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中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (04) : 220 -228. doi: 10.3877/cma.j.issn.2095-1221.2024.04.004

论著

氯普鲁卡因通过调控circRNA-ZKSCAN1表达抑制肝癌Huh-7细胞体外生长和转移的研究
曾聿理1, 雷发容1,(), 肖慧1, 邱德亮1, 谢静1, 吴寻2   
  1. 1. 610100 成都市龙泉驿区第一人民医院麻醉科
    2. 610100 成都市龙泉驿区第一人民医院普外科
  • 收稿日期:2023-12-28 出版日期:2024-08-01
  • 通信作者: 雷发容
  • 基金资助:
    四川省卫生和计划生育委员会科研课题(16PJ091)

Chloroprocaine inhibits the growth and metastasis of liver cancer Huh-7 cells in vitro by regulating the expression of circRNA-ZKSCAN1

Yuli Zeng1, Farong Lei1,(), Hui Xiao1, Deliang Qiu1, Jing Xie1, Xun Wu2   

  1. 1. Department of Anesthesiology, the First People's Hospital of Longquanyi District, Chengdu 610100, China
    2. Department of General Surgery, the First People's Hospital of Longquanyi District, Chengdu 610100, China
  • Received:2023-12-28 Published:2024-08-01
  • Corresponding author: Farong Lei
引用本文:

曾聿理, 雷发容, 肖慧, 邱德亮, 谢静, 吴寻. 氯普鲁卡因通过调控circRNA-ZKSCAN1表达抑制肝癌Huh-7细胞体外生长和转移的研究[J]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 220-228.

Yuli Zeng, Farong Lei, Hui Xiao, Deliang Qiu, Jing Xie, Xun Wu. Chloroprocaine inhibits the growth and metastasis of liver cancer Huh-7 cells in vitro by regulating the expression of circRNA-ZKSCAN1[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(04): 220-228.

目的

探讨氯普鲁卡因(CP)对人肝癌细胞Huh-7生物学行为的影响及其对circRNA-ZKSCAN1的调控作用。

方法

同剂量CP处理Huh-7细胞,后续实验处理:pcDNA、pcDNA-circRNA-ZKSCAN1分别转染至Huh-7细胞,si-NC、si-circRNA-ZKSCAN1分别转染至Huh-7细胞后用CP (10 mmol/L)处理;qRT-PCR法检测circRNA-ZKSCAN1表达量;CCK-8、平板克隆形成实验、划痕实验、Transwell小室分别检测细胞增殖、克隆形成、迁移及侵袭能力;Western blot检测各组细胞E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)蛋白的表达情况。两组间比较使用t检验,多组间比较使用单因素方差分析,进一步两两比较采用SNK-q检验。

结果

与对照组相比,CP低、中、高剂量干预后,细胞增殖抑制率[(24.20 ± 2.21)%、(48.25 ± 4.42)%、(65.19 ± 5.49)%比(0.00 ± 0.00)%]、E-cadherin蛋白水平、circRNA-ZKSCAN1表达量[1.51 ± 0.13、2.11 ± 0.22、2.92 ± 0.27比1.00 ± 0.00]升高(P < 0.05),细胞克隆形成数、侵袭细胞数[(99.96 ± 7.03)、(79.87 ± 4.96)、(58.64 ± 3.99)比(124.01 ± 10.98)个]减少(P < 0.05),划痕愈合率[(53.71 ± 4.19)%、(40.61 ± 4.58)%、(24.13 ± 2.22)%比(67.15 ± 5.17)%]、N-cadherin蛋白水平降低(P < 0.05),且呈剂量依赖性;与转染pcDNA相比,转染pcDNA-circRNA-ZKSCAN1后细胞增殖抑制率[(57.19 ± 5.27)%比(7.13 ± 0.54)%]、E-cadherin蛋白水平升高(P < 0.05),细胞克隆形成数、划痕愈合率[(31.11 ± 3.01)%比(68.07 ± 4.76)%]、侵袭数[(66.33±5.84)比(126.12±11.74)个]以及N-cadherin蛋白水平降低(P < 0.05);转染si-circRNA-ZKSCAN1可减低CP对Huh-7细胞生物学行为的作用。

结论

CP可通过上调circRNA-ZKSCAN1表达而抑制肝癌Huh-7细胞生长、转移。

Objective

To investigate the effect of chloroprocaine (CP) on the biological behavior of human hepatoma cell Huh-7 and its regulation on circZKSCAN1.

