Influences of CircRHOT1 on proliferation,apoptosis and immune escape of breast cancer cells by regulating miR-187-3p/SOX4 axis
10.3969/j.issn.1000-484X.2024.03.023
- VernacularTitle:CircRHOT1调节miR-187-3p/SOX4轴对乳腺癌细胞增殖、凋亡和免疫逃逸的影响
- Author:
Pei SUN
1
;
Zhenwei JIANG
;
Qian LIU
Author Information
1. 武汉市普仁医院检验科,武汉 430080
- Keywords:
CircRHOT1;
miR-187-3p/SOX4 axis;
Breast cancer;
Proliferation;
Apoptosis;
Immune escape
- From:
Chinese Journal of Immunology
2024;40(3):577-585
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To study the influences of circular RNA ras homolog family member T1(CircRHOT1)on the prolifera-tion,apoptosis and immune escape of breast cancer cells by regulating the miR-187-3p/sex-determining region Y-box protein 4(SOX4)axis.Methods:Real-time quantitative PCR and Western blot were used to detect the expressions of CircRHOT1,miR-187-3p and SOX4 in human normal breast epithelial cells MCF-10A and breast cancer cells MCF-7,Hs-578T and MDA-MB-231 in vitro;MCF-7 cells cultured in vitro were randomly separated into control group,CircRHOT1 knockdown(transfected with CircRHOT1 siRNA plasmid)group,miR-187-3p mimics(transfected with miR-187-3p mimics)group,co-transfection negative control(transfected with empty plasmid and miR-187-3p inhibitor negative control)group,and CircRHOT1 knockdown+miR-187-3p inhibitor(transfected with CircRHOT1 siRNA plasmid and miR-187-3p inhibitor)group.After grouping and transfection,real-time quantitative PCR and Western blot were used to detect the expressions of CircRHOT1,miR-187-3p and SOX4 in each group;cell proliferation in each group was detected by EdU staining and plate colony formation assay;cell apoptosis in each group was detected by flow cytometry;im-munofluorescence staining was used to detect the ratio of apoptosis-related proteins Bax and Bcl-2(Bax/Bcl-2)expressions in each group.The cells of each group were co-cultured with human peripheral blood lymphocytes and transfected into groups,flow cytometry was used to detect the proportion of activated CD8+T cells in human peripheral blood lymphocytes in each group;the killing rate of human peripheral blood lymphocytes to MCF-7 cells in each group was detected by MTT assay.Dual-luciferase reporter assay was used to detect the targeted regulation of miR-187-3p and miR-187-3p on SOX4 by CircRHOT1 in MCF-7 cells.Results:Compared with human normal breast epithelial cells MCF-10A,human breast cancer MCF-7,Hs-578T and MDA-MB-231 cells had obviously higher protein and mRNA expression of CircRHOT1 and SOX4(P<0.05),and obviously lower expression of miR-187-3p(P<0.05).Com-pared with control group,the protein and mRNA expression of SOX4,EdU positive rate and colony formation rate of cells in Circ-RHOT1 knockdown group and miR-187-3p mimics group decreased(P<0.05),the expression of miR-187-3p,apoptosis rate,Bax/Bcl-2,the proportion of activated CD8+T cells in human peripheral blood lymphocytes and the killing rate on MCF-7 cells increased(P<0.05).Compared with CircRHOT1 knockdown group,there was no obvious difference in the expression of CircRHOT1 in the CircRHOT1 knockdown+miR-187-3p inhibitor group(P>0.05),the protein and mRNA expression of SOX4,EdU positive rate and colony formation rate of cells increased(P<0.05),the expression of miR-187-3p,apoptosis rate,Bax/Bcl-2,the proportion of activated CD8+T cells in human peripheral blood lymphocytes and the killing rate on MCF-7 cells decreased(P<0.05);there was no obvious difference in the indexes of cells in co-transfection negative control group(P>0.05).Conclusion:Knockdown of CircRHOT1 can reduce the expression of SOX4 by up-regulating miR-187-3p,thereby attenuating the proliferative activity of breast cancer cells and promoting their apoptosis.It can also inhibit the activation of CD8+T cells and enhance their cancer cell cytotoxicity,thereby weakening immune evasion of breast cancer cells.
