| Objective: Non-coding RNA is considered to be a "dark substance" in the human genome.Recent studies have found that it regulates the expression of genes at various levels such as epigenetics,transcription and post-transcription,which in turn affects tumorigenesis and development.LncRNA LINC00473(LNC473)has recently been reported to be involved in the regulation of the progression of various tumors such as gastric cancer,lung cancer,and liver cancer.However,the biological characteristics and mechanism of LNC473 in colorectal cancer are still unclear.Methods: 1.Bioinformatics: LNC473,hsa-miR-574-5p and hsa-miR-15b-5p,APAF1 mRNA were screened by cluster analysis of CRC Chip expression profile,then TCGA and GEO data sets were further extracted to compare the expression of each indicator and analyze the correlation between APAF1 mRNA and the prognosis of CRC patients.The pathways of LNC473 co-expressing mRNA were enriched by KEGG and the target genes were identified by cluster analysis.2.Tissue samples: 20 pairs of CRC clinical tissue samples were collected,and the expression of LNC473,hsa-miR-574-5p and hsa-miR-15b-5p and APAF1 mRNA were detected by qPCR and RT-PCR.Then the samples were further expanded to 157 pairs,in situ hybridization and immunohistochemistry were used to detect the expression of three RNAs and APAF1 protein respectively.Parallel linear regression was used to analyze the correlation among expression of each index.Further,Pearson chi-square test and unconditional logistic regression analysis were used to analyze the correlation between the expression of each index and clinicopathological parameters,log-rank single factor Kaplan-Meier method and adjusted multivariate COX regression were further used to analyze the correlation between the expression of each index and the prognosis of CRC patients.3.Basic experiments: qPCR,RT-PCR,Western blot,immunofluorescence,CCK8,colony formation and cell flow cytometry were used to compare the changes of CRC cells' phenotype and function.And the method of binding ISH and IF was used to co-localize the three RNAs with APAF1 protein in CRC cells.4.Mechanism validation: Dual luciferase reporter assay was used to further verify the specific binding ofhsa-miR-574-5p and hsa-miR-15b-5p to APAF1-IRES,and RNA pull down assay was used to confirm the specific sponging of LNC473 and hsa-miR-574-5p,hsa-miR-15b-5p.5.Xenograft mice in vivo: The effects of LNC473 and APAF1-IRES on the proliferation of CRC cells were verified by BALB/c nude mice.Results: 1.Firstly,LNC473 was significantly down-regulated based on the expression profile of 3 paired CRC chips,reversely,the expression of hsa-miR-574-5p and hsa-miR-15b-5p were up-regulated.The results were confirmed in TCGA or GEO dataset,6 CRC cell lines and 20 pairs of CRC tissue.In addition,the results of CRC patients' large 157-pair-sample showed that the three RNAs were mainly expressed in the cytoplasm,and the difference in expression of CRC compared to adjacent tissues was further verified,and the expression of LNC473 was lower in CRC,which was negatively correlated with the expression of hsa-miR-574-5p and hsa-miR-15b-5p,and predicting a poor prognosis.In addition,the expression of the three RNAs was also related to the clinicopathological parameters.2.Secondly,in order to further clarify the function of LNC473 regulating hsa-miR-574-5p or hsa-miR-15b-5p expression,which was correlated with biological characteristics of CRC cells,we overexpressed and silenced LNC473,and the expression of hsa-miR-574-5p and hsa-miR-15b-5p was negatively regulated.In addition,when LNC473 was up-regulated,the ability of CRC cell proliferation and colony formation was inhibited,cell proliferation index was decreased and apoptosis level was increased.while LNC473 was down-regulated,hsa-miR-574-5p and hsa-miR-15b-5p expression