{"title":"血液外泌体标志物miRNA-30d-5p：在肝细胞癌细胞干性和吉西他滨耐药性中的作用和调节机制。","authors":"Biao Tang, Longhui Xie, Xin Tang, Junjie Tian, Shaofei Xiao","doi":"10.1016/j.mcp.2023.101924","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Cancer stem cells (CSCs) are different from regular cancer cells because of their self-renewal feature and differentiation potential, which establishes the backbone of the vital role of CSCs in the progress and drug resistance of hepatocellular carcinoma (HCC). The objective of this study was to evaluate the effects of blood exosome-derived miRNA-30d-5p on the stemness and gemcitabine resistance of HCC cells and the underlying mechanisms.</p></div><div><h3>Methods</h3><p>The expression data of HCC-related miRNAs and mRNAs were downloaded from TCGA database and analyzed for differences. Employing the databases of starBase, TargetScan, miRDB, and mirDIP, we conducted target gene prediction upstream of mRNA. The expression of miRNA-30d-5p and SOCS3 mRNA was assayed by qRT-PCR, and the binding between them was validated by dual luciferase assay. CCK-8 was employed to evaluate cell viability and the IC<sub>50</sub> value of gemcitabine. Cells were subjected to a sphere-forming assay to assess their ability to form spheres. Western blot was applied to evaluate the levels of cell surface marker proteins (Nanog, CD133, and Oct4) and exosome markers (CD9, CD81, and FLOT1).</p></div><div><h3>Results</h3><p>Bioinformatics analysis found that SOCS3 expression was down-regulated in HCC. qRT-PCR showed that SOCS3 expression was notably lower in HCC cell lines than in normal liver cell WRL68. At the cellular functional level, SOCS3 overexpression inhibited the viability, sphere-forming ability, stemness, and gemcitabine resistance of HCC cells. Bioinformatics analysis demonstrated that miRNA-30d-5p was the upstream regulator of SOCS3 and highly expressed in HCC tissues and cells. Dual luciferase assay demonstrated that miRNA-30d-5p could bind SOCS3. Rescue experiments showed that upregulating SOCS3 could reverse the effects of miRNA-30d-5p overexpression on the viability, sphere-forming ability, and gemcitabine sensitivity of HCC cells.</p></div><div><h3>Conclusions</h3><p>Blood exosome-derived miRNA-30d-5p promoted the stemness and gemcitabine resistance of HCC cells by repressing SOCS3 expression. Hence, the miRNA-30d-5p/SOCS3 axis might be a therapeutic target for chemotherapy resistance and a feasible marker for the prognosis of HCC patients.</p></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"71 ","pages":"Article 101924"},"PeriodicalIF":2.3000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Blood exosome marker miRNA-30d-5p: Role and regulation mechanism in cell stemness and gemcitabine resistance of hepatocellular carcinoma\",\"authors\":\"Biao Tang, Longhui Xie, Xin Tang, Junjie Tian, Shaofei Xiao\",\"doi\":\"10.1016/j.mcp.2023.101924\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Cancer stem cells (CSCs) are different from regular cancer cells because of their self-renewal feature and differentiation potential, which establishes the backbone of the vital role of CSCs in the progress and drug resistance of hepatocellular carcinoma (HCC). The objective of this study was to evaluate the effects of blood exosome-derived miRNA-30d-5p on the stemness and gemcitabine resistance of HCC cells and the underlying mechanisms.</p></div><div><h3>Methods</h3><p>The expression data of HCC-related miRNAs and mRNAs were downloaded from TCGA database and analyzed for differences. Employing the databases of starBase, TargetScan, miRDB, and mirDIP, we conducted target gene prediction upstream of mRNA. The expression of miRNA-30d-5p and SOCS3 mRNA was assayed by qRT-PCR, and the binding between them was validated by dual luciferase assay. CCK-8 was employed to evaluate cell viability and the IC<sub>50</sub> value of gemcitabine. Cells were subjected to a sphere-forming assay to assess their ability to form spheres. Western blot was applied to evaluate the levels of cell surface marker proteins (Nanog, CD133, and Oct4) and exosome markers (CD9, CD81, and FLOT1).</p></div><div><h3>Results</h3><p>Bioinformatics analysis found that SOCS3 expression was down-regulated in HCC. qRT-PCR showed that SOCS3 expression was notably lower in HCC cell lines than in normal liver cell WRL68. At the cellular functional level, SOCS3 overexpression inhibited the viability, sphere-forming ability, stemness, and gemcitabine resistance of HCC cells. Bioinformatics analysis demonstrated that miRNA-30d-5p was the upstream regulator of SOCS3 and highly expressed in HCC tissues and cells. Dual luciferase assay demonstrated that miRNA-30d-5p could bind SOCS3. Rescue experiments showed that upregulating SOCS3 could reverse the effects of miRNA-30d-5p overexpression on the viability, sphere-forming ability, and gemcitabine sensitivity of HCC cells.