Effect of temozolomide on cell proliferation in TJ905 glioma stem cells with Livin over-expression

doi:10.3877/cma.j.issn.2095-123X.2020.02.008

Abstract

Abstract:

Objective

To analyze the effect of cell proliferation after temozolomide (TMZ) intervention in TJ905 cells and stem cells with Livin gene overexpression. To explore the mechanism for TMZ to Livin and Caspase-3/7 in glioma TJ905 cells and stem cells.

Methods

CD133 positive TJ905 glioma cells were separated by immunomagnetic beads to isolate glioma stem cells, to establish the overexpression cell models of Livin gene with lentivirus transfection technology. Livin over-expression cell models were intervened by different concentration of TMZ for 48 h. Cell counting kit-8 assay, flow cytometry, reverse transcription-PCR were used to analyze cell cycle to investigate cell proliferation and to detect the expression of Livin and Caspase-3/7 respectively, following treatment with various concentrations of TMZ (0, 25, 50, 100, 200 and 400 μmol/L) for 48 h.

Results

(1)The expression of Livin gene in glioma TJ905 cells was significantly higher than that of TJ905 stem cells (P<0.05); (2)TMZ could inhibit the proliferation of glioma TJ905 cells and stem cells, and the inhibitory effect increased with the increase of drug concentration; (3)TMZ could inhibit the expression of Livin gene and induce the expression of Caspase- 3/7. With the increase of drug concentration, the inhibitory effect gradually increased, and the difference was statistically significant (P<0.05); (4)After Livin gene transfection, the cell cycle G0/G1 of TJ905 stem cellsincreased significantly, while TJ905 cell cycle increased slightly in G2/M phase. after TMZ intervention, TJ905 stem cell overexpression group stopped in S and G2/M phase, while that in control group was in S phase. TJ905 cellsoverexpression group stopped in S phase in the overexpression group, while G2/M in the control group was induced by low concentration, the induction effect was weakened with the increase of drug concentration, and the differences were statistically significant (P<0.05).

Conclusion

TMZ can inhibit the expression of Livin gene, induce the expression of Caspase-3/7, and regulate the changes of cell cycle to reduce the proliferation activity and promote apoptosis.

Key words: Livin, Caspase-3/7, TJ905 stem cells, Temozolomide, Cell proliferation

Genhua Li, Song Feng, Guangkui Han, Wenyuan Ma, Chen Liu, Lingsheng Kong, Feng Jin. Effect of temozolomide on cell proliferation in TJ905 glioma stem cells with Livin over-expression[J]. Chinese Journal of Brain Diseases and Rehabilitation(Electronic Edition), 2020, 10(02): 99-105.

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Figures/Tables 8

References 15

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基因	引物序列
Homo-Livin	Forward：5 ’-GCTGTCAGTTCCTGCTCCGGTC-3 ’
	Reverse：5 ’-CAGGGGCTGCGTCTTCCGGTTC-3 ’
Homo-Caspase-3	Forward：5 ’-GAAGCGAATCAATGGACTCTGG-3 ’
	Reverse：5 ’-GTTTGCTGCATCGACATCTGTAC-3 ’
Homo-Caspase-7	Forward：5 ’-CCTCGTTTGTACCGTCCCTCTT-3 ’
	Reverse：5 ’-GGCATCTTTGTCTGTTCCGTTTC-3 ’
Homo-GAPDH	Forward：5 ’-GGAGCGAGATCCCTCCAAAAT-3 ’
	Reverse：5 ’-GGCTGTTGTCATACTTCTCATGG-3 ’

基因	引物序列
Homo-Livin	Forward：5 ’-GCTGTCAGTTCCTGCTCCGGTC-3 ’
	Reverse：5 ’-CAGGGGCTGCGTCTTCCGGTTC-3 ’
Homo-Caspase-3	Forward：5 ’-GAAGCGAATCAATGGACTCTGG-3 ’
	Reverse：5 ’-GTTTGCTGCATCGACATCTGTAC-3 ’
Homo-Caspase-7	Forward：5 ’-CCTCGTTTGTACCGTCCCTCTT-3 ’
	Reverse：5 ’-GGCATCTTTGTCTGTTCCGTTTC-3 ’
Homo-GAPDH	Forward：5 ’-GGAGCGAGATCCCTCCAAAAT-3 ’
	Reverse：5 ’-GGCTGTTGTCATACTTCTCATGG-3 ’

TMZ浓度	ACC		CSC
TMZ浓度	CON	OE	CON	OE
0 μmol/L（Control）	1.202±0.045	1.347±0.068	0.650±0.020	0.753±0.018
25 μmol/L	1.157±0.065	1.263±0.066	0.635±0.019	0.673±0.021^a
50 μmol/L	1.033±0.063^a	1.150±0.079^a	0.528±0.016^a	0.555±0.012^a
100 μmol/L	0.901±0.032^a	1.121±0.098^a	0.453±0.011^a	0.474±0.021^a
200 μmol/L	0.863±0.022^a	1.094±0.082^a	0.437±0.021^a	0.445±0.014^a
400 μmol/L	0.816±0.021^a	0.982±0.098^a	0.383±0.019^a	0.403±0.017^a
F值	32.642	6.895	111.962	188.866
P值	<0.001	0.003	<0.001	<0.001

TMZ浓度	ACC		CSC
TMZ浓度	CON	OE	CON	OE
0 μmol/L（Control）	1.202±0.045	1.347±0.068	0.650±0.020	0.753±0.018
25 μmol/L	1.157±0.065	1.263±0.066	0.635±0.019	0.673±0.021^a
50 μmol/L	1.033±0.063^a	1.150±0.079^a	0.528±0.016^a	0.555±0.012^a
100 μmol/L	0.901±0.032^a	1.121±0.098^a	0.453±0.011^a	0.474±0.021^a
200 μmol/L	0.863±0.022^a	1.094±0.082^a	0.437±0.021^a	0.445±0.014^a
400 μmol/L	0.816±0.021^a	0.982±0.098^a	0.383±0.019^a	0.403±0.017^a
F值	32.642	6.895	111.962	188.866
P值	<0.001	0.003	<0.001	<0.001

TMZ浓度	ACC-OE	CSC-OE	t值	P值	ACC-CON	CSC-CON	t值	P值
0 μmol/L（Control）	564.113±21.951	84.315±1.808	-37.732	<0.001	245.777±14.176^b	27.025±3.895^b	-25.773	<0.001
25 μmol/L	391.114±6.444^a	59.191±2.032^a	-85.083	<0.001	124.651±8.176^a	12.366±1.684^a	-23.297	<0.001
50 μmol/L	370.328±11.541^a	58.190±1.582^a	-46.410	<0.001	100.874±10.516^a	8.502±1.286^a	-15.101	<0.001
100 μmol/L	315.832±10.691^a	46.150±2.042^a	-42.917	<0.001	82.033±7.470^a	7.842±0.524^a	-17.159	<0.001
200 μmol/L	223.033±9.489^a	38.270±1.779^a	-33.131	<0.001	59.741±6.982^a	7.710±0.454^a	-12.881	<0.001
400 μmol/L	169.745±8.608^a	29.555±2.523^a	-27.068	<0.001	28.807±3.895^a	6.694±0.697^a	-9.691	0.001
F值	373.502	283.050			206.544	52.685
P值	<0.001	<0.001			<0.001	<0.001

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