| Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors of the digestive system.There is extensive information and substance exchange between liver cancer cells and tumor microenvironment in the process of occurrence and development.In recent years,studies have shown that tumor cells can release a large number of 30-150nm diameter microvesicles,called exosomes,into the microenvironment during development and progression.Exosomes have a double-layer membrane structure and can stably carry proteins,RNA and other substances to participate in the transmission of information between tumor cells.Studies have confirmed that exosomes secreted by tumor cells can extensively participate in the transformation of microenvironment before tumor cell metastasis,the process of drug resistance in tumor cells and the reaction mediating the metabolic coupling between tumor cells and stromal cells.Therefore,the study of exosomes in tumor cells has attracted the attention of many scholars.However,the process of tumor cell exosome release has not been fully revealed at present.This study focused on the molecular mechanism of the exosome release in HCC.Long noncoding RNAs(lncRNAs)are a class of noncoding RNAs with a length of more than 200 nucleotides,which usually do not have the function of coding proteins.The regulatory role of LncRNA in tumor formation has been paid more and more attention,but the regulatory mechanism of LncRNA in exosome release process in liver cancer cells remains to be explored.In recent years,it has been found that HOX transcriptional antisense RNA(HOTAIR),as an oncogenic gene,can be involved in the occurrence and development of a variety of tumors.Our previous studies confirmed that HOTAIR could affect the occurrence of autophagy in liver cancer cells by promoting the expression of ATG3 and ATG7 genes.The process of autophagy flow in HCC cells is closely related to the production and final destination of intracellular exosomes.Therefore,this study focus on whether HOTAIR can affect the release of exosomes in liver cancer cells and the specific regulatory molecular mechanism,from which we use gene enrichment analysis,nanoparticle tracking analysis technology,transmission electron microscope,fluorescence co-localization analysis and other relevant research methods of exosome to revealed the molecular mechanism of HOTAIR in regulating the release of exosomes in liver cancer cells,and provided a theoretical basis for the occurrence and development of liver cancerMaterials and Methods:1.RNA sequencing data of 374 liver cancer samples were extracted from the TCGA database,and the samples were divided into high and low expression groups according to the median expression value of HOTAIR.Then,31 gene sets involved in exosome release were selected for gene enrichment analysis(GSEA).The expressions of core regulatory genes such as Rab35,SNAP23 and VAMP3 were compared and analyzed.2.HepG2 liver cancer cell lines with high expression of IncRNA and HOTAIR were constructed by liposome transfection.Meanwhile,real-time PCR was used to detect the transfection efficiency3.Exosome was extracted using exosome extraction kit,and the effects of HOTAIR on exosome release in liver cancer cells were confirmed by western blot detection of exosome markers CD63 and Tsg101,nanoparticle tracking analysis technology and transmission electron microscope4.Fluorescence confocal microscopy was used to determine the effect of HOTAIR on the distribution of CD63,a surface marker of the polycystic vesicle,and SNAP23,a cell membrane releasing molecule,in liver cancer cells.The influence of overexpression of HOTAIR on distribution of multivesicular bodies(MVBs)was investigated by transmission electron microscope5.Real-time PCR was used to screen and detect Rab5,Rab7,Rab11,Rab27a,Rab27b and Rab35,which may be regulated by HOTAIR;fluorescence confocal microscopy was used to determine the localization of CD63 and Rab35,which are the surface markers of polycystic bodies in hepatocellular carcinoma cells;co-transfection assay was used to verify that HOTAIR regulates the release of extracellular secretions from hepatocellular carcinoma cells through Rab356.The co-localization of t-SNAP23 and v-VAMP3 in the SNARE complex of exosome releasing protein regulated by HOTAIR in liver cancer cells was determined by fluorescence confocal microscopy7.The effect of overexpression of HOTAIR on the phosphorylation level of SNAP23 protein was detected by phos-tag SDS-PAGE gel electrophoresis8.Western blot was used to detect the effect of overexpression of HOTAIR on the mTOR pathway,and rapamycin was used as an inhibitor to verify the regulation of HOTAIR on SNAP23 through the mTOR pathway9.Statistical analysis:Each experiment was repeated for 3 times,all values were expressed as means± SEM,and the software SPSS17.0 was used for Student's t-test*P<0.05 and**P<0.01 was considered to be statistically significantResults:Experiment 1:GSEA analysis showed that the concentration of exosome releasing genes in the HOTAIR group with relatively high expression was positive compared with the low expression group,and the NES value was 1.548,p value was 0.028.In addition,the expressions of Rab35,SNAP23 and VAMP3 in liver cancer tissues were significantly higher than those in normal tissues(P<0.05).HOTAIR highly expressed HepG2 cell line was constructed with a transfection efficiency of 34 times.Exosome morphology was observed by transmission electron microscope.The nanoparticle tracking analysis technology verified that the extraction vesicle diameter was between 50-100nm.Western blot was used to detect the expressions of markers CD63 and Tsg101 in the extracted exosome protein.The secretion of exosomes by hepatocellular carcinoma cells in the overexpressed HOTAIR group increasedExperiment 2:Overexpression of HOTAIR could significantly promote the proliferation of CD63,the MVB surface marker of polycystis.Moreover,overexpression of HOTAIR can promote the co-localization of CD63 and SNAP23 protein that regulates exosome release on the cell membrane surface.Through transmission electron microscopy,HOTAIR can promote the diffusion of MVB vesicles in cytoplasm.Real-time PCR and western blot results showed that HOTAIR could significantly up-regulate Rab35 protein expression.Moreover,the fluorescence co-localization experiment showed that HOTAIR could promote the binding of CD63 and Rab35.Co-transfection experiments showed that HOTAIR promotes exosome release through Rab3 5Experiment 3:Single fluorescence experiment showed that HOTAIR could promote SNAP23 molecule distribution to cell membrane;Fluorescence co-localization experiments showed that HOTAIR could significantly promote the co-localization of SNAP23 and VAMP3 in the SNARE protein complex of exosomes regulated by cell membrane.The Phos-tag SDS-PAGE phosphorylated protein gel electrophoresis experiment showed that HOTAIR could significantly activate SNAP23 phosphorylation level.Western blot analysis showed that HOTAIR could promote mTOR phosphorylation.Rapamycin rescue experiments showed that HOTAIR activated SNAP23 phosphorylation levels through the mTOR pathway;The nanoparticle tracking analysis technique verified the regulatory effect of HOTAIR exosome release through the mTOR pathwayConclusion:1.There was a functional correlation between HOTAIR expression and exosome release in TCGA liver cancer clinical tissue samples.In addition,the expressions of Rab35,SNAP23 and VAMP3 were increased in liver cancer tissues.HOTAIR can promote the release of exosomes in liver cancer cells.HOTAIR promotes the transport of MVB to the cell membrane2.HOTAIR regulates the release of exosomes in liver cancer cells by up-regulating the expression of Rab35 protein,thereby achieving the docking process between MVB and cell membrane3.HOTAIR promoted the membrane surface regulation of exosome releasing molecule SNAP23 and co-localization with VAMP3,thus promoting the formation of SNARE protein complex;In addition,HOTAIR can activate SNAP23's phosphorylation level through mTOR pathway,thereby promoting SNAP23 to play a role in promoting exosome release. |
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