| Objective: Microarray microarrays were used to analyse small RNAs(mirco RNAs,miRNA)differentially expressed in plasma exosomes of primary colorectal cancer(CRC)patients and colorectal cancer liver metastasis(CRLM)patients,combined with The Cancer Genome Atlas(TCGA)database was used to screen candidate miRNAs,and q PCR was used to detect their expression levels in the samples and analyse their potential to become markers for monitoring colorectal cancer liver metastasis.The effects of the candidate miRNAs on the biological functions of colorectal cancer cell lines were initially investigated by constructing cell models and performing in vitro experiments.Methods:(1)Using Agilent microarray technology,plasma exosomal miRNA differential expression profiles were constructed for six primary CRC patients and six CRLM patients using the criteria of difference fold change(FC)>1 and difference significance(P-value,P)< 0.05;a sample set containing 590 samples from the TCGA database was used,which including 500 primary samples and 90 distant metastasis(m CRC)samples,the top 20 miRNAs meeting the conditions were screened to constitute the dataset using P < 0.05 and |log2FC|>0.3 as the threshold values,and the above 20 miRNAs were screened again by LASSO logistic regression analysis model to obtain the set of miRNAs with the best effect on distinguishing whether metastasis occurred or not The two data sets were intersected and combined with the results of regression analysis to screen out the suitable candidate miRNAs,and the candidate miRNA target genes were analyzsed using the database and GO/KEGG analysis was performed on the target genes.(2)Plasma exosomes and total plasma exosomal RNA were extracted using kits,identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blot(WB),and reverse transcribed for RNA;realtime fluorescence quantitative PCR(q RT-PCR)was used to further detect miR-146a-5p expression in 30 pairs of primary colorectal cancer patients and liver metastases in Patients’ plasma exosomes were examined for miR-146a-5p expression,while patients’ total plasma RNA was extracted and reverse transcribed and q PCR was performed to detect plasma miR-146a-5p expression;the relationship between plasma and plasma exosomal miR-146a-5p was analysed and its diagnostic efficacy was analysed.(3)Metastatic colorectal cancer cells Lo Vo cells and primary colorectal cancer cells HCT116 cells were transfected using miR-146a-5p analogues and blank control(NC)to establish a cell model of miR-146a-5p.qRT-PCR was used to detect miR-146a-5p expression levels in different subgroups of cells after transfection to verify the transfection efficiency.The effects of miR-146a-5p on the proliferation,migration,invasion and apoptosis of colorectal cancer cells were verified by cell proliferation assay,apoptosis assay and cell invasion and migration assay.Results:(1)After exosome identification,the constructed exosomal miRNA gene chips were analysed and among the eligible miRNAs,13 miRNAs were upregulated and 99 miRNAs were down-regulated in CRLM patients compared to CRC patients,resulting in a dataset containing 112 miRNAs;the tissue miRNA data from TCGA,after removing miRNAs not expressed in > After removing the miRNAs that were not expressed in >10% of CRC samples,20 miRNAs that were eligible for expression differences between CRC and m CRC samples were screened again by LASSO logistic regression analysis to obtain 8 miRNAs that had the best grouping effect on CRC and m CRC;the 8 miRNAs obtained from the screening were compared with the aforementioned The eight screened miRNAs were intersected with the aforementioned dataset containing 112 miRNAs to obtain three candidate miRNAs,including miR-146a-5p and miR-625-3p,which were down-regulated in transfer,and miR-200-5p,which was upregulated in expression.target gene analysis of the three candidate miRNAs revealed a total of 2722 target genes;GO/KEGG pathway analysis of the target genes showed that candidate miRNA target genes were enriched in cancer-related pathways.(2)The results of the exosome detection: the vesicles were round or oval with a bilayer-like structure under transmission electron microscopy,and the nanoparticle tracking analysis suggested that the diameter of the extracted extracellular vesicles was about 30-150 nm.The western blot results showed that the vesicle surface marker proteins CD63,CD9 and TSG101 were positive,which were consistent with the exosome characteristics.q RT-PCR The q RT-PCR results showed that miR-146a-5p expression was reduced in plasma and plasma exosomes of CRLM samples compared to CRC samples,which was consistent with the microarray results.By correlation analysis,there was no correlation between plasma exosomal miR-146a-5p and plasma miR-146a-5p expression levels(R=-0.054,P=0.72);using ROC curves for diagnostic efficacy analysis,plasma exosomal miR-146a-5p(AUC=0.588,P<0.01)was superior to Plasma miR-146a-5p(AUC=0.588,P<0.01)was superior to plasma miR-146a-5p(AUC=0.588,P<0.01).(3)As identified by q PCR,the expression level of miR-146a-5p in the overexpression group was higher than that in the NC group,indicating that the cells were successfully transfected and ready for the next step of study.The CCK8 assay showed that the proliferation ability of the miR-146a-5p overexpression group was reduced compared to the control group;the scratch assay showed that the migration ability of the miR-146a-5p overexpression group was reduced compared to the control group;Annexin V-FITC/PI fluorescence double-staining assay detected the apoptosis rate,and the early apoptosis rate increased in the overexpression group compared with the control group;Transwell invasion assay results show reduced invasion ability in miR-146a-5p overexpression group compared to control group.Conclusions:(1)Multiple miRNAs with differential expression between patients with primary and metastatic colorectal cancer were verified by analysis of plasma exosome microarrays in combination with tissue microarrays.(2)Plasma exosome miR-146a-5p is differentially expressed in patients with primary and liver metastases from colorectal cancer,with significantly down-regulated expression in patients with metastases,and has a higher diagnostic efficacy for CRLM compared to plasma miR-146a-5p,and has the potential to become a marker for monitoring liver metastases in patients with colorectal cancer.(3)The expression level of miR-146a-5p had an effect on the ability of colorectal cancer cells to migrate,invade and proliferate,and its increased expression inhibited migration,invasion and proliferation and induced their early apoptosis. |
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