| Objective:Breast cancer is the most common malignancy in women.Despite advances in early diagnosis and treatment,breast cancer remains the highest mortality among women in developed countries.Therefore,research on the pathogenesis of breast cancer is still very necessary.This is very helpful for the prognosis,diagnosis and even treatment of breast cancer patients.There is increasing evidence that breast cancer stem cells(BCSCs)can affect tumor progression,metastasis,and even lead to resistance to conventional treatments.Studies have shown that BCSCs have the ability to self-renew,can be in the form and function On the formation of a variety of cells,including cell lines resistant to treatment and metastasis.However,the specific mechanism of self-renewal capacity of BCSCs is not clear,which also affects the diagnosis and treatment of breast cancer patients.Notch,Hh and Wnt pathways are essential signal pathways for maintaining the normal physiological processes of tissues.However,loss of normal regulation of these pathways in the breast may result in the conversion of normal stem cells into CSCs.Wnt signaling pathway plays an important role in regulating breast cancer stem cell self-renewal.Activation of Wnt/?-catenin signaling can lead to the proliferation of stem and progenitor cells,leading to increased breast cancer volume.LncRNAs constitute a class of non-coding RNAs of more than 200 nucleotides in length,and lncRNAs have been found to be abnormally expressed in a variety of cancers and are associated with cancer progression.Abnormal lncRNA expression is a potential biomarker for cancer diagnosis,prognosis,and targeted therapy.However,the regulation of lncRNAs in the self-renewal of breast cancer stem cells is not clear.According to the current research,the mechanism of action of lncRNA is mainly epigenetic regulation,transcription regulation,and post-transcriptional regulation.Post-transcriptional regulation includes the lncRNA binding to miRNA and miRNA and further affecting miRNA-mediated gene silencing.Therefore,in this study,successful MCF-7 MS breast carcinoma cells and MCF-7 breast cancer cells were analyzed by LncRNAs microarray analysis.The differentially expressed lncRNAs were analyzed,and the lncRNA SPRY4 with the highest expression and highest differential expression was selected.-IT1,and its role in the regulation of stem-cell stem-cell self-renewal ability,and the mechanism by which SPRY4-IT1 binds to cisRNA and miR-6882-3p and influences Wnt pathway on the regulation of breast cancer stem cell stemness and self-renewal ability To provide new ideas and inspiration for the study of the treatment of breast cancer.Methods:1.Breast cancer stem cell induction: MCF-7 breast cancer cells were induced by MCF-7 breast cancer cells in a serum-free medium suspension condition.2.Flow cytometry: Flow cytometry was used to detect the expression of the cell surface stem markers CD24 and CD44.3.LncRNAs microarray expression profile: The expression of lncRNA in MCF-7 MS breast cancer stem cells and MCF-7 breast cancer cells induced by LncRNAs microarray analysis was analyzed.4.Bioinformatics analysis:(1)TCGA database: The RNA-Seq data for breast cancer patients were downloaded from the TCGA database.CD44+CD24-positive breast cancer patients(n=255)and non-CD44+CD24-patients(n=847)were selected for statistics.mRNA expression;(2)Target gene prediction website: We searched for miRNAs that bind to lncRNAs in mirDB database and SEGAL database,and used RNAhybrid website to bind energy;We searched for miRNAs that bind to miRNAs in mirDB database and DIANA database,and used RNAhybrid website to predict their binding energies.5.Lentivirus transfection: construction of sh-lncRNA using lentivirus transfection MCF-7 MS breast cancer stem cells,oe-lncRNA MCF-7 breast cancer cells,and sh-miRNA MCF-7 breast cancer cells and oe-miRNAs MCF-7 MS breast cancer stem cells.6.qRT-PCR and Western Blot: qRT-PCR and Western Blot methods were used to detect the expression of RNA and protein levels of TCT7L2 and Wnt1 in breast cancer stem cells such as OCT4,Nanog,CD44 and c-Myc.7.Luciferase Report Analysis: The luciferase reporter assay was used to test the binding of the miRNA and the 3' UTR of the target gene.8.Tumor xenografts in nude mice: Cells from control and experimental groups were inoculated on the fat pad of right arm of BALB/C-nu(4-6 weeks old)mice,and the tumor bearing mice were observed and recorded.The time of tumor formation and the number of tumors were calculated and the rate of tumor formation was calculated;the weight change,tumor volume and tumor weight of tumor-bearing mice were examined.Result: 1.Induction of MCF-7 MS cells: MCF-7 breast cancer cells can be enriched to form MCF-7 MS breast adenocarcinoma cells in serum-free non-adherent suspension cultures.Compared with the former,MCF-7 cells show CD44.