Establishment of the Wilms’ tumor gene 1 low and over-expression models in breast cancer cells

doi:10.3877/cma. j. issn.1674-0807.2013.04.001

Abstract

Abstract:

Objective

To construct small interfering RNA (siRNA) and double strands RNA(dsRNA) by RNA interference (RNA interference, RNAi) and RNA activation (RNA activating, RNAa),and establish the low and over-expression model of Wilms’ tumor gene 1 (WT1) in breast cancer cells.

Methods

Three sequences (siRNA-516, siRNA-803 and siRNA-1029) established by foreign scholars were adopted to construct WT1 siRNA; one sequence (dsRNA-319)which demonstrated to be able to up-regulate WT1 expression was adopted to construct WT1 dsRNA. siRNA and dsRNA were transfected into MDA-MB-321 and MCF-7 cells by Lipofectamine^TM 2000,respectively. WT1 siRNA screening experiment contained six groups:WT1 siRNA-516, WT1 siRNA-803, WT1 siRNA-1029, negative control, transfection reagent and blank groups, and the points of time for observation were at 24, 48 and 72 h after transfection, respectively. WT1 dsRNA screening experiments contained three groups: WT1 dsRNA-319, negative control and blank group, and the point of time for observation was at 48, 72 and 96 h after transfection respectively. Quantitative real-time PCR(qRT-PCR)and Western Blotting were performed to select siRNA and dsRNA with obvious impacts on WT1 expression. The one-way ANOVA was used for statistical analysis.

Results

Three WT1 siRNAs (WT1 siRNA-516, WT1 siRNA-803 and WT1 siRNA-1029) were successfully constructed and transfected into MDAMB-231 cells with transfection efficiency ＞90%. WT1 siRNAs could effectively inhibit the expression of WT1 mRNA and protein, among which siRNA-1029 could inhibit the WT1 mRNA expression at 48 h after transfection most significantly (0.49±0.02 for WT1 siRNA-1029 group vs 1.00±0.08 for blank group,P=0.00;the protein expression was also decreased dramatically). WT1 dsRNA-319 could increase the expression of WT1 mRNA in MCF-7 cell with transfection efficiency ＞90%. The most significant impact was achieved in 50 μmol/L WT1 dsRNA-319 group at 96 h after transfection (319.06±14.84 for WT1 dsRNA-319 group vs 1.00±0.07 for blank group, P=0.00; the protein expression was also increased remarkably).

Conclusion

The low expression WT1 model in MDA-MB-231 cells and the over-expression WT1 model in MCF-7 cells are successfully established, which provides a basis for subsequent study on WT1 biological behaviors in breast cancer.

Key words: Wilms’ tumor gene 1, RNA interference, RNA activating, Breast neoplasms

Xiao-wei QI, Xin-hua YANG, Yi ZHANG, Lin-jun FAN, Fan ZHANG, Li CHEN, Zhen-ning TANG, Yan ZHANG, Xiao-ning YANG, Bei-ge ZONG, Bao-quan HU, Ming-hao WANG, Jun JIANG. Establishment of the Wilms’ tumor gene 1 low and over-expression models in breast cancer cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2013, 07(04): 238-244.

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https://zhrxbzz.cma-cmc.com.cn/EN/Y2013/V07/I04/238

Figures/Tables 9

References 21

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siRNA	靶点	序列	荧光标记
WT1 siRNA-516	外显子4	5′-GACAAUUUAUACCAAAUGATT-3′	FAM
WT1 siRNA-803	外显子7	5′-GCUGUCCCACUUACAGAUGdTdT-3′	FAM
WT1 siRNA-1029	外显子10	5′-AAGCCCUUCAGCUGUCGGUdTdT-3′	FAM
WT1 siRNA-NC		5′-CGUACGCGGAAUACUUCGAdTdT-3′	FAM

siRNA	靶点	序列	荧光标记
WT1 siRNA-516	外显子4	5′-GACAAUUUAUACCAAAUGATT-3′	FAM
WT1 siRNA-803	外显子7	5′-GCUGUCCCACUUACAGAUGdTdT-3′	FAM
WT1 siRNA-1029	外显子10	5′-AAGCCCUUCAGCUGUCGGUdTdT-3′	FAM
WT1 siRNA-NC		5′-CGUACGCGGAAUACUUCGAdTdT-3′	FAM

dsRNA	序列	末端标记
WT1 dsRNA-319	5′-FGACUCACUGCUUACCUGAA[dT][dT] -3′	HPLC
WT1 dsRNA-319	5′-RUUCAGGUAAGCAGUGAGUC[dT][dT] -3′	HPLC
WT1 dsRNA-NC	5′-ACUACUGAG UGACAGUAGA[dT][dT] -3′	FAM
WT1 dsRNA-NC	5′-UCUACUGUCACUCAGUAGU[dT][dT] -3′	FAM

dsRNA	序列	末端标记
WT1 dsRNA-319	5′-FGACUCACUGCUUACCUGAA[dT][dT] -3′	HPLC
WT1 dsRNA-319	5′-RUUCAGGUAAGCAGUGAGUC[dT][dT] -3′	HPLC
WT1 dsRNA-NC	5′-ACUACUGAG UGACAGUAGA[dT][dT] -3′	FAM
WT1 dsRNA-NC	5′-UCUACUGUCACUCAGUAGU[dT][dT] -3′	FAM

组别	实验次数	WT1 mRNA 相对表达量
组别	实验次数	24 h^a	48 h^b	72 h^b
空白细胞组	3	1.00±0.08	1.00±0.08	1.00±0.01
脂质体组	3	0.94±0.04	0.94±0.04	0.85±0.02
阴性对照组	3	1.02±0.18	0.91±0.09	0.93±0.02
WT1 siRNA-516组	3	0.75±0.05	0.49±0.03^c	0.55±0.02^c
WT1 siRNA-803组	3	0.66±0.05	0.45±0.04^c	0.58±0.01^c
WT1 siRNA-1029组	3	0.71±0.06	0.49±0.02^c	0.70±0.01^c
F值		9.60	63.35	345.71
P值		0.00	0.00	0.00

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