Mechanism of differentiation antagonizing non-protein-coding RNA regulating proliferation of breast cancer cells

doi:10.3877/cma.j.issn.1674-0807.2023.01.005

Abstract

Abstract:

Objective

To investigate the potential mechanism of differentiation antagonizing non-protein-coding RNA (DANCR) in breast cancer cells.

Methods

Fresh primary breast cancer samples and paired adjacent normal tissue samples were obtained from 26 patients in the Zhongda Hospital, Southeast University from January 2017 to January 2018. None of the patients received neoadjuvant therapy. DANCR expression was detected by qRT-PCR in breast cancer tissues and adjacent normal tissue, and in some cell lines (MCF-7, MDA-MB-231, SK-BR-3 and MCF-10A). MDA-MB-231 cells were transfected with negative control lentivirus and short hairpin RNA (shRNA) lentivirus respectively to obtain three groups of cells: negative control group, shRNA (381) group and shRNA (766) group. MTT, Transwell assay and flow cytometry were used to evaluate the proliferation, migration, apoptosis and cell cycle of MDA-MB-231 cells. Western blot analysis was used to detect the expression of cyclin D1 and cyclin-dependent kinase kinase 4 (CDK4) in three cell lines. Paired t test was used to compare the expression of DANCR between breast cancer tissue and adjacent normal tissue. Using the median value of DANCR expression (1.344) as a cutoff, patients were divided into two groups: high DANCR expression group and low DANCR expression group. Fisher’s exact test was used to compare the clinicopathological characteristics between two groups. The expression level of DANCR among four cell lines, cell survival rate, migrated cell number, apoptotic rate, percentage of cells in different cell cycle and the expression levels of cyclin D1 and CDK4 in MDA-MB-231 cells among three groups were compared by one-way analysis of variance. The LSD method was used for pairwise comparison.

Results

(1) The expression of DANCR in breast cancer tissue was significantly higher than that in adjacent normal tissue(2.02±0.33 vs 1.07±0.03, t=-2.875, P=0.008). Among the 26 patients, 13 patients showed DANCR high expression and 13 patients showed low expression. There was a significant difference in TNM stage and ER expression between two groups (P=0.002, 0.047). (2)The expression of DANCR in breast cancer cell line MCF-7, SK-BR-3, MDA-MB-231 and normal epithelial cell line MCF-10A was 1.46±0.23, 2.33±0.22, 2.14±0.08 and 1.00±0.09, respectively, indicating a significant difference (F=35.656, P<0.001). Compared with MCF-10A cells, the expression of DANCR in SK-BR-3 cells and MDA-MB-231 cells was significantly higher (both P<0.001). (3)The expression of DANCR was 1.00±0.07, 0.08±0.01 and 0.28±0.02 in negative control, shRNA(381) and shRNA(766) groups, respectively, indicating a significant difference (F=340.619, P<0.001). DANCR expression in shRNA(381) and shRNA(766) groups was significantly lower compared with negative control group (both P<0.001). (4)The optical density of cells was 1.226±0.092, 0.985±0.097 and 0.611±0.032 in negative control, shRNA(381) and shRNA(766) groups, respectively. The survival rate of cells were (100.00±0.00)%, (87.10±9.52)% and (53.13±4.57)% in negative control, shRNA(381) and shRNA(766) groups, respectively, indicating a significant difference (F=31.511, P=0.001). The survival rate of cells in shRNA(766) group was significantly decreased compared with negative control group (P=0.005). (5)The number of migrated cells was 880.00±78.46, 56.00±2.82 and 76.66±25.50 in negative control, shRNA(381) and shRNA(766) groups, respectively, indicating a significant difference (F=118.550, P=0.001). The migrated cells in shRNA(381) and shRNA(766) groups was significantly decreased compared with negative control group (both P<0.001). (6) The apoptotic rate of cells was (18.03±0.33)%, (18.10±0.11)% and(18.22±0.08)% in negative control, shRNA(381) and shRNA(766) groups, respectively, indicating a significant difference (F=0.445, P=0.677). (7) There a significant difference in the proportion of cells in G₀/G₁ phase, S phase and G₂/M phase among three groups (F=84.482, 40.702, 93.691, P=0.002, 0.007, 0.002). The shRNA(381) and shRNA(766) groups had a significantly higher proportion of cells in G₀/G₁ phase and a significantly lower proportion of cells in G₂/M phase compared with negative control group (P=0.001, 0.014; P=0.009, 0.001). Only ShRNA (381) group had a significantly higher proportion of cells in S phase compared with negative control group (P=0.009). (8) The expression of cyclin D1 in negative control, shRNA(381) and shRNA(766) groups was 1.03±0.09, 0.91±0.03 and 0.47±0.03, respectively, and the expression of CDK4 was 1.06±0.02, 0.85±0.04 and 0.53±0.02, respectively. Both indicated a significant difference among groups (F=48.241, 33.479, P=0.005, 0.009). The expression of cyclin D1 and CDK4 in shRNA(766) group was significantly lower than that in negative control group (P=0.003, 0.004). The expression of CDK4 in ShRNA(381) group was significantly lower than that in negative control group (P=0.028).

Conclusions

DANCR is highly expressed in breast cancer tissue and breast cancer cell lines. Inhibition of DANCR expression results in the decrease of cyclin D1 and CDK4 expression, cell cycle arrest in G₀/G₁ phase and inhibition of proliferation and migration of breast cancer cells.

Key words: Breast neoplasms, RNA, Long non-coding, Proliferation, Migration, Cell cycle

Lifei Han, Jianxin Lyu, Baocai Wang, Xinhua Cao, Xiao Ma, Haolin Hu, Yanan Zhang. Mechanism of differentiation antagonizing non-protein-coding RNA regulating proliferation of breast cancer cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2023, 17(01): 21-29.

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Figures/Tables 9

References 27

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名称	上游引物	下游引物
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA
DANCR	GCCACTATGTAGAGGGTTTC	ACCTGCGCTAAGAACTGAGG

名称	上游引物	下游引物
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA
DANCR	GCCACTATGTAGAGGGTTTC	ACCTGCGCTAAGAACTGAGG

步骤	反应程序	循环数	温度	时间
1	预变性	1	95 ℃	5 min
2	循环反应	40	95 ℃	10 s
		40	60 ℃	30 s
3	溶解曲线	1	95 ℃	15 s
			60 ℃	60 s
			95 ℃	15 s

步骤	反应程序	循环数	温度	时间
1	预变性	1	95 ℃	5 min
2	循环反应	40	95 ℃	10 s
		40	60 ℃	30 s
3	溶解曲线	1	95 ℃	15 s
			60 ℃	60 s
			95 ℃	15 s

分组	例数^a	年龄		TNM分期			组织学分级			Ki67表达		ER表达		HER-2表达
分组	例数^a	<60岁	≥60岁	Ⅰ期	Ⅱ期	Ⅲ期	1级	2级	3级	<30%	≥30%	阴性	阳性	阳性	阴性
DANCR高表达组	13	6	7	0	4	9	0	7	6	5	8	9	4	7	6
DANCR低表达组	13	7	6	4	8	1	1	5	7	8	5	3	10	8	5
P值		1.000^b		0.002^b			0.695^b			0.434^b		0.047^b		0.336^b

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