Effects of circRASSF2 targeting miR-1343-3p on the proliferation and apoptosis of breast cancer MDA-MB-231 cells

doi:10.11958/20221254

Abstract

Abstract:

Objective To explore the effect of circRASSF2 on the proliferation and apoptosis of breast cancer MDA-MB-231 cells and its molecular mechanism. Methods Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect circRASSF2 and miR-1343-3p expression levels in 37 breast cancer tissue samples and adjacent tissue samples. Breast cancer MDA-MB-231 cells were divided into the si-circRASSF2 group, the si-NC group, the miR-1343-3p group, the miR-NC group, the si-circRASSF2+anti-miR-1343-3p group and the si-circRASSF2+anti-miR-NC group. Tetramethylazolium salt colorimetry method (MTT) was used to detect cell viability. Clone formation experiment was used to detect the number of cell clones. Annexin V-FITC/PI double staining method was used to detect cell apoptosis. The relative expression levels of Cleaved-caspase 3 and Cleaved-caspase 9 proteins were detected by Western blot assay. Dual luciferase reporter experiment was used to detect targeting relationship between circRASSF2 and miR-1343-3p. Results Compared with paracancer tissue samples, the expression level of circRASSF2 was increased and the expression level of miR-1343-3p was decreased in breast cancer tissue samples (P<0.01). Compared with the si-NC group, the OD value of MDA-MB-231 cells was decreased in the si-circRASSF2 group, the number of cell clone formation was decreased, and the apoptosis rate was increased (P<0.05). Compared with the miR-NC group, the OD value of MDA-MB-231 cells was decreased in the miR-1343-3p group, the number of cell clone formation was decreased, and the apoptosis rate was increased (P<0.05). Compared with the miR-NC group, WT-circRASSF2 luciferase activity decreased in the miR-1343-3p group (P<0.05). Compared with the si-circRASSF2+anti-miR-NC group, the OD value and the number of cell clone formation were increased, and the apoptosis rate was decreased in the si-circRASSF2+anti-miR-1343-3p group (P<0.05). Conclusion The inhibition of circRASSF2 expression can up-regulate miR-1343-3p, thus inhibiting the proliferation of breast cancer MDA-MB-231 cells and promoting apoptosis.

Key words: breast neoplasms, cell proliferation, apoptosis, gene expression regulation, circRASSF2, miR-1343-3p

CLC Number:

R737.9

Figures/Tables 14

Tab.1 Comparison of circRASSF2 and miR-1343-3p expression levels between breast cancer tissue and paracancer tissue

组织	circRASSF2	miR-1343-3p
癌旁组织	1.00±0.02	1.00±0.05
乳腺癌组织	4.03±0.21	0.37±0.03
t	89.518^＊＊	60.709^＊＊

Fig.1 Cell clonogenesis ability in the si-NC group and the si-circRASSF2 group

Tab.2 Comparison of cell proliferation between the si-NC group and the si-circRASSF2 group

组别	circRASSF2	OD₄₉₀	克隆形成数/个
si-NC组	1.00±0.06	0.71±0.06	101.25±8.96
si-circRASSF2组	0.34±0.02	0.34±0.03	48.79±2.34
t	19.219^＊＊	9.992^＊＊	9.815^＊＊

Fig.2 Apoptosis of the si-NC group and the si-circRASSF2 group

Fig.3 Expression levels of apoptosis related proteins in the si-NC group and the si-circRASSF2 group

Tab.3 Comparison of apoptosis rate and related protein expression levels between the si-NC group and the si-circRASSF2 group

组别	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
si-NC组	8.75±0.13	0.27±0.04	0.20±0.02
si-circRASSF2组	23.89±3.21	0.75±0.09	0.54±0.06
t	8.156^＊＊	8.486^＊＊	10.304^＊＊

Fig.4 Binding sequence of circRASSF2 and miR-1343-3p

Fig.5 circRASSF2 targeting regulates the expression of miR-1343-3p

Fig.6 Clonal formation and apoptosis in the miR-NC group and the miR-1343-3p group

Fig.7 Expression of apoptosis related proteins in the miR-NC group and the miR-1343-3p group

Tab.4 Comparison of proliferation and apoptosis between the miR-NC group and the miR-1343-3p group

组别	miR-1343-3p	OD₄₉₀	克隆形成数/个	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
miR-NC组	1.01±0.02	0.70±0.05	99.87±8.43	7.48±0.51	0.25±0.03	0.19±0.02
miR-1343-3p组	3.21±0.13	0.42±0.03	50.23±2.45	19.96±2.03	0.73±0.08	0.48±0.05
t	28.357^＊＊	9.058^＊＊	9.794^＊＊	10.317^＊＊	9.821^＊＊	10.793^＊＊

Tab.5 Proliferation and apoptosis in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

组别	miR-1343-3p	OD₄₉₀	克隆形成数/个	细胞凋亡率/%	Cleaved-caspase 3	Cleaved-caspase 9
si-circRASSF2+anti-miR-NC组	1.00±0.03	0.39±0.02	40.78±3.51	27.45±3.11	0.75±0.09	0.52±0.06
si-circRASSF2+anti-miR-1343-3p组	0.28±0.02	0.65±0.04	82.63±8.74	12.13±0.98	0.32±0.04	0.24±0.02
t	35.040^＊＊	10.373^＊＊	7.696^＊＊	8.140^＊＊	7.460^＊＊	7.545^＊＊

