腺病毒介导的SPRY1过表达对肝细胞癌凋亡和自噬的影响
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关键词：癌，肝细胞;细胞凋亡；自噬；斑点蛋白酪氨酸激酶底物1；原癌基因蛋白质 c-myc ;原癌基因蛋白质bcl-2中图分类号：R735.7 文献标志码：A DOI：10.11958/20252541
Abstract： ObjectiveTo investigate the efect and mechanismof adenovirus-mediated overexpressionof Sprouty RTK signaling antagonist1（SPRY1）onproliferation，apoptosisandautophagy inhumanhepatocelularcarcinoma （HCC）cells. MethodsTherecombinantadenovirus vectorAd5-SPRY1was constructedandusedto infect human HCCcellines Huh7 and Hep3B.Cels were divided into the control group （infected with Ad5-GFP）and the SPRY1 overexpression group （infected withAd5-SPRY1）.SPRY1 mRNA expresion was detectedbyqPCR.Western blot assaywas used toanalyze the protein levels of SPRY1， c -MYC，BCL2 and autophagy-related proteins LC3 I/I . Cell apoptosis was measured by flow cytometry with Annexin V-PE/7-AAD double staining.Autophagosome formation was observed by transmision electron microscopy（TEM），andautophagicfluxwasassessed using theAd-mCherry-GFP-LC3Breportersystem.ResultsAfter48 hours of Ad5-SPRY1 infection in Huh7and Hep3Bcels，theexpression levelsof SPRY1mRNAand protein were higher than those in the control group and the Ad5-GFP group （ P<0.05 ）.Flow cytometry analysis showed that after 24 hours of infection in Huh7and Hep3Bcels，the apoptosisrateintheAd-SPRY1 group waslowerthanthatinthecontrol groupand the Ad5-GFP group（ P<0.05 ）.Western blot results showed that the expression levels of c-MYC and BCL2 proteins in the Ad5-SPRY1 group were higher than those in the control group and the Ad5-GFP group （ P<0.05 ）. Transmission electron microscopy observation revealed alarge numberofdouble-membrane autophagosome structures in the cyoplasmof the Ad 5- SPRY1 group.Westernblotresults indicatedthatthe expressonlevelofLC3I protein intheAd5-SPRY1groupwas higher than that in the control group and the Ad5-GFP group P<0.05 ）. Ad-mCherry-GFP-LC3B fluorescence assay showed that the numberof yellow spots inthe Ad5-SPRY1 group was higher than that in the control groupand pcDNA3.1 group （ P< 0.05）.ConclusionAdenovirus-mediated SPRY1overexpression inhibitsapoptosis and promotes autophagy in HCCcells， possibly through upregulation of c -MYC and BCL2 and enhancement of LC3-mediated autophagic flux.SPRY1 may represent a novel therapeutic target for hepatocellular carcinoma.
Key words：carcinoma，hepatocellular; apoptosis; autophagy; SPla and RyR domain-containing proteinl; protooncogene proteins c-myc;proto-oncogene proteins bcl-2
肝癌是全球范围内死亡率最高的恶性肿瘤之一，其疾病负担在我国尤为严峻。（剩余13722字）