Effects of miR-661 on proliferation, migration and invasion of SW620 cells by targeting HRTA1

doi:10.3877/cma.j.issn.2095-1221.2021.03.003

Abstract

Abstract:

Objective

To investigate the effect of miR-661 on the proliferation, migration and invasion of colon cancer cells by targeting high temperature requirement A1 (HTRA1) .

Methods

The colon cancer tissue and their paired para-carcinoma colon tissue of 85 patients in People's Hospital of Langfang City from March 2016 to March 2018 were collected. The expression level of miR-661 and HTRA1 mRNA in colon cancer tissue, para-carcinoma colon tissue, colon cancer cells (SW620, SW480, HCT116, LoVo) and colonic epithelial cells (NCM460) were detected by real-time fluorescence quantitative PCR (RT-qPCR) and the expression level of HTRA1 protein was detected by Western blot. The SW620 cells were divided into anti-miR-NC group (transfected with anti-miR-NC) , anti-miR-661 group (transfected with anti-miR-661) , pcDNA group (transfected with pcDNA) , pcDNA-HTRA1 group (transfected with pcDNA-HTRA1) , anti-miR-661+si-NC group (transfected with anti-miR-661 and si-NC) , and anti-miR-661+si-HTRA1 group (transfected with anti-miR-661 and si-HTRA1) and control (Con) group. The relationship between miR-661 and HTRA1 was determined by Target Scan online prediction, luciferase reporter gene experiments, and Western blot. The proliferation, migration and invasion ability of SW620 cells were detected by CCK-8 method and Transwell test. The independent sample t test was used to compare the two groups, and the One-way ANOVA was used to compare multiple groups. The relationship between miR-661 or HTRA1 expression and clinicopathological parameters was analyzed by c² test.

Results

Compared with para-carcinoma colon tissue, the expression of miR-661 in colon cancer tissues was increased, while the expression of HTRA1 mRNA and protein were significantly decreased (P < 0.05) . Compared with NCM460 cells, the expression of miR-661 in SW620, SW480, HCT116, LoVo cells were increased, while the expression of HTRA1 mRNA and protein in these cells were significantly decreased (P < 0.05) . miR-661 may negatively regulate HTRA1 expression. Compared with the Con group and anti-miR-NC group, cell proliferation activity (0.49±0.04 vs 0.76±0.06, 0.72±0.07, P < 0.05) , migration rate (48.93±5.28 vs 98.67±8.57, 95.14±9.36, P < 0.05) and invasion ability (36.48±4.87 vs 88.48±8.64, 81.65±8.36, P < 0.05) of SW620 cells in the anti-miR-661 group were decreased, but there was no significantly difference between Con group and anti-miR-NC group (P > 0.05) . Compared with the Con group and pcDNA group, cell proliferation activity (0.55±0.05 vs 0.76±0.06, 0.74±0.07, P < 0.05) , migration rate (56.87±5.32 vs 101.65±9.87, 98.69±9.33, P < 0.05) and invasion ability (43.28±4.37 vs 87.21±8.98, 85.91±8.42, P < 0.05) of SW620 cells in the pcDNA-HTRA1 group were also decreased (P < 0.05) , but there was no significantly difference between Con group and anti-miR-NC group (P > 0.05) . Compared with the anti-miR-661+si-NC group, the cell proliferation activity (0.48±0.04 vs 0.62±0.06, P < 0.05) , migration rate (47.58±4.72 vs 82.36±8.21, P < 0.05) and invasion ability (36.98±3.66 vs 71.54±7.16, P < 0.05) of SW620 cells in the anti-miR-661+si-HTRA1 group were increased (P < 0.05) .

Conclusions

miR-661 was highly expressed and HTRA1 was lowly expressed in colon cancer. Inhibiting miR-661 may up-regulate HTRA1 and then inhibit the proliferation, migration and invasion of colon cancer cells.

Key words: miR-661, HTRA1, Colon cancer, Cell proliferation, Migration, Invasion

Feng Zhong, Xiaocheng Zhang. Effects of miR-661 on proliferation, migration and invasion of SW620 cells by targeting HRTA1[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(03): 146-154.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2021.03.003

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Figures/Tables 17

References 19

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基因	序列（5'→3'）	预期扩增产物长度（bp）
miR-661	上游GTGCCTGGGTCTCTGGCCT	72
miR-661	下游GTGCCTGGGTCTCTGGCCT	72
U6（内参）	上游CTCGCTTCGGCAGCACA	94
U6（内参）	下游AACGCTTCACGAATTTGCGT	94
HTRA1	上游CAGCACCCAAAGGTCAATGC	113
HTRA1	下游CAGAGTCATCGTGCTCCCAA	113
GAPDH	上游ACCACAGTCCATGCCATCAC	450
GAPDH	下游TCCACCACCCTGTTGCTGTA	450

基因	序列（5'→3'）	预期扩增产物长度（bp）
miR-661	上游GTGCCTGGGTCTCTGGCCT	72
miR-661	下游GTGCCTGGGTCTCTGGCCT	72
U6（内参）	上游CTCGCTTCGGCAGCACA	94
U6（内参）	下游AACGCTTCACGAATTTGCGT	94
HTRA1	上游CAGCACCCAAAGGTCAATGC	113
HTRA1	下游CAGAGTCATCGTGCTCCCAA	113
GAPDH	上游ACCACAGTCCATGCCATCAC	450
GAPDH	下游TCCACCACCCTGTTGCTGTA	450

	癌旁组织	结肠癌组织	± Sd	t值	P值
miR-661	1.00±0.10	3.14±0.36	2.14±0.28	-52.374	< 0.001
HTRA1 mRNA	1.00±0.07	0.55±0.05	0.45±0.06	44.191	< 0.001
HTRA1蛋白	0.71±0.07	0.49±0.04	0.22±0.05	25.499	< 0.001

	癌旁组织	结肠癌组织	± Sd	t值	P值
miR-661	1.00±0.10	3.14±0.36	2.14±0.28	-52.374	< 0.001
HTRA1 mRNA	1.00±0.07	0.55±0.05	0.45±0.06	44.191	< 0.001
HTRA1蛋白	0.71±0.07	0.49±0.04	0.22±0.05	25.499	< 0.001

临床病理特征		例数	miR-661		χ²值	P值	HTRA1		χ²值	P值
临床病理特征		例数	低表达（n=30）	高表达（n=55）	χ²值	P值	低表达（n=50）	高表达（n=35）	χ²值	P值
性别					0.353	0.552			1.586	0.208
	男	49	16	33			26	23
	女	36	14	22			24	12
年龄					0.035	0.851			2.209	0.137
	<55	38	13	25			19	19
	≥55	47	17	30			31	16
临床分期					17.872	0.001			43.540	0.000
	Ⅰ	21	14	7			3	18
	Ⅱ	16	8	8			4	12
	Ⅲa	19	3	16			17	2
	Ⅲb	17	3	14			15	2
	Ⅳ	12	2	10			11	1
分化程度					22.949	0.000			49.387	0.000
	高	22	19	3			2	20
	中	24	8	16			10	14
	低	39	3	36			38	1
淋巴结转移					44.350	0.000			52.545	0.000
	无	38	28	10			6	32
	有	47	2	45			44	3

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