| BackgroundHepatocellular carcinoma(HCC),as one of the most common cancers in the world,has significantly increased the global burden of cancer.Although the incidence and mortality of HCC have declined in China and Japan,they have been on the rise in recent years in North America and some European regions,and HCC remains a global health challenge.Viruses,including Hepatitis B virus(HBV)and Hepatitis C virus(HCV)infection,alcoholism and metabolic syndrome are typical risk factors.Despite great advances in both diagnosis and treatment,the inability to detect HCC early and prevent tumor recurrence remains a great challenge,and most patients with HCC eventually die from multiple organ metastases.Therefore,it is critical to address these issues.LIM domain and actin binding 1(LIMA1)is an actin-binding protein,also known as Epithelial protein lost in neoplasm(EPLIN),has been widely studied,and its role has also expanded from the initial regulation of cytoskeleton dynamics to gene regulation,lipid metabolism,cell cycle,and angiogenesis/lymphangiogenesis related to cancer progression.LIMA1 can regulate the life cycle by interacting with proteins related to the regulatory cytoskeleton,and participate in the control of the malignant biological behavior and progression of specific solid tumors.All cells in the tumor microenvironment(TME)participate in a complex regulatory network together.Carcinoma-associated fibroblasts(CAFs),as the main stromal cells that affect tumor progression,have been confirmed that various factors play a role in tumor proliferation,angiogenesis,metastasis and chemotherapy resistance by releasing various factors into the TME.Meanwhile,the new regulatory mechanism of exosomes secreted by CAFs in regulating tumor progression has received increasing attention.Exosomes are a kind of extracellular vesicles with a diameter of 30-150 nm and a lipid bilayer structure,which carry a large amount of important information,including lipids,nucleic acids,and proteins.Moreover,exosomal miRNA can participate in the regulation of tumor microenvironment and become relevant participants in intercellular communication.Therefore,we first proposed a scientific hypothesis of "upstream exosomal miRNA→LIMA1→downstream unknown protein(signaling pathway)→changes in biological behavior of HCC" and speculated that exosomal miRNA was derived from CAFs in HCC tissues.By combining GEO and miRNA related databases,we identified miR-20a-5p that targets LIMA1 expression.Therefore,we designe this study to explore the role of LIMA1 in the occurrence and development of HCC and its downstream pathways,and to verify the relationship between miR-20a-5p,CAFs and exosomes and their effects on HCC progression.AimsTo investigate the role and molecular mechanism of LIMA1 in the proliferation,invasion and metastasis of HCC,as well as the role of miR-20a-5p targeting LIMA1 expression in the process of HCC proliferation,invasion and metastasis,and to verify the source of miR-20a-5p in HCC and its relationship with CAFs and exosomes.Methods1.Quantitative real-time PCR(qRT-PCR),western blot and immunohistochemistry(IHC)were used to detect the expression of LIMA1 in HCC tumor tissues and paired adjacent normal tissues.The expression of LIMA1 in human liver cancer cell lines(SMMC7721,Huh7,YY8103,Hep3 B,Focus,Hep G2 and HCCLM3)and normal liver cell lines(MIHA,WRL68)was detected,and the relationship between the expression of LIMA1 and clinicopathological features and prognosis of patients was also determined.2.The overexpression plasmid was transfected into YY8103 and HCCLM3 cell lines with relatively low LIMA1 level,which were divided into overexpression group(OE group)and negative control group(NC group),and the targeted sh RNA was used to effectively knock down LIMA1 level in Huh7 cell lines with relatively high LIMA1 level,and they were divided into knockdown group(sh-LIMA1 group)and negative control group(sh-NC group).CCK-8 assay,plate cloning assay,Ed U assay and transwell assay were used to detect the proliferation,invasion and metastasis of HCC cells in each group.Immunofluorescence and western blot were used to detect the expression levels of epithelial-mesenchymal transformation(EMT)related marker proteins in each group of HCC cells.3.YY8103 cells transfected with LIMA1 overexpression plasmid and negative control were injected into the flanks and tail veins of nude mice,the relationship between LIMA1 and HCC cell proliferation and metastasis was further verified by measuring tumor size and volume,HE staining and immunohistochemistry(IHC).4.LIMA1-interacting proteins were identified by online database,co-immunoprecipitation and immunofluorescence,and the signaling pathways of LIMA1 function in HCC cells were explored.5.Combined with miRNA related databases and Target Scan databases,qRT PCR,double Luciferase experiment and function recovery experiment,verify the miR-20a-5p expression targeting LIMA1,and wound healing assay,colony formation assay,Ed U assay,transwell assay,immunofluorescence and western blot were used to detect the proliferation,invasion and metastasis ability of miR-20a-5p in HCC cells and the expression level of EMT-related marker proteins.6.The expression of miR-20a-5p in normal liver cell lines,liver cancer cell lines,carcinoma-associated fibroblasts(CAFs)and normal fibroblasts(NFs)were detected by qRT-PCR;exosomes labeled with cell membrane staining kit PKH-67 and CAFs transfected with miR-20a-5p mimic labeled with CY3 fluorescent dye,and then co-cultured with Huh7 cells,and observed whether