Effect of circDNMT1 regulating miR-377-3p/PUM1 axis on proliferation,apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells

LIN Ming-li; ZHENG Mei-ling; LU Hong-quan; LIN Yao-ting

doi:10.11659/jjssx.12E022130

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Effect of circDNMT1 regulating miR-377-3p/PUM1 axis on proliferation,apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells

Basic Research | 更新时间：2024-01-22

- Effect of circDNMT1 regulating miR-377-3p/PUM1 axis on proliferation,apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 33, Issue 1, Pages: 1-7(2024)
- 作者机构：
  
  1.儋州市人民医院检验科，海南儋州 571700
  2.海南西部中心医院肿瘤内科，海南儋州 571700
  3.儋州市人民医院普外科，海南儋州 571700
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.12E022130
  CLC： R737.9
- Published：25 January 2024，
  
  Received：26 December 2022，
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林明利,郑美玲,卢宏全等.circDNMT1调节miR-377-3p/PUM1轴对三阴性乳腺癌细胞增殖、凋亡和上皮-间质转化的影响[J].局解手术学杂志,2024,33(01):1-7.

LIN Ming-li,ZHENG Mei-ling,LU Hong-quan,et al.Effect of circDNMT1 regulating miR-377-3p/PUM1 axis on proliferation,apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(01):1-7.
林明利,郑美玲,卢宏全等.circDNMT1调节miR-377-3p/PUM1轴对三阴性乳腺癌细胞增殖、凋亡和上皮-间质转化的影响[J].局解手术学杂志,2024,33(01):1-7. DOI： 10.11659/jjssx.12E022130.

LIN Ming-li,ZHENG Mei-ling,LU Hong-quan,et al.Effect of circDNMT1 regulating miR-377-3p/PUM1 axis on proliferation,apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(01):1-7. DOI： 10.11659/jjssx.12E022130.

摘要

目的

探讨circDNMT1调节miR-377-3p/PUM1轴对三阴性乳腺癌（TNBC）细胞增殖、凋亡和上皮-间质转化（EMT）的影响。

方法

收集儋州市人民医院2018年至2021年收治的24例TNBC患者的TNBC组织和其邻近正常组织，采用qRT-PCR、Western blot法分别检测组织及小鼠正常乳腺上皮细胞系HC11和TNBC细胞系4T1、Eph41424、JC中circDNMT1表达、miR-377-3p表达、PUM1蛋白表达。将4T1细胞分为4T1组（未转染）、si-NC组（转染si-NC）、si-DNMT1组（转染si-DNMT1）、si-DNMT1+anti-NC组（同时转染si-DNMT1和anti-NC）、si-DNMT1+anti-miR-377-3p组（同时转染si-DNMT1和anti-miR-377-3p），qRT-PCR检测各组4T1细胞中circDNMT1、miR-377-3p表达；CCK-8检测各组4T1细胞增殖情况；流式细胞术检测各组4T1细胞凋亡情况；Western blot检测各组4T1细胞PUM1、EMT相关蛋白及凋亡相关蛋白表达；TargetScan网站预测miR-377-3p与circDNMT1、PUM1的结合位点，双荧光素酶报告验证miR-377-3p与circDNMT1、PUM1的靶向关系。BALB/c小鼠接种4T1细胞后随机分为空白对照组（注射等量生理盐水）、阴性对照组（尾静脉注射si-NC）、DNMT1沉默组（尾静脉注射si-DNMT1）、联合对照组（尾静脉注射si-DNMT1和anti-NC）、联合沉默组（尾静脉注射si-DNMT1和anti-miR-377-3p），记录小鼠肿瘤质量，采用苏木精-伊红染色观察肿瘤形态变化。

结果

在TNBC组织和细胞中circDNMT1、PUM1上调，miR-377-3p下调，且在4T1细胞中表达差异最显著，因此本研究选择4T1细胞进行后续实验。与4T1组、si-NC组比较，si-DNMT1组4T1细胞中miR-377-3p表达、4T1细胞凋亡率及Bax、cleaved caspase-3、E-cadherin蛋白表达水平显著升高（

0.05），circDNMT1水平、PUM1蛋白表达水平、24 h和48 h OD值、Bcl-2蛋白表达水平、N-cadherin蛋白表达水平、Vimentin蛋白表达水平均显著减少/降低（

0.05）；与si-DNMT1组、si-DNMT1+anti-NC组比较，si-DNMT1+anti-miR-377-3p组4T1细胞中miR-377-3p表达、4T1细胞凋亡率、Bax蛋白表达水平、cleaved caspase-3蛋白表达水平、E-cadherin蛋白表达水平显著降低（

0.05），PUM1蛋白表达水平、24 h和48 h OD值、Bcl-2蛋白表达水平、N-cadherin蛋白表达水平、Vimentin蛋白表达水平均显著增加/升高（

0.05）。与miR-NC+WT-circDNMT1组比较，miR-377-3p mimic+WT-circDNMT1组细胞荧光素酶活性显著降低（

0.05）；与miR-NC+WT-PUM1组比较，miR-377-3p mimic+WT-PUM1组细胞荧光素酶活性显著降低（

0.05）。空白对照组、阴性对照组小鼠肿瘤细胞排列密集，边界清晰；DNMT1沉默组、联合对照组肿瘤细胞排列疏松，胞核固缩，细胞碎片增多；联合沉默组小鼠肿瘤细胞排列比较密集，边界趋于清晰。与空白对照组、阴性对照组比较，DNMT1沉默组小鼠肿瘤质量显著减少（

