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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2023, Vol. 17 ›› Issue (01) : 21 -29. doi: 10.3877/cma.j.issn.1674-0807.2023.01.005

论著

分化拮抗非蛋白编码RNA调控乳腺癌细胞增殖的机制研究
韩丽飞1, 吕建鑫1, 王宝偲1, 曹欣华1, 马骁1, 胡浩霖1,†(), 张亚男1   
  1. 1. 210009　南京，东南大学附属中大医院乳腺中心
  • 收稿日期:2022-01-26 出版日期:2023-02-01
  • 通信作者: 胡浩霖
  • 基金资助:
    国家自然科学基金青年科学基金资助项目(81502287)

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

Lifei Han1, Jianxin Lyu1, Baocai Wang1, Xinhua Cao1, Xiao Ma1, Haolin Hu1,†(), Yanan Zhang1   

  1. 1. Breast Disease Center, Zhongda Hospital, Southeast University, Nanjing 210009, China
  • Received:2022-01-26 Published:2023-02-01
  • Corresponding author: Haolin Hu
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引用本文：

韩丽飞, 吕建鑫, 王宝偲, 曹欣华, 马骁, 胡浩霖, 张亚男. 分化拮抗非蛋白编码RNA调控乳腺癌细胞增殖的机制研究[J]. 中华乳腺病杂志(电子版), 2023, 17(01): 21-29.

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.

目的

研究分化拮抗非蛋白编码RNA(DANCR)在乳腺癌中可能的作用机制。

方法

使用定量RT-PCR检测DANCR在2017年1月至2018年1月东南大学附属中大医院收治的26例未行新辅助治疗的乳腺癌患者癌组织和癌旁正常组织中以及正常乳腺上皮细胞(MCF-10A)和不同乳腺癌细胞系(MCF-7、MDA-MB-231、SK-BR-3)中的表达水平。分别用阴性对照、短发夹RNA(shRNA)慢病毒载体转染MDA-MB-231细胞，得到阴性对照组、shRNA(381)和shRNA(766)3组细胞。分别采用MTT、Transwell实验、流式细胞技术检测3组细胞增殖、迁移、凋亡和细胞周期的差别。采用Western blot检测3组细胞中细胞周期蛋白D1(cyclin D1)和周期蛋白依赖性激酶4(CDK4)表达的变化。乳腺癌患者癌组织和癌旁正常组织中DANCR表达量的组间比较采用配对t检验。以DANCR表达中位数1.344作为截止值，将患者分为DANCR高表达组和DANCR低表达组，采用Fisher精确概率法比较2组患者临床病理特征的差别。4种细胞系中DANCR表达量的组间比较、3组MDA-MB-231细胞存活率、细胞迁移数、凋亡细胞比例、细胞周期数比例和cyclin D1和CDK4蛋白表达量的比较均采用单因素方差分析，进一步两两比较采用LSD法。

结果

(1)DANCR在乳腺癌组织中的表达量高于癌旁正常组织，差异具有统计学意义[2.02±0.33比1.07±0.03，t＝－2.875, P＝0.008)]。26例患者中DANCR高表达组13例，DANCR低表达组13例，2组患者的TNM分期、ER表达比较，差异具有统计学意义(P＝0.002、0.047)。(2)乳腺癌细胞系MCF-7、SK-BR-3、MDA-MB-231和正常乳腺上皮细胞系MCF-10A中DANCR的表达量分别为1.46±0.23、2.33±0.22、2.14±0.08和1.00±0.09，组间比较差异具有统计学意义(F＝35.656，P<0.001)。与正常乳腺上皮细胞MCF-10A相比，SK-BR-3细胞系和MDA-MB-231细胞系中的DANCR表达量均显著增加(P均<0.001)。(3)DANCR表达量在阴性对照组、shRNA(381)和shRNA(766)3组细胞中分别为1.00±0.07、0.08±0.01和0.28±0.02，组间比较差异有统计学意义(F＝340.619，P<0.001)。与阴性对照组相比，shRNA(381)和shRNA(766)细胞中的DANCR表达量均降低(P均<0.001)。(4)3组细胞的吸光度值分别为1.226±0.092、0.985±0.097和0.611±0.032，细胞存活率分别为(100.00±0.00)%、(87.10±9.52)%和(53.13±4.57)%，组间比较差异具有统计学意义(F＝31.511，P＝0.001)。与阴性对照组相比，shRNA(766)细胞的细胞存活率降低(P＝0.005)。(5)3组的细胞迁移数分别为(880.00±78.46)个、(56.00±2.82)个和(76.66±25.50)个，组间比较差异具有统计学意义(F＝118.550，P＝0.001)。与阴性对照组相比，shRNA(381)和shRNA(766)细胞的细胞迁移数均降低(P均<0.001)。(6)3组细胞的凋亡率分别为(18.03±0.33)%、(18.10±0.11)%和(18.22±0.08)%，3组间比较差异无统计学意义(F＝0.445，P＝0.677)。(7)3组细胞G0/G1期、S期、G2/M期细胞比例比较，差异具有统计学意义(F＝84.482、40.702、93.691，P＝0.002、0.007、0.002)；与阴性对照组相比，shRNA(381)和shRNA(766)细胞的G0/G1期细胞比例更高，G2/M期细胞比例更低(P＝0.001、0.014；P＝0.009、0.001)，S期细胞比例仅shRNA(381)高于阴性对照组(P＝0.009)。(8)3组细胞cyclin D1表达量分别为1.03±0.09、0.91±0.03和0.47±0.03，CDK4表达量分别为1.06±0.02、0.85±0.04和0.53±0.02，组间比较差异均有统计学意义(F＝48.241、33.479，P＝0.005、0.009)。shRNA(766)细胞的cyclin D1和CDK4表达量均低于阴性对照组(P＝0.003、0.004)。shRNA(381)细胞仅CDK4表达量低于阴性对照组(P＝0.028)。

