| Objective:Screening and identifying the stem cell properties related genes of colorectal cancer,and exploring the effect and mechanism of lncRNA-cCSC1 on the biological behavior of colorectal cancer stem cells.Method:1)Download pre-processed RNA-Seq data for colorectal cancer stem-like and non-stem-like tissue subtypes at TCGA(https://cancergenome.nih.gov/)and match each other with TCGA identification code.Differential expression analysis was performed on the data using the Limma algorithm,and lncRNAs with differential expression were selected for clustering analysis.The 10 lncRNAs with the highest expression levels and 22 classical stem markers were selected for correlation matrix analysis to select the key lncRNAs with the highest correlation and named lncRNA-cCSC1 according to the lncRNA relevant naming rules.The chromosomal position of lncRNA-cCSC1 was identified through the related database,and its full length and cell localization were verified.The related software predicts and analyzes its protein coding ability to determine whether it has the characteristics of lncRNA.2)QRT-PCR was used to detect the expression of lncRNA-cCSC1 in colorectal cancer tissues and adjacent normal tissues,and the relationship between the expression of lncRNA-cCSC1 and clinicopathological characteristics and overall prognosis was analyzed with clinical pathological data.QRT-PCR was used to continue to detect the expression of lncRNA-cCSC1 in six common colorectal cancer cell lines and human normal intestinal epithelial cells,and HCT116 and HT29 cells with the most significant expression were selected for subsequent experiments.Then CD133~+CD44~+and CD133~-CD44~-cells were sorted from these two cell lines by magnetic-activated cell sorting method,and the sorting purity was detected by flow cytometry.QRT-PCR was used to detect the expression of lncRNA-cCSC1 in CD133~+CD44~+and sphere cells.CD133~-CD44~-and adherent cells were used as control groups.3)Cell lines that stable knockdown or over expressed lncRNA-cCSC1(sh-cCSC1 and oe-cCSC1 groups)were constructed in HCT116 and HT29through lentivirus infection,and their unloaded lentivirus groups were used as control groups(Cont group).The transfection was observed by inverted fluorescence microscopy,and the transfection efficiency was verified by QRT-PCR.The proportion of CD133~+CD44~+after interfering lncrna-ccsc1expression was detected by flow cytometry.The protein and mRNA expressions of stemness markers CD133,CD44,SOX2,CXCR4 and Nanog in the two groups of cells were detected by Western blot and QRT-PCR,respectively.Sphere formation assays were used to observe the difference in the number and size of stem cell microspheres formed between the two groups of cells before and after interference with lncRNA-cCSC1 expression.Apoptosis experiments were used to detect the sensitivity of the two groups of cells to 5-FU,a classic chemotherapy drug for colorectal cancer.Subcutaneous tumor formation experiments in nude mice were used to detect the volume difference of xenograft tumors formed after interfering with lncRNA-cCSC1 expression.4)Gene enrichment analysis(GSEA)was used to detect the enrichment of lncRNA-cCSC1 and classical stemness pathways.Then CD133~+CD44~+cells were selected from the Cont group,sh-cCSC1 group and oe-cCSC1 group,and the effects of interfering lncRNA-cCSC1expression on the expression of key proteins Gli1 and SMO in the Hedgehog pathway were detected by Western blot.After inhibition of Hedgehog pathway activity with SMO protein inhibitor Cyclopamine,protein expression levels of stemness markers CD133 and CD44 were detected by Western blot.Results:1)From the data of 644 CRC patients in TCGA database,a total of1,227 genes with significant differential expression were screened by Limma,including 416 mRNAs,209 miRNAs,and 602 lncRNAs.The key gene lncRNA-cCSC1 was selected through cluster analysis and correlation matrix analysis,and it was significantly correlated with CD133(PROM1),CD44,SOX2,CXCR4,Nanog and other stemness markers.According to the ensmble database,lncRNA-cCSC1 