+ CD24-high proportion of cell populations,and high expression of stem cell markers OCT4,CD44,Nanog,HIF-2?,and c-Myc.2.Expression of lncRNA SPRY4-IT1 in MCF-7 and MCF-7 MS cells: Compared with MCF-7 cells,SPRY4-IT1 was highly expressed in MCF-7 MS cells;CD44+CD24-mammary glands in TCGA database.The expression of SPRY4-IT1 in cancer patients(n=255)was higher than that in non-CD44+CD24-breast cancer patients(n=847),and SPRY4-IT1 was positively correlated with the stemness indicators HIF-1?,HIF-2?,and ALDH1A1.3.SPRY4-IT1 regulates the stemness and self-renewal ability of breast cancer stem cells: After silencing SPRY4-IT1 in MCF-7 MS,the expression of stem indexes SOX2,OCT4,Nanog,CD44 and c-Myc decreased,CD44+CD24-The proportion of cells decreased,and overexpression of SPRY4-IT1 in MCF-7 resulted in decreased expression of the stemness indices SOX2,OCT4,Nanog,and CD44.4.The binding of SPRY4-IT1 and miR-6882-3p: miR-6882-3p was found to bind to SPRY4-IT1 in mirDB database and SEGAL database,and the binding energy was predicted to be-31.5 kcal/mol using RNAhybrid website.The two have a combination.The expression of miR-6882-3p was decreased after overexpression of SPRY4-IT1 in MCF-7 cells,and the expression of miR-6882-3p was significantly increased in MCF-7 MS cells after silencing SPRY4-IT1.After overexpression of miR-6882-3p in MCF-7 MS cells,the dryness indexes of Nanog,CD44,OCT4 and c-Myc were significantly decreased;miR-6882-3p was silenced in MCF-7 cells,and the dryness indicators of Nanog,CD44,OCT4 and c-Myc increased significantly5.SPRY4-IT1 as a ceRNA affects the binding of miR-6882-3p and TCF7L2 to regulate the Wnt pathway: The combination of miR-6882-3p and TCF7L2(TCF4)was discovered in the mirDB database and the DIANA database,and the binding energy of the two was predicted using the RNAhybrid website.It is-26.5 kcal/mol,which proves that the two are combined.After overexpression of miR-6882-3p by MCF-7 MS,TCF7L2 and Wnt1 were significantly decreased,while miR-6882-3p,TCF7L2 and Wnt1 were increased in MCF-7 cells.Luciferase reporter assays revealed that miR-6882-3p had a significant inhibitory effect on the relative activity of luciferase,demonstrating the combination of the two.In the TCGA database,compared with other breast cancer patients,TCF7L2 expression was higher in CD44+CD24-positive breast cancer patients,and there was a positive correlation between SPRY4-IT1 and TCF7L2.After overexpression of SPRY4-IT1 in MCF-7 cells,Wnt1 and TCF7L2 were significantly increased.After silencing of SPRY4-IT1 in MCF-7 MS cells,Wnt1 and TCF7L2 were significantly decreased.6.Animal experiments verify the effect of SPRY4-IT1 expression on the stemness and self-renewal ability of breast cancer stem cells: compared with nude mice of control group,transplanted tumors of nude mice of sh-SPRY4-IT1 MCF-7 MS cell group The growth rate was significantly slower and the tumor weight was significantly reduced.The expression of SPRY4-IT1,stemness index OCT4,Nanog,CD44 and c-Myc and Wnt1 and TCF7L2 were significantly decreased,and the expression of miR-6882-3p was significantly increased.Compared with the nude mice of the control group,the growth rate of the transplanted tumors of the nude mice of the oe-SPRY4-IT1 MCF-7 cell group was significantly faster,the tumor weight was significantly increased,and the SPRY4-IT1,dryness index OCT4,Nanog,CD44 and The expression of c-Myc and Wnt1 and TCF7L2 were significantly increased,and the expression of miR-6882-3p was significantly decreased.Conclusion: 1.SPRY4-IT1 and miR-6882-3p have the effect of regulating the stemness of breast cancer stem cells.2.SPRY4-IT1 binds to miR-6882-3p as ceRNA and reduces the inhibitory effect of miR-6882-3p on TCF7L2,which in turn activates the Wnt/?-catenin signaling pathway to regulate the stemness of breast cancer stem cells. |
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