage of cells in S phase was significantly decreased(P<0.05),and the percentage of cells in G0/G1 phase was significantly increased(P<0.05).(6)By MTS assay,we found that after induction of apoptosis by cisplatin,the survival rate of miR-181a-5p overexpressing cells was significantly higher than that of the control group,while the survival rate of miR-181a-5p low expressing cells was significantly lower than that of the control group.(7)Flow cytometry revealed that the apoptosis rate of SMMC-7721 cells and RBE cells was not significantly different after miR-181a-5p overexpression(P>0.05),while miR-181 a was down-regulated.After down-regulated expression of miR-181a-5p,the apoptotic rate of SMMC-7721 cells and RBE cells was significantly higher than that of the control group(P<0.05).After apoptosis induction,the apoptosis rate of SMMC-7721 cells and RBE cells overexpressing miR-181a-5p was significantly lower than that of the control group(P<0.05),while SMMC-7721 cells expressing miR-181a-5p were down-regulated.The apoptotic rate of RBE cells was significantly higher than that of the control group(P<0.05).(8)Western blot results showed that overexpression of miR-181a-5p in HCC cell line SMMC-7721 significantly decreased the expression levels of cleaved Caspase 3 and Bax proteins,and increased the expression levels of Bcl-2 and Bcl-xL proteins;while silencing miR-181a-5p,the results were reversed.In the ICCA cell line RBE cells,overexpression of miR-181a-5p significantly reduced the expression levels of cleaved Caspase 3 and Bax proteins,and increased the expression of Bcl-2 protein.The result of silencing miR-181a-5p was reversed.Conclusion(1)In this study,we performed differential expression analysis of HCC and ICCA expression profiles in GEO database,and constructed miRNA-transcription factors coregulatory networks of in HCC and ICCA tissues based on the bioinformatics analysis results,respectively.And we found the key genes in the co-regulatory network of HCC and ICCA are both enriched in the pathways related to tumor proliferation and apoptosis,such as "p53 signaling pathway" and "cell cycle pathway" based on GO function annotation and KEGG enrichment analysis.(2)Through the comprehensive bioinformatics analysis of HCC and ICCA,we screened and identified the possible common miR-181a-5p→EGR1/FOS regulatory pathway in HCC and ICCA,and confirmed that the expression levels of EGR1 and FOS in HCC and ICCA tissues were significantly lower than that in adjacent tissues,while the expression levels of miR-181a-5p in HCC and ICCA tissues were significantly higher than that in adjacent tissues.(3)We verified indirectly the regulatory relationship between miR-181a-5p and EGR1 and FOS through vitro experiments,and confirmed that miR-181a-5p might down-regulate the expression of EGR1 and FOS,thereby promoting the proliferation of PLC cells by promoting DNA synthesis and cell cycle differentiation from G1 phase to S phase.(4)We confirmed that miR-181a-5p might down-regulate the expression of EGR1 and FOS,thereby activating Bcl-2-mediated anti-apoptotic pathway and inhibiting apoptosis of PLC cells.In summary,we performed a three-part work to construct a miR-TF co-regulatory network of PLC,and exprlore the role of hsa-miR-181a-5p→EGR1/FOS in promoting PLC proliferation and anti-apoptosis from the tissue,cell and molecular levels,which provided new clues for comprehensive treatment of PLC and the discovery of markers. |
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