was found on the 12th band of the24th arm of chromosome 12.The full length of lncRNA-cCSC1 was verified by RACE experiments to be 2290 bp.The result of FISH assay showed that lncRNA-cCSC1 was mainly expressed in the cytoplasm.The combined analysis of 4 different protein-coding ability prediction software showed that lncRNA-cCSC1 did not have protein-coding ability,which accorded with the characteristics of lncRNA.2)QRT-PCR results showed that the expression of lncRNA-cCSC1 in colorectal cancer tissues was significantly higher than that in normal adjacent tissues(P<0.01),and the expression abundance was significantly correlated with the degree of tumor invasion,lymph node metastasis,and TNM staging(P<0.05),and there were no significant correlations with clinicopathological factors such as patient age,gender,tumor location,size,degree of differentiation,and serum tumor marker levels(P>0.05).Kaplan-Meier survival analysis showed that the high expression of lncRNA-cCSC1 resulted in a poor overall survival(OS)and disease-free survival(DFS)in patients.In addition,the expression level of lncRNA-cCSC1 mRNA in colorectal cancer cell lines was significantly higher than that of human normal intestinal epithelial cells,and the highest expression was in HCT116 and HT29 cells.The proportion of CD133~+CD44~+cells selected from these two cells by magnetic-activated cell sorting method was 83.36%and 90.93%,and the expression abundance of lncRNA-cCSC1 in CD133~+CD44~+cells and spheroid cells was significantly higher than CD133~-CD44~-cells and adherent cells,P<0.05.3)Through fluorescence microscopy,the transfection efficiency in lncRNA-cCSC1 knockout group(sh-ccsc1)and control group(Cont)was more than 90%,and QRT-PCR verified that the mRNA transcription level in sh-cCSC1 group was significantly lower than that in Cont group.After knocking down lncRNA-cCSC1,compared with the Cont group,the proportion of CD133~+CD44~+cells in the sh-cCSC1 group was significantly reduced,while the protein expression abundance of stem cell surface markers CD133,CD44,SOX2,CXCR4,and the stem cell transcription factor Nanog and the mRNA transcription level were significantly reduced,and the number of stem cell microspheres formed was also significantly reduced.The sensitivity of CD133~+CD44~+cells to the 5-FU chemotherapy drug was significantly increased,and the ability of proliferation,metastasis,and invasion was weakened.The volume of subcutaneous xenograft in nude mice decreased significantly(P<0.05).In the lncRNA-cCSC1overexpression group(oe-cCSC1)and the control group(Cont),the transfection efficiency was greater than 90%.After verification by QRT-PCR,the mRNA transcription level in the oe-cCSC1 group was significantly higher than Cont group.Compared with the Cont group,the proportion of CD133~+CD44~+cells in the oe-cCSC1 group was significantly increased,the protein expression abundance of stem cell surface markers CD133,CD44,and the stem cell transcription factor Nanog was significantly increased,and the number of stem cell microspheres also significantly increased,P<0.05.4)GSEA analysis showed that the affected genes after targeted differential expression of lncRNA-cCSC1 were mainly concentrated in the Hedgehong signaling pathway.In the sorted CD133~+CD44~+cells,compared with the Cont group,the expression of SMO and Gli1 protein in sh-cCSC1 group was significantly reduced after lncRNA-cCSC1knockdown;otherwise,the expression levels of SMO and Gli1 in the oe-cCSC1 group were significantly increased,P<0.05.After treatment with the SMO protein inhibitor Cyclopamine,compared with the Cont group,the expression levels of SMO and Gli1 proteins in the inhibitor group were significantly reduced,while the expression levels of stem cell surface markers CD133 and CD44 were also reduced,P<0.05.Conclusion:LncRNA-cCSC1 may be involved in the regulation of colorectal cancer stem cell properties through the Hedgehog signaling pathway,thereby mediating changes in various tumor biological behaviors. |
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