were up-regulated,which promoted the proliferation and colony forming ability of CRC cells,increased the cell proliferation index,and decreased the level of apoptosis.3.In order to determine the target genes of LNC473/hsa-miR-574-5p and hsa-miR-15b-5p,the result of function-enriched showed that co-expressed mRNAs of LNC473 were mainly related to CRC cells,ribosomes,and apoptosis-related pathways.And a significant down-regulation of the apoptotic enzyme activator APAF1 was screened in these pathway-related factors,and its specifically low expression in CRC was also found in the TCGA dataset,online platform,6 CRC cell lines,and 20 pairs or157 pairs of CRC tissue.In addition,It was further validated that APAF1 significantly prolonged the prognosis of CRC patients based on GEO,TCGA datasets and large-sample CRC patients.4.To clarify the specific mechanism of LNC473 positivelyregulating APAF1 via hsa-miR-574-5p and hsa-miR-15b-5p,we first predicted the secondary structure of internal ribosome entry sites(IRES)in the APAF1-5'UTR region and it could bind to hsa-miR-574-5p,hsa-miR-15b-5p stably(mfe: 25.1 kcal/mol and24.2 kcal/mol).The results of further dual luciferase reporter gene assay in 293 T cell line showed that the expression of luciferase was significantly decreased when APAF1-IRES wild-type plasmid and hsa-miR-574-5p or hsa-miR-15b-5p were co-transfected,and the synergistic effect of hsa-miR-574-5p and hsa-miR-15b-5p was more obvious,while the luciferase activity was restored when APAF1-IRES mutant plasmid was co-transfected with hsa-miR-574-5p or hsa-miR-15b-5p,and further qPCR results showed when the expression levels of hsa-miR-574-5p and hsa-miR-15b-5p were up-regulated,the mRNA level of APAF1 was decreased,and similar result was further confirmed in the HCT116 cell line.5.In addition,based on RNA and protein localization experiments,LNC473,hsa-miR-574-5p and hsa-miR-15b-5p and APAF1 protein were mainly expressed in cytoplasm.And LNC473 could up-regulate APAF1 mRNA and protein expression,which could be reversed by hsa-miR-574-5p and hsa-miR-15b-5p.It was also predicted that the RNA secondary structure of LNC473 could stably bind to hsa-miR-574-5p and hsa-miR-15b-5p(mfe: 38.1kcal/mol and 23.2kcal/mol).Results of RNA pull down in293 T cell lines showed that hsa-miR-574-5p and hsa-miR-15b-5p dragged down by LNC473 wild-type probes were increased significantly compared to NC probes after incubating with miRNAs respectively.While the amount of hsa-miR-574-5p and hsa-miR-15b-5p that LNC473 mutant probe dragged down was recovered.In addition,after overexpressing of LNC473,the amount of hsa-miR-574-5p and hsa-miR-15b-5p was significantly reduced.And the similar results were further confirmed in the HCT116 cell line.6.Finally,the results in BALB/c nude mice showed that APAF1-IRES inhibited the proliferation of CRC cells,and tumor suppressor effect of APAF1-IRES was further enhanced after simultaneously expressing LNC473.Conclusion: 1.LNC473 was downregulated following a negative correlation with hsa-miR-574-5p and miR-15b-5p,and the direct target of APAF1 was upregulated in CRC cells and paired CRC tissues.2.Elevated expression of LNC473 significantly prolonged DFS and OS of CRC patients,however,higher expression of hsa-miR-574-5p and hsa-miR-15b-5p was significantly associated with poor prognosis.3.Enforcedexpression of LNC473 acting as ceRNAs remarkably inhibited CRC cell proliferation and promoted apoptosis by competitive sponging hsa-miR-574-5p and hsa-miR-15b-5p,and vice versa.4.hsa-miR-574-5p and hsa-miR-15b-5p competitively inhibited APAF1IRES-mediated translation regulated by the expression of LNC473 sharing its miRNA response elements(MREs)in CRC cells.5 LNC473 suppressed tumorigenesis by exposing APAF1-IRE in BALB/c nude mice. |
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