0.05）；与DNMT1沉默组、联合对照组比较，联合沉默组小鼠肿瘤质量显著增加（

0.05）。

结论

沉默circDNMT1可能通过上调miR-377-3p来抑制PUM1表达，进而抑制TNBC细胞增殖和EMT，促进细胞凋亡。

Abstract

Objective

To investigate the effects of circDNMT1 on the proliferation，apoptosis and epithelial-mesenchymal transition （EMT） of triple-negative breast cancer （TNBC） cells by regulating miR-377-3p/PUM1 axis.

Methods

The TNBC tissues and adjacent normal tissues of 24 patients with TNBC treated in Danzhou People's Hospital from 2018 to 2021 were collected. qRT-PCR and Western blot were used to detect the expression of circDNMT1，miR-377-3p，and PUM1 protein in tissue and mouse normal breast epithelial cell line HC11 and TNBC cell lines 4T1，Eph41424，and JC. 4T1 cells were divided into the 4T1 group （untransfected），the si-NC group （transfected with si-NC），the si-DNMT1 group （transfected with si-DNMT1），the si-DNMT1+anti-NC group （simultaneously transfected with si-DNMT1 and anti-NC），and the si-DNMT1+anti-miR-377-3p group （simultaneously transfected with si-DNMT1 and anti-miR-377-3p）. qRT-PCR was used to detect the expression of circDNMT1 and miR-377-3p of 4T1 cells in each group； CCK-8 was used to detect the proliferation of 4T1 cells in each group； flow cytometry was used to detect the apoptosis of 4T1 cells in each group； Western blot was used to detect the expression of PUM1，EMT-related proteins and apoptosis-related proteins of 4T1 cells in each group； TargetScan website was used to predict the binding sites of miR-377-3p with circDNMT1 and PUM1； dual luciferase report was used to verify the targeting relationships of miR-377-3p with circDNMT1 and PUM1. After inoculation with 4T1 cells，BALB/c mice were randomly divided into the blank control group （injected with equal amount of normal saline），the negative control group （injected with si-NC via tail vein），the DNMT1 silencing group （injected with si-DNMT1 via tail vein），the combined control group （injected with si-DNMT1 and anti-NC via tail vein），and the combined silencing group （injected with si-DNMT1 and anti-miR-377-3p via tail vein），the tumor massess of mice were recorded and the morphological changes of tumors were observed by hematoxylin-eosin staining.

Results

circDNMT1 and PUM1 were up-regulated in TNBC tissues and cells，and miR-377-3p was down-regulated.The expression difference was most obvious in 4T1 cells，so 4T1 cells were selected for subsequent experiments. Compared with the 4T1 group and the si-NC group，the expression of miR-377-3p，the apoptosis rate of 4T1 cells，the expression levels of Bax，cleaved caspase-3 and E-cadherin protein of 4T1 cells in the si-DNMT1 group were significantly increased （

0.05），the circ-DNMT1 level，the expression level of PUM1 protein，OD values at 24 hours and 48 hours，the expression level of Bcl-2，N-cadherin，Vimentin protein were significantly decreased （

0.05）. Compared with the si-DNMT1 group and the si-DNMT1+anti-NC group，the expression of miR-377-3p，the apoptosis rate of 4T1 cells，the expression levels of Bax，cleaved caspase-3 and E-cadherin proteins of 4T1 cells in the si-DNMT1+anti-miR-377-3p group were significantly decreased （

0.05），the expression level of PUM1 protein，OD values at 24 hours and 48 hours，the expression levels of Bcl-2，N-cadherin，Vimentin proteins were significantly increased （

0.05）. Compared with the miR-NC+WT-circDNMT1 group，the cell luciferase activity in the miR-377-3p mimic+WT-circDNMT1 group was significantly decreased （

0.05）； compared with the miR-NC+WT-PUM1 group，the cell luciferase activity in the miR-377-3p mimic+WT-PUM1 group was significantly decreased （

0.05）. The tumor cells in the blank control group and the negative control group were densely arranged with clear boundary； the tumor cells in the DNMT1 silencing group and the combined control group were loosely arranged，the nuclei were pyknotic，and the cell fragments were increased； the tumor cells in the combined silencing group were densely arranged and the boundaries tended to be clear. Compared with the blank control group and the negative control group，the tumor mass of mice in the DNMT1 silencing group was significantly decreased （

0.05）. Compared with the DNMT1 silencing group and the combined control group，the tumor mass of mice in the combined silencing group was significantly increased （

0.05）.

Conclusion

Silencing circDNMT1 may inhibit the expression of PUM1 by up-regulating miR-377-3p，thereby inhibiting the proliferation and EMT of TNBC cells，and promoting cell apoptosis.

关键词

circDNMT1miR-377-3p三阴性乳腺癌细胞增殖细胞凋亡上皮-间质转化

Keywords

circDNMT1miR-377-3ptriple-negative breast cancercell proliferationcell apoptosisepithelial-mesenchymal transition

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