结论

DANCR在乳腺癌组织和乳腺癌细胞系中高表达，抑制DANCR表达则cyclinD1和CDK4表达下降，细胞周期阻滞于G0/G1期，从而抑制乳腺癌细胞的增殖和迁移。

关键词: 乳腺肿瘤, RNA，长链非编码, 增殖, 迁移, 细胞周期
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
表1 分化拮抗非蛋白编码RNA的引物序列
名称 上游引物 下游引物
GAPDH GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAA
DANCR GCCACTATGTAGAGGGTTTC ACCTGCGCTAAGAACTGAGG
表2 实时荧光定量PCR反应程序
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
表3 26例DANCR高表达与低表达患者临床病理特征比较(例)
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
<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   1.000b 0.002b 0.695b 0.434b 0.047b 0.336b
图1 shRNA转染后3组MDA-MB-231细胞的形态　a~c图分别表示阴性对照、shRNA (381)和shRNA (766)3组细胞的明场图片(　×200)；d~f图分别表示阴性对照、shRNA (381)和shRNA (766)3组细胞的荧光图片(　×200)注：DANCR为分化拮抗非蛋白编码RNA
图2 分化拮抗非蛋白编码RNA敲低后MDA-MB-231细胞迁移能力的变化　a~c图分别为阴性对照、shRNA (381)和shRNA (766)分化拮抗非蛋白编码RNA敲降MDA-MB-231细胞迁移实验结果(Diff-Quik染色　×200)
表4 分化拮抗非蛋白编码RNA敲低后MDA-MB-231细胞凋亡和周期比较(%)
组别 凋亡率 G0/G1期 G2/M期 S期
阴性对照组 18.02±0.33 35.21±1.44 20.87±0.10 43.90±1.32
shRNA 381 18.10±0.11 49.01±0.90 16.60±0.71 34.43±1.54
shRNA 766 18.22±0.08 40.70±0.75 11.38±0.97 47.93±1.72
F值 0.445 84.482 93.600 40.702
P值 0.677 0.002 0.002 0.007
图3 分化拮抗非蛋白编码RNA敲低后MDA-MB-231细胞凋亡的变化　a~c图分别为阴性对照、shRNA (381)和shRNA (766)分化拮抗非蛋白编码RNA敲降MDA-MB-231细胞凋亡检测结果注：PI为碘化丙啶，Annexin V为膜联蛋白；Q1为坏死细胞百分率；Q2为晚期凋亡细胞百分率；Q3为正常细胞百分率；Q4为早期凋亡细胞百分率
图4 分化拮抗非蛋白编码RNA敲低后MDA-MB-231细胞周期的变化　a~c图分别表示阴性对照、shRNA (381)和shRNA (766)分化拮抗非蛋白编码RNA敲降MDA-MB-231细胞周期检测结果
图5 分化拮抗非蛋白编码RNA敲低后MDA-MB-231中细胞cyclin D1和CDK4的表达变化注：CDK4为周期蛋白依赖性激酶4；cyclin D1为细胞周期素D1；β-actin为内参蛋白；NC为阴性对照
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