沉默Ki-67基因对乳腺癌MCF-7/DOX细胞多柔比星耐药性的影响

doi:10.16139/j.1007-9610.2023.03.013

摘要/Abstract

摘要：

目的：研究沉默Ki-67基因对乳腺癌细胞多柔比星(doxorubicin，DOX)耐药性的影响。方法：研究采用MCF-7/DOX细胞和脂质体瞬时转染法，用小干扰RNA(small interfering RNA，siRNA)转染和阴性对照(negative control, NC)siRNA转染体外培养乳腺癌MCF-7细胞。转染48 h后采用反转录PCR（RT-PCR）检测Ki-67 mRNA的表达。后续实验用干扰效率最高的Ki- 67 siRNA。共分为3个组：MCF-7空白对照(CON组)、MCF-7 NC siRNA转染(NC组)和MCF-7 siRNA转染(siRNA组)。用RT-PCR和蛋白质印迹法检测3组细胞的Ki-67 mRNA表达和相关蛋白质表达。用不同浓度的DOX处理3组MCF-7/DOX细胞，MTT法检测细胞增殖。结果：特异性siRNA成功转染MCF-7/DOX细胞后，荧光显微镜下观察，见siRNA均匀分布在细胞质内，转染效率达85%以上。转染MCF-7 siRNA后，不同程度降低了MCF-7 /DOX细胞 Ki-67 mRNA表达，Ki-67蛋白的表达水平显著降低，抑制率为78.51%，与NC组和CON组比较，差异具有统计学意义(P<0.05)。MTT 检测结果示siRNA干扰Ki-67mRNA表达后，MCF-7/DOX细胞的体外增殖能力被显著抑制。siRNA组MCF-7/DOX细胞对DOX的半抑制浓度为(7.45±0.18) μmol/L，显著低于NC组(55.19±2.86) μmol/L和CON组(56.43±4.22) μmol/L，相对于CON组的耐药逆转倍数达7.69倍(P<0.05)。siRNA组不同浓度DOX对细胞的抑制作用显著高于NC组和CON组(P<0.05)。结论：siKi-67能有效抑制Ki-67基因的表达，抑制乳腺癌MCF-7/ DOX细胞增殖，以及部分逆转乳腺癌MCF-7/DOX细胞对DOX的耐药性。

关键词: 乳腺癌, Ki-67, 多柔比星, 凋亡, 耐药性

Abstract:

Objective To study the effect of Ki-67 gene silencing on doxorubicin (DOX) resistance in breast cancer MCF-7 cells. Methods Liposome transient transfection method and breast cancer MCF-7 cells cultured in vitro were used combined with the transfection of small interfering RNA (siRNA) and negative control (NC) siRNA. The expression of Ki-67 mRNA was detected by RT-PCR after 48 h of transfection. Ki-67 siRNA with the highest interference efficiency was used in subsequent experiments including three groups: control (CON) group without transfection, NC group transfected with MCF-7 negative control siRNA and siRNA group with MCF-7 transfected with Ki-67 siRNA. The expression of Ki-67 mRNA and related proteins in three groups were detected by RT-PCR and Western blot. MCF-7/DOX cells were treated with DOX at different concentrations. Proliferation of MCF-7/DOX cells was detected by MTT assay. Results After the specific siRNA was successfully transfected into MCF-7 /DOX cells, it was observed under fluorescence microscope that siRNA was evenly distributed in the cytoplasm, and transfection efficiency was more than 85%. Results of transfection of MCF-7 siRNA cells showed that Ki-67 mRNA expression in MCF-7 cells reduced Ki-67 at varying degrees. The expression of Ki-67 protein reduced significantly with the inhibition of 78.51% and the statistically significant difference compared with that in NC group and CON group (P < 0.05). MTT showed that the proliferation ability of MCF-7/DOX cells in vitro was inhibited significantly after Ki-67 mRNA expression interfered by siRNA. The half maximal inhibitory concentration (IC₅₀) of DOX to MCF-7/DOX cells in siRNA group was (7.45±0.18) μmol/L, lower significantly than (55.19±2.86) μmol/L in NC group and (56.43±4.22) μmol/L in CON group. The reversal of drug resistance was 7.69 times compared with that in CON group (P < 0.05). The inhibitory effect of DOX at different concentrations on the cells in siRNA group was significantly higher than that in NC group and CON group with statistically significant difference (P < 0.05). Conclusions It was indicated that siKi-67 could inhibit both the expression of Ki-67 gene effectively and the proliferation of MCF-7/DOX cells, and partially reverse the DOX resistance of MCF-7/DOX cells.

Key words: Breast cancer, Ki-67, Doxorubicin, Apoptosis, Resistance

中图分类号:

R737.9

董军, 崔凤鸣, 刘军. 沉默Ki-67基因对乳腺癌MCF-7/DOX细胞多柔比星耐药性的影响[J]. 外科理论与实践, 2023, 28(03): 254-259.

DONG Jun, CUI Fengming, LIU Jun. Effects of silencing Ki-67 gene on doxorubicin resistance of breast cancer MCF-7/DOX cells[J]. Journal of Surgery Concepts & Practice, 2023, 28(03): 254-259.

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参考文献 14

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Gene	Primer location	Primer sequence（5′-3′）
Ki-67	Upstream	ACTCCAGTTGCCAGTGAT
Ki-67	Downstream	GACTTCGGCTGATAGACA
β-Actin	Upstream	GTGGGGCGCCCCAGGCACCA
β-Actin	Downstream	CTCCTTAATGTCACGCACGATTTC

Group	24 h	48 h	72 h
CON group	0.448±0.045	0.616±0.018	0.724±0.032
NC group	0.456±0.023	0.600±0.018	0.705±0.024
siRNA group	0.365±0.036^a)b)	0.250±0.031^a)b)	0.214±0.023^a）b)
P value	<0.05	<0.05	<0.05

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