</p></div><div><h3>Conclusions</h3><p>Blood exosome-derived miRNA-30d-5p promoted the stemness and gemcitabine resistance of HCC cells by repressing SOCS3 expression. Hence, the miRNA-30d-5p/SOCS3 axis might be a therapeutic target for chemotherapy resistance and a feasible marker for the prognosis of HCC patients.</p></div>\",\"PeriodicalId\":49799,\"journal\":{\"name\":\"Molecular and Cellular Probes\",\"volume\":\"71 \",\"pages\":\"Article 101924\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2023-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular and Cellular Probes\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0890850823000336\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and Cellular Probes","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0890850823000336","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Blood exosome marker miRNA-30d-5p: Role and regulation mechanism in cell stemness and gemcitabine resistance of hepatocellular carcinoma
Background
Cancer stem cells (CSCs) are different from regular cancer cells because of their self-renewal feature and differentiation potential, which establishes the backbone of the vital role of CSCs in the progress and drug resistance of hepatocellular carcinoma (HCC). The objective of this study was to evaluate the effects of blood exosome-derived miRNA-30d-5p on the stemness and gemcitabine resistance of HCC cells and the underlying mechanisms.
Methods
The expression data of HCC-related miRNAs and mRNAs were downloaded from TCGA database and analyzed for differences. Employing the databases of starBase, TargetScan, miRDB, and mirDIP, we conducted target gene prediction upstream of mRNA. The expression of miRNA-30d-5p and SOCS3 mRNA was assayed by qRT-PCR, and the binding between them was validated by dual luciferase assay. CCK-8 was employed to evaluate cell viability and the IC50 value of gemcitabine. Cells were subjected to a sphere-forming assay to assess their ability to form spheres. Western blot was applied to evaluate the levels of cell surface marker proteins (Nanog, CD133, and Oct4) and exosome markers (CD9, CD81, and FLOT1).
Results
Bioinformatics analysis found that SOCS3 expression was down-regulated in HCC. qRT-PCR showed that SOCS3 expression was notably lower in HCC cell lines than in normal liver cell WRL68. At the cellular functional level, SOCS3 overexpression inhibited the viability, sphere-forming ability, stemness, and gemcitabine resistance of HCC cells. Bioinformatics analysis demonstrated that miRNA-30d-5p was the upstream regulator of SOCS3 and highly expressed in HCC tissues and cells. Dual luciferase assay demonstrated that miRNA-30d-5p could bind SOCS3. Rescue experiments showed that upregulating SOCS3 could reverse the effects of miRNA-30d-5p overexpression on the viability, sphere-forming ability, and gemcitabine sensitivity of HCC cells.
Conclusions
Blood exosome-derived miRNA-30d-5p promoted the stemness and gemcitabine resistance of HCC cells by repressing SOCS3 expression. Hence, the miRNA-30d-5p/SOCS3 axis might be a therapeutic target for chemotherapy resistance and a feasible marker for the prognosis of HCC patients.
期刊介绍：
MCP - Advancing biology through–omics and bioinformatic technologies wants to capture outcomes from the current revolution in molecular technologies and sciences. The journal has broadened its scope and embraces any high quality research papers, reviews and opinions in areas including, but not limited to, molecular biology, cell biology, biochemistry, immunology, physiology, epidemiology, ecology, virology, microbiology, parasitology, genetics, evolutionary biology, genomics (including metagenomics), bioinformatics, proteomics, metabolomics, glycomics, and lipidomics. Submissions with a technology-driven focus on understanding normal biological or disease processes as well as conceptual advances and paradigm shifts are particularly encouraged. The Editors welcome fundamental or applied research areas; pre-submission enquiries about advanced draft manuscripts are welcomed. Top quality research and manuscripts will be fast-tracked.
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