special marker signals were detected in the cultured cells through fluorescence confocal microscopy to verify whether miR-20a-5p is transferred from CAFs to hepatocellular carcinoma cells by exosomes.And Ed U assay,wound healing assay and transwell assay were used to detect the effect of CAFs-derived exosomes carrying miR-20a-5p on the proliferation,invasion and metastasis of hepatocellular carcinoma cells.Nude mice inoculated with Huh7 cells were treated with phosphate buffer salt solution(PBS)and CAFs-derived exosomes with or without miR-20a-5p transfection.The effect of CAFs-derived exosomal miR-20a-5p on the proliferation of HCC cells was detected by measuring tumor size and volume.Results1.The expression of LIMA1 is low in HCC tissues and HCC cell lines,and HCC patients with high expression of LIMA1 have better overall survival(OS)and recurrence-free survival(RFS).2.LIMA1 overexpression significantly inhibited the proliferation,invasion and metastasis of HCC cells,while LIMA1 knockdown enhanced the proliferation,invasion and metastasis of HCC cells;In addition,the expression of EMT marker Vimentin was decreased in the LIMA1 overexpression group but increased in the LIMA1 knockdown group;however,the expression level of E-cadherin,another EMT marker,showed an opposite change.3.Tumor growth in mice injected with LIMA1 overexpressing cells was significantly lower than that in mice injected with negative control cells.HE staining and IHC results showed that increased LIMA1 levels in vivo clearly abolished Ki67 and Vimentin expression but promoted E-cadherin expression.In addition,the number of metastatic nodules in the lungs of mice inoculated with LIMA1 overexpressing cells was significantly less than that of negative control group.4.It was confirmed that there was an endogenous interaction between LIMA1 and BMI1 in HCC cells through searching online bioinformatics databases such as Bio Grid,Genemania and Hitredict,as well as co-immunoprecipitation(Co-IP)and immunofluorescence,and LIMA1 regulates BMI1 degradation through ubiquitin-proteasome system(UPS).Overexpression of LIMA1 increased the cytosolic localization of β-catenin.However,LIMA1 knockdown cells retained more β-catenin in the nucleus,and CCND1,CD44,JUN and TCF1,the downstream molecules ofβ-catenin pathway,were significantly reduced in LIMA1 overexpression cells but increased in LIMA1 knockdown cells.Moreover,in LIMA1-knockdown HCC cells,BMI1 knockdown reduced nuclear β-catenin localization and partially reversed the inhibitory effects of LIMA1 on HCC cell proliferation,migration,invasion and EMT.5.The expression of miR-20a-5p was significantly increased in HCC tissues,and was negatively correlated with the expression level of LIMA1.Moreover,HCC patients with high miR-20a-5p expression had worse OS and RFS.Luciferase reporter assay showed that miR-20a-5p mimic transfection significantly reduced the luciferase activity of the LIMA1 wild type group,but not the LIMA1 mutant group.Functional studies showed that miR-20a-5p knockdown inhibited the proliferation,invasion,metastasis and EMT process of HCC cells.Knockdown of miR-20a-5p increased the expression level of LIMA1 and the retention of cytoplasmic β-catenin.In addition,the expression level of β-catenin downstream molecules decreased with the reduction of miR-20a-5p.Functional recovery experiments showed that the oncogenic effect of miR-20a-5p was partially mediated by LIMA1.6.The level of miR-20a-5p in CAFs was significantly higher than that in normal liver cell lines,liver cancer cell lines and NFs.Huh7 cells were co-cultured with NFs and CAFs with or without GW4869 pretreatment.The results showed that the level of miR-20a-5p in Huh7 cells co-cultured with CAFs without GW4869 pretreatment was significantly increased,and the proliferation of Huh7 cells was also significantly increased.Exosomes derived from CAFs and NFs were labeled with PKH-67 and co-cultured with Huh7 cells.Fluorescence showed that PKH-67-labeled exosomes were taken up by Huh7 cells and increased the level of miR-20a-5p in Huh7 cells.The CY3-labeled miR-20a-5p mimic was transfected into CAFs and co-cultured with Huh7 cells,and CY3 fluorescence was detected in the co-cultured Huh7 cells.In addition,the expression level of LIMA1 in Huh7 cells treated with exosomes obtained from CAFs transfected with miR-20a-5p mimic was significantly decreased,while the expression level of LIMA1 in Huh7 cells treated with exosomes obtained from CAFs transfected with miR-20a-5p inhibitor was significantly increased.At the same time,CAFs-derived exosomes carrying miR-20a-5p mimics could promote the proliferation,migration and invasion of Huh7 cells,while CAFs-derived exosomes carrying miR-20a-5p inhibitors had the opposite effect.In vivo experiments showed that treatment of CAFs-derived exosomes without miR-20a-5p mimic significantly promoted tumor growth compared with mice treated with PBS solution,while a stronger tumor growth effect was observed in the group of mice treated with CAFs-derived exosomes transfected with miR-20a-5p mimic.ConclusionLIMA1 is a new tumor suppressor of HCC,which is negatively regulated by exosomal miR-20a-5p released by CAFs,and hinders the proliferation,migration and invasion of HCC by regulating BMI1 degradation and inhibiting the activation of β-catenin signaling pathway.It is suggested that the therapeutic methods targeting LIMA1 or CAFs,exosomes and circulating miR-20a-5p may be helpful for the diagnosis and treatment of HCC. |
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