Effects of silencing Ki-67 gene on doxorubicin resistance of breast cancer MCF-7/DOX cells

Abstract

Abstract:

ObjectiveTo study the effect of Ki-67 gene silencing on doxorubicin (DOX) resistance in breast cancer MCF-7 cells.MethodsLiposome transient transfection method and breast cancer MCF-7 cells culturedin vitrowere 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.ResultsAfter 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%.Resultsof 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 cellsin vitrowas 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).ConclusionsIt 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

CLC Number:

R737.9

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.

Figures/Tables8

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

Tab 1

Fig 1

Fig 2

Fig 3

Fig 4

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)
Pvalue	<0.05	<0.05	<0.05

Tab 2

Fig 5

Fig 6

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