| BackgroundAs a malignant tumor,lung cancer ranks first in the world in terms of morbidity and mortality.It has seriously endangered the quality of life and health of human beings,and has been on the rise year by year in recent years.NSCLC accounts for 80-85%of lung cancer cases,and most of them are adenocarcinomas.In recent years,some progress has been made in the diagnosis and treatment of NSCLC,but the overall survival rate has not changed significantly.Data show that the 5-year survival rate of NSCLC patients is only about 13%.Selenium is an essential trace element in human body and a component of many enzymes in human body.It has many biological effects such as protecting liver,anti-cancer,protecting cardiovascular,regulating hormone secretion and so on.The earliest selenium supplement used by people is inorganic selenium(mainly sodium selenite and sodium selenite).Inorganic selenium has similar action dose and toxicity dose,low safety and biological activity,and has been replaced by selenium nanoparticles(SeNPs),which are highly bioactive and non-toxic.Compared with selenomethionine,selenium-methyl selenocysteine and sodium selenite,SeNPs have low toxicity and high anti-cancer effect,so they have been widely concerned.The anti-cancer mechanism of SeNPs has been proposed,including promoting cell apoptosis,inhibiting cell proliferation,regulating redox status,inactivating carcinogens,stimulating immune system and inhibiting angiogenesis.SeNPs promote cancer cell apoptosis,which is considered to be one of the main molecular mechanisms of its anti-cancer.In recent years,cancer researchers have found a series of cell death patterns different from apoptosis,which are considered to play an important regulatory role in the development of cancer.However,whether SeNPs are involved in these cell death remains to be clarified.This study is divided into three parts:(1)Biosynthesis,characterization and inhibition of SeNPs on NSCLC;(2)Screening and identification of key genes related to SeNPs based on RNA-seq detection;(3)SeNPs promotes autophagy in non-small cell lung cancer.The three parts are progressively related to each other.The previous part of the follow-up study is based on each other and complements each otherMethods1.SeNPs were synthesized by the biological method of Escherichia coli,characterized and identified by ultraviolet-visible absorption spectroscopy,measured the size and shape of SeNPs by transmission electron microscopy,and determined the elemental composition of synthetic particles by energy spectrometer to determine the purity and characterization of synthetic products2.X-ray and synthetic SeNPs were used to treat non-small cell lung cancer cell line A549.MTT assay,caspase-3 activity detection kit and immunofluorescence assay were used to detect the inhibitory effect of SeNPs on A549 cells3.The transcripts of A549 cells in blank control group,X-ray group,SeNPs group and X-ray+SeNPs group were quantified by RNA-seq technique,and key genes with differentially expressed genes were screened and identified4.QRT-PCR,Western-blotting and ELISA techniques were used to verify the differential expression of key genes among groups5.Western-blotting,confocal laser microscopy and electron microscopy were used to determine the difference of autophagy between groups6.The molecular mechanism of SeNPs inhibiting A549 cell proliferation was determined by using key protein inhibitors as probesResultsPart ? Biosynthesis,Characterization and Inhibitory Effect of SeNPs on Non-small Cell Lung CancerSeNPs were synthesized by Escherichia coli and characterized by ultraviolet-visible absorption spectroscopy.X-ray and electron microscopy images show that the synthesized SeNPs have a new structure of hexagonal ring with diffraction ring pattern.The main element component of the synthesized nanoparticles was selenium,which was determined by energy dispersive spectrometry.On this basis,it was found that the newly synthesized SeNPs had a strong inhibitory effect on the proliferation of A549 cells,but a relatively weak inhibitory effect on normal control cells.At the same time,SeNPs can up-regulate caspase-3 activity and promote cell death in A549 cells These results suggest that up-regulation of apoptosis in A549 cells by SeNPs may be the main factor that inhibits cell proliferationPart ?.Screening and identification of key genes of SeNPs-related non-small cell lung cancer based on RNA-seq detectionIn this part,non-small cell lung cancer A549 cells were divided into four groups blank control group,X-ray group,SeNPs group and X-ray+SeNPs group.RNA was extracted from each group of cells for RNA-seq analysis,and a series of differentially expressed genes were identified.Through GO and KEGG cluster analysis of differentially expressed gene data,it was found that some genes were involved in regulating important cellular functions,such as cell necrosis,autophagy,apoptosis and inflammation.Preliminary verification by qRT-PCR,ELISA and Western-blotting showed that some inflammation-related genes,such as COX-2 and iNOS,were significantly up-regulated in A549 cells treated with SeNPs.The results suggest that the regulation of SeNPs on the secretion of inflammatory factors may be a key factor in promoting apoptosis and inhibiting tumorigenesisPart ? SeNPs promotes autophagy in non-small cell lung cancerAutophagy is a new programmed cell death mode discovered and confirmed in recent years.It is characterized by ATG-dependent signaling pathway,accompanied by a large number of autophagic bodies and autophagic lysosomes.In the second part,a series of differentially expressed autophagy-related genes(ATG)were identified by RNA-seq.In this part,ATG5 and BECN1 were significantly up-regulated in A549 cells stimulated by SeNPs,while LC3-?/LC-? ratio was significantly increased in A549 cells stimulated by SeNPs,while SQSTM1 expression was significantly decreased by electronic display.The number of autophagic lysosomes was observed by microscopy and the co-localization of LAMP1 and LC3 was analyzed by laser confocal technique.These classical autophagy experiments confirmed that SeNPs could promote the autophagy of A549 cells.After treated with autophagy inhibitors,inflammatory factor iNOS inhibitors and apoptotic inhibitors,it was found that autophagy inhibitors could significantly block the inhibition of proliferation of A549 cells by SeNPs.These results suggest that the inhibition of A549 by SeNPs is partly achieved by up-regulating autophagyConclusionSeNPs synthesized in this study have a new structure of hexagonal ring with diffraction ring pattern.The newly synthesized SeNPs inhibit the proliferation of A549 cells and regulate the expression of a large number of genes.Some of the controlled genes are related to cell death such as apoptosis,autophagy and necrosis,and some inflammatory factors are also confirmed to be regulated by SeNPs Experiments showed that the inhibition of A549 by SeNPs was partly achieved by up-regulating autophagy.This study suggests that SeNPs may inhibit the proliferation of A549 cells not only because it promotes apoptosis,but also through other death modes,such as autophagy.The results of this study will enable people to understand the anti-cancer mechanism of SeNPs more thoroughly and provide a theoretical basis for the follow-up application of SeNPs in clinical anti-cancer research. |
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