Fig.8 Clonal formation and apoptosis in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

Fig.9 Expression of apoptosis related proteins in the si-circRASSF2+anti-miR-NC group and the si-circRASSF2+anti-miR-1343-3p group

References 15

[1]	赵晶, 吴楠, 张世超, 等. 乳腺癌靶向治疗新进展[J]. 中华肿瘤杂志, 2020, 42(5):353-361.
	ZHAO J, WU N, ZHANG S C, et al. New advances in targeted therapy for breast cancer[J]. Chin J Oncol, 2020, 42(5):353-361. doi:10.3760/cma.j.cn112152-112152-20190919-00614.
[2]	MEISEL J L, VENUR V A, GNANT M, et al. Evolution of targeted therapy in breast cancer:where precision medicine began[J]. Am Soc Clin Oncol Educ Book, 2018, 38(1):78-86. doi:10.1200/EDBK_201037.
[3]	ZHANG H D, JIANG L H, SUN D W, et al. CircRNA:a novel type of biomarker for cancer[J]. Breast Cancer, 2018, 25(1):1-7. doi:10.1007/s12282-017-0793-9.
[4]	TIAN L, CAO J, JIAO H, et al. CircRASSF2 promotes laryngeal squamous cell carcinoma progression by regulating the miR-302b-3p/IGF-1R axis[J]. Clin Sci, 2019, 133(9):1053-1066. doi:10.1042/CS20190110.
[5]	YANG L, BI T, ZHOU S, et al. CircRASSF2 facilitates the proliferation and metastasis of colorectal cancer by mediating the activity of Wnt/β-catenin signaling pathway by regulating the miR-195-5p/FZD4 axis[J]. Anticancer Drugs, 2021, 32(9):919-929. doi:10.1097/CAD.0000000000001084.
[6]	LI Y, ZHAO Z, SUN D, et al. Novel long noncoding RNA LINC02323 promotes cell growth and migration of ovarian cancer via TGF-beta receptor 1 by miR-1343-3p[J]. J Clin Lab Anal, 2021, 35(2):e23651. doi:10.1002/jcla.23651.
[7]	ZHANG X, DU L, HAN J, et al. Novel long non-coding RNA LINC02323 promotes epithelial-mesenchymal transition and metastasis via sponging miR-1343-3p in lung adenocarcinoma[J]. Thorac Cancer, 2020, 11(9):2506-2516. doi:10.1111/1759-7714.13562.
[8]	QIN Y, SUN W, WANG Z, et al. ATF2-induced lncRNA GAS8-AS1 promotes autophagy of thyroid cancer cells by targeting the miR-187-3p/ATG5 and miR-1343-3p/ATG7 axes[J]. Mol Ther Nucleic Acids, 2020, 22:584-600. doi:10.1016/j.omtn.2020.09.022.
[9]	ZHOU Y, MA G, PENG S, et al. Circ_0000520 contributes to triple-negative breast cancer progression through mediating the miR-1296/ZFX axis[J]. Thorac Cancer, 2021, 12(18):2427-2438. doi:10.1111/1759-7714.14085.
[10]	SANG Y, CHEN B, SONG X, et al. circRNA_0025202 regulates tamoxifen sensitivity and tumor progression via regulating the miR-182-5p/FOXO3a axis in breast cancer[J]. Mol Ther, 2019, 27(9):1638-1652. doi:10.1016/j.ymthe.2019.05.011.
[11]	XIE R, TANG J, ZHU X, et al. Silencing of hsa_circ_0004771 inhibits proliferation and induces apoptosis in breast cancer through activation of miR-653 by targeting ZEB2 signaling pathway[J]. Biosci Rep, 2019, 39(5):BSR20181919. doi:10.1042/BSR20181919.
[12]	ZHONG W, BAO L, YUAN Y, et al. CircRASSF2 acts as a prognostic factor and promotes breast cancer progression by modulating miR-1205/HOXA1 axis[J]. Bioengineered, 2021, 12(1):3014-3028. doi:10.1080/21655979.2021.1933300.
[13]	CHEN X, WANG J, XIE F, et al. Long noncoding RNA LINC01559 promotes pancreatic cancer progression by acting as a competing endogenous RNA of miR-1343-3p to upregulate RAF1 expression[J]. Aging (Albany NY), 2020, 12(14):14452-14466. doi:10.18632/aging.103487.
[14]	WANG L, BO X, YI X, et al. Exosome-transferred LINC01559 promotes the progression of gastric cancer via PI3K/AKT signaling pathway[J]. Cell Death Dis, 2020, 11(9):723-737. doi:10.1038/s41419-020-02810-5.
[15]	QI J, WANG Z, ZHAO Z, et al. EIF3J-AS1 promotes glioma cell growth via up-regulating ANXA11 through sponging miR-1343-3p[J]. Cancer Cell Int, 2020, 20(1):428-441. doi:10.1186/s12935-020-01487-2.