| Objective:Breast cancer is the number one malignant tumor among women in the world.Since the 21st century,the incidence of breast cancer has been increasing rapidly,with 2.26 million new cases of breast cancer worldwide by 2020,including 420,000 new cases in China.Breast cancer stem cells(BCSCs)are a very small but important population of breast cancer cells.Their strong self-renewal ability and multidirectional differentiation potential are important factors of breast cancer heterogeneity,and are also widely considered to be the root cause of breast cancer development,recurrence,metastasis and drug resistance.Therefore,it is of great theoretical and clinical significance to study the biological properties and molecular regulatory mechanisms of BCSCs.Long non-coding RNA(lncRNA)is involved in many important regulatory processes such as X chromosome silencing,genomic imprinting,chromatin modification,transcriptional activation,transcriptional interference,and intranuclear transport,etc.Abnormal expression or function of lncRNA is closely related to tumor development,and the study of its molecular mechanism of regulating BCSCs is particularly important.LncRNA ST8SIA6-AS1 has been found to be highly expressed in numerous cancers,including liver,lung,and colon cancers,but the expression and regulation in breast cancer have been rarely reported.The main objective of this study was to investigate the role and molecular mechanism of lncRNA ST8SIA6-AS1 in the maintenance of stemness in BCSCs and to provide a theoretical basis for the development of therapeutic regimens targeting BCSCs.Methods:Analysis of ST8SIA6-AS1,miR-410-3p,COL3A1 expression and prognosis in breast cancer patients using bioinformatics tools;detection of differential expression of target genes or RNAs in cells using quantitative real-time PCR(qRT-PCR)and western blot(WB);construction of human breast cancer cell models with knockdown or overexpression of target genes using lentiviral packaging technology;the cell counting kit-8(CCK8)was used to detect cell proliferation and resistance to chemotherapeutic agents paclitaxel and doxorubicin;the Transwell assay was used to detect cell migration;detection of cell colony formation using clone formation assays;the ability of stem cells to form microspheres was examined using a sphere-forming assay;the regulatory relationship between ST8SIA6-AS1,miR-410-3p and COL3A1 was verified using a luciferase reporter assay and reversion assay;the in vivo tumorigenic ability of tumor cells was examined using a nude mouse xenograft tumor model.Results:(1)ST8SIA6-AS1 contributes to the maintenance of stemness characteristics of BCSCs:ST8SIA6-AS1 was highly expressed in breast cancer patients,and the overall survival(OS),disease-free survival(DFS)and distant metastases-free survival(DMFS)of breast cancer patients in the high ST8SIA6-AS 1 expression group were worse;ST8SIA6-AS1 was highly expressed in breast cancer cell lines and BCSCs;knockdown of ST8SIA6-AS1 significantly reduced the cell proliferation,drug resistance,cell migration,clone formation,stem cell sphere formation,and stemness marker expression of BCSCs;in contrast,overexpression of ST8SIA6-AS1 significantly enhanced the cell proliferation,drug resistance,cell migration,clone formation,stem cell sphere formation,and stemness marker expression of BCSCs.(2)COL3A1 is involved in regulating stemness characteristics of BCSCs:COL3 A1 was highly expressed in breast cancer patients,and the overall survival(OS)and disease-free survival(DFS)of breast cancer patients in the high COL3A1 expression group was worse;COL3A1 was highly expressed in breast cancer cell lines and BCSCs;knockdown or overexpression of ST8SIA6-AS1 is followed by diminished or enhanced COL3 A1 expression;knockdown of COL3 A1 significantly diminished the cell proliferation,drug resistance,cell migration,clone formation,stem cell sphere formation,and stemness marker expression of BCSCs;in contrast,overexpression of COL3A1 significantly enhanced the cell proliferation,drug resistance,cell migration,clone formation,stem cell sphere formation,and stemness marker expression in BCSCs;overexpression of COL3A1 in cell lines with knockdown of ST8SIA6-AS1 resulted in significant restoration of the cell migration,clone formation,stem cell sphere formation,and expression of stemness markers in BCSCs;knockdown of COL3A1 in cell lines overexpressing ST8SIA6-AS1 significantly impaired the cell migration,clone formation,stem cell sphere formation,and stemness marker expression of BCSCs.(3)miR-410-3p is involved in regulating the stemness characteristics of BCSCs:miR-410-3p was lowly expressed in breast cancer patients,and the overall survival(OS)of breast cancer patients in the low miR-410-3p expression group was worse;miR-4103p was lowly expressed in breast cancer cell lines and BCSCs;the expression level of miR-410-3p was significantly increased after overexpression of miR-410;the cell proliferation ability,drug resistance ability,cell migration ability,clone formation ability,stem cell sphere formation ability,and stemness marker expression of BCSCs were significantly diminished after overexpression of miR-410.(4)ST8SIA6-AS1/miR-410-3p/COL3A1 axis regulates the stemness characteristics of BCSCs:the expression of miR-410-3p was subsequently enhanced or diminished after knockdown or overexpression of ST8SIA6-AS1;the expression of miR-410-3p was also enhanced or diminished after knockdown or overexpression of COL3A1;the expression of ST8SIA6-AS1 and COL3A1 was significantly attenuated after overexpression of miR-410;lower luciferase activity of wild-type ST8SIA6-AS1 and COL3 A1 group after transfection with miR-410-3p mimic compared to the control;restoration of COL3A1 expression,cell migration capacity,clone formation capacity,stem cell sphere formation capacity,and stemness marker expression in BCSCs after transfection of miR-410-3p inhibitor in cell lines with knockdown of ST8SIA6-AS1 compared to controls;ST8SIA6-AS1 and COL3A1 promote cellular tumorigenesis in vivo,and miR-410-3p inhibits cellular tumorigenesis in vivo.Conclusion:(1)ST8SIA6-AS1 and COL3A1 were highly expressed in breast cancer and BCSCs,while miR-410-3p was lowly expressed,and both were associated with poor prognosis in breast cancer patients.(2)ST8SIA6-AS1 positively regulates the sternness characteristics of BCSCs.(3)COL3A1 contributes to the maintenance of stemness characteristics in BCSCs and is a downstream regulator of ST8SIA6-AS1.(4)miR-410-3p negatively regulates the sternness characteristics of BCSCs.(5)ST8SIA6-AS1/miR-410-3p/COL3A1 has an axial regulatory relationship and regulates the sternness characteristics of BCSCs. |
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