Methods

Huh-7 cells were treated with different doses of CP, and subsequent experimental treatments: pcDNA and pcDNA-circZKSCAN1 were transfected into Huh-7 cells, respectively. si-NC and si-circZKSCAN1 were transfected into Huh-7 cells and treated with CP (10 mmol/L). QRT-PCR detected the expression of circZKSCAN1. CCK-8, plate clone formation assay, scratch assay, and Transwell chamber were used to detect cell proliferation, clone formation, migration and invasion abilities, respectively. Western blot was used to detect the expression of E-cadherin and N-cadherin. T test was used for comparison between the two groups, One-way analysis of variance was used for statistical comparison among multiple groups, and the SNK-q method was used for further pairwise statistical comparison.

Results

After the treatment of CP, the inhibition rate of cell proliferation [ (24.20 ± 2.21) %, (48.25 ± 4.42) %, (65.19 ± 5.49) % vs (0.00 ± 0.00) %], the protein level of E-cadherin, and the expression of circZKSCAN1 [1.51 ± 0.13, 2.11 ± 0.22, 2.92 ± 0.27 vs 1.00 ± 0.00] were increased (P < 0.05), while the number of cell clones and invasive cells [ (99.96 ± 7.03), (79.87 ± 4.96), (58.64 ± 3.99) vs (124.01 ± 10.98) pcs] was decreased (P < 0.05), and the wound healing rate [ (53.71 ± 4.19) %, (40.61 ± 4.58) %, (24.13 ± 2.22) % vs (67.15 ± 5.17) %], the protein level of N-cadherin were decreased (P < 0.05), and they were dose-dependent. After transfection of pcDNA-circZKSCAN1, the inhibition rate of cell proliferation [ (57.19 ± 5.27) % vs (7.13 ± 0.54) %] and the protein level of E-cadherin were higher than those of transfected pcDNA (P < 0.05), while the number of clones and invasive cells [ (31.11 ± 3.01) % vs (68.07 ± 4.76) %] was lower than those of transfected pcDNA (P < 0.05), and the wound healing rate [ (66.33 ± 5.84) vs (126.12 ± 11.74) pcs], the protein level of N-cadherin was lower than that of transfected pcDNA (P < 0.05). Transfection of si-circZKSCAN1 could reduce the effect of CP on the biological behavior of Huh-7 cells.

Conclusion

CP could inhibit the growth and metastasis of liver cancer Huh-7 cells by up-regulating the expression of circZKSCAN1.

表1 qRT-PCR引物序列信息
图1 倒置显微镜下观察CP肝癌Huh-7细胞克隆形成的影响(结晶紫染色)注:a图为对照组,b图为CP低剂量组,c图为CP中剂量组,d图为CP高剂量组
表2 CP对肝癌Huh-7细胞增殖的影响( ± s
图2 倒置显微镜下观察CP对肝癌Huh-7细胞迁移(×40)、侵袭(结晶紫染色,×200)的影响
图3 CP对肝癌Huh-7细胞迁移、侵袭蛋白E-cadherin、N-cadherin表达的影响
表3 CP对肝癌Huh-7细胞迁移、侵袭的影响( ± s
图4 circRNA在对照组和CP高剂量组的表达热图
图5 CP处理后的肝癌Huh-7细胞中的差异表达circRNA的火山图
表4 CP对肝癌Huh-7细胞circRNA-ZKSCAN1表达( ± s
图6 circRNA-ZKSCAN1过表达对肝癌Huh-7细胞移、侵袭相关蛋白表达的影响
图7 倒置显微镜下观察circRNA-ZKSCAN1过表达对肝癌Huh-7细胞克隆形成(结晶紫染色)、迁移(×40)、侵袭(结晶紫染色,×200)的影响
表5 circRNA-ZKSCAN1过表达对肝癌Huh-7细胞增殖、迁移、侵袭及相关蛋白表达的影响( ± s
图8 倒置显微镜下观察下调circRNA-ZKSCAN1表达逆转了CP对肝癌Huh-7细胞克隆形成(结晶紫染色)、迁移(×40)、侵袭(结晶紫染色,×200)的影响
图9 下调circRNA-ZKSCAN1表达逆转了CP对肝癌Huh-7细胞迁移、侵袭相关蛋白表达的作用
表6 下调circRNA-ZKSCAN1表达逆转了CP (10 mmol/L)对肝癌Huh-7细胞增殖、迁移和侵袭的作用( ± s
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