| Triple-negative breast cancer（TNBC）is a type of malignant tumor with strong invasiveness and poor prognosis.Due to the insensitivity to molecular targeted therapy and endocrine therapy,anthracycline-based chemotherapy remains the major treatment for TNBC.In order to reduce the body damage caused by toxic and side effects,dietary supplements are proposed as a new adjuvant chemotherapy regimen,of which underlying molecular mechanisms have not been fully elucidated.Therefore,the tendency of treatments for TNBC is to seek for safe and effective adjuvant chemotherapy drugs with easy obtained properties and low side effects.Chitosan oligosaccharides（COS）is a dietary supplement that has been proven to effectively inhibit the proliferation of tumor cells by a large number of experiments.However,the high dose of it commonly used in vitro restricts further application in human body.Therefore,it is of great significance to investigate the intervention and impact of COS at a low concentration and the combination with chemotherapy drug doxorubicin（DOX）on the TNBC model.Whether this regulatory effect has an effect on suppression effect of DOX,and what is the mechanism,has not yet been carried out in depth.In this experiment,the combination of COS and DOX was constructed,and the anti-tumor activity was investigated on MDA-MB-231 human breast cancer cells with high metastatic characteristics.Furthermore,RNA sequencing（RNA-seq）technology was used to mine and identify the molecular targets of its anti-tumor effect.The main research work and results are as follows:（1）Characterization of COS components.liquid chromatography-mass spectrometry（LC-MS）was used to characterize the COS used in this experiment.The results showed that the degree polymerization of COS was 2～4.（2）Inhibitory effect evaluation of COS on cell proliferation.CCK-8 assay was performed to detect cell viability of COS alone and the combination therapy with DOX.The results showed that low concentrations of COS（0～1000μg/mL）alone was found no cytotoxicity.However,in the combination therapy,the IC50 concentration（2.5μg/m L）of DOX could enhance the original half inhibition effect of DOX in a dose-dependent manner（p<0.001）by the momentary preincubation of COS.The optimized preincubation time of COS was 4 h,and the optimized monomer of COS was chitotriose.Despite concentration of chitotriose was as low as 6.25μM,the inhibition rate was reduced by 16.49%compared with DOX group（p<0.001）.The estimated lowest cell viability was 19.44%±1.91 with 100μM chitotriose.From this,the optimal regimen for combined therapy was determined to be chitotriose（4 h,100μM）+DOX（2.5μg/m L）.（3）Effects on cell migration,cell morphology changes,and cellular uptake with COS.Transwell assay was measured and the results showed that COS and its monomer chitotriose could both significantly inhibit cell migration.Optical microscope was used and showed that MDA-MB-231 cells turned mesenchymal-epithelial transition（MET）with momentary preincubation of COS or chitotriose.Besides,cells changed into shorter cluster detected by field emission scanning electron microscopy（FESEM）.Confocal laser scanning microscopy（CLSM）and flow cytometry analysis were adopted to explore qualitative and quantitative evidence for cellular uptake of the DOX.The results showed that COS or chitotriose preincubation could significantly promote the entry of DOX into cell nucleus in a time-dependent manner（p<0.001）,and mean fluorescence intensity（MFI）of chitotriose+DOX was significantly increased compared to COS+DOX group（p<0.01）.Thus,COS and chitotriose could both promote the rapid enrichment of DOX into cell nucleus of MDA-MB-231,and synergistic inhibitory effect of chitotriose was superior to COS.（4）RNA-seq analysis for target genes.chitotriose+DOX under the optimized factors was used as combined group,the medium group as control,DOX group as positive control,and chitotriose incubation for 1,2,4 h as the time series.The sequencing was of good quality and high credibility.Heat map of differential expression genes（DEGs）in time series showed that2 h was a distinct differential expression pattern within 4 h,indicating that 2 h may be a key node for gene expression.KEGG pathways where DEGs between chitotriose 4 h group and CTL group were mainly enriched in Pathways in cancer,Human papillomavirus infection,PI3K-Akt signaling pathway,Focal adhesion and Breast cancer.DEGs enriched in the mainly pathways were analyzed in the order of fold change.The results showed the significantly DEGs were TMEM61,MYC,GADD45A,FOS,JUN,CDKN1A,RASGRP1,FGL2,COLQ,BMP4,EFNA4,EFNA1 and FRAT1.Venn diagram displayed the overlapping genes between comparison groups of chitotriose preincubation for 4 h and combined treatment.424 DEGs were first screened and protein interactions were then predicted by protein-protein interaction（PPI）network analysis.EGR1 were then identified as the most crucial protein related to various other proteins therein,which could be speculated as the most significant regulatory factor.（5）Functional validation results of target gene:qRT-PCR method was performed to verify the DEGs above.Chitotriose group significantly up-regulated（p<0.001）mRNA levels of TMEM61,MYC,GADD45A,FOS,JUN,CDKN1A and RASGRP1,while significantly down-regulated（p<0.001）that of FGL2,COLQ,BMP4,EFNA4,EFNA1 and FRAT1.Among them,TMEM61（up-regulated by 12.62 times）and FGL2（down-regulated by 11.36 times）acted as the key role in chitotriose preincubation effect.After the key target gene EGR1 was silenced through small interfering RNA（siRNA）method,the transcription and protein levels of related gene GADD45A was decreased with it.Meanwhile,cell viability experiments showed that silencing EGR1 gene expression could weaken the synergistic suppression effect,suggesting that chitotriose-induced upregulation of EGR1 was the key regulatory gene in combined therapy.In summary,chitotriose momentary preincubation enhanced antitumor activity of doxorubicin via upregulation of EGR1 expression in MDA-MB-231 cells. |
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