Effects of LINC00839 on proliferation，apoptosis，and migration of colorectal cancer cells by regulating the miR-17-5p/WEE1 axis

doi:10.3877/cma.j.issn.2095-2015.2024.06.003

Abstract

Abstract:

Objective

To investigate the effects of long non-coding RNA LINC00839 on the proliferation，apoptosis，and migration of colorectal cancer （CRC） cells by regulating the microRNA-17-5p（miR-17-5p）/serine/threonine protein kinase 1 （WEE1） axis.

Methods

The expression levels of LINC00839，miR-17-5p，and WEE1 mRNA in CRC cell lines were detected to screen the optimal cell line.LS513 cells were separated into Control group，LINC00839 knockdown negative control group （si-NC group），LINC00839 knockdown group （si-LINC00839 group），LINC00839 knockdown+miR-17-5p inhibition negative control group （si-LINC00839+NC inhibitor group），and LINC00839 knockdown+miR-17-5p inhibitor expression group （si-LINC00839+miR-17-5p inhibitor group）. qRT-PCR was applied to detect the expression levels of LINC00839，miR-17-5p，and WEE1 mRNA in cells. Dual luciferase reporter gene experiment was applied to verify the targeting relationship between LINC00839，miR-17-5p，and WEE1. MTT was applied to detect cell viability. Edu was applied to detect cell proliferation. Scratch experiment was applied to detect the migration ability of cells. Flow cytometry was applied to detect cell apoptosis. Western blot was applied to detect protein levels of WEE1，PCNA，Bax，Bcl-2，caspase-3，E-cadherin，and N-cadherin.

Results

In the CRC cell line，LINC00839 and WEE1 mRNA were highly expressed，while miR-17-5p was low expressed. LS513 cells were selected for the experiment. Dual luciferase reporter gene experiment confirmed a targeted relationship between miR-17-5p and LINC00839，WEE1. Knocking down LINC00839 up-regulated miR-17-5p expression and down-regulated WEE1 expression. Knocking down LINC00839 greatly reduced LS513 cell viability，Edu positive rate，scratch healing rate，and expression of PCNA，Bcl-2，N-cadherin，and WEE1 proteins （P＜0.05），while greatly increased the expression of Bax，caspase-3，and E-cadherin proteins （P＜0.05）. Inhibiting the expression of miR-17-5p was able to reverse the inhibitory effect of LINC00839 knockdown on the malignant biological behavior of LS513 cells （P＜0.05）.

Conclusion

LINC00839 is up-regulated in CRC cells，and knocking down LINC00839 may inhibit CRC cell proliferation and migration by regulating the miR-17-5p/WEE1 axis，promoting cell apoptosis.

Key words: Colorectal cancer, Long non-coding RNA LINC00839, MicroRNA-17-5p/serine/threonine protein kinase 1 axis, Proliferation, Apoptosis, Migration

Guoqiang Wang, Gang Zhang, Jianpo Tang, Yuguo Zhang, Yongjiang Yang. Effects of LINC00839 on proliferation，apoptosis，and migration of colorectal cancer cells by regulating the miR-17-5p/WEE1 axis[J]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2024, 14(06): 491-499.

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URL: https://zhxhbyyxzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-2015.2024.06.003

https://zhxhbyyxzz.cma-cmc.com.cn/EN/Y2024/V14/I06/491

Figures/Tables 15

基因	上游（5'-3'）	下游（5'-3'）
LINC00839	GCCCATACGGACCTACTGC	CAATCTGGGCCTGAACTGTG
miR-17-5p	CGGCGGCAAAGTGCTTACAG	GTGCAGGGTCCGAGGT
WEE1	GCTTGCCCTCACAGTGGTATG	CCGAGGTAATCTACCCTGTCT
GAPDH	TGCACCACCAACTGCTTAGC	GGCATGGACTGTGGTCATGAG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

细胞	LINC00839/GAPDH	miR-17-5p/U6	WEE1 mRNA
CCD-18Co	0.99±0.06	1.03±0.09	1.00±0.08
HCT116	1.37±0.12^a	0.51±0.04^a	1.63±0.14^a
LS513	2.13±0.19^a	0.27±0.01^a	3.47±0.29^a
LoVo	1.85±0.17^a	0.62±0.05^a	2.15±0.23^a
HT29	1.96±0.20^a	0.49±0.03^a	2.94±0.27^a
SW480	1.57±0.12^a	0.85±0.07^a	1.76±0.18^a
F值	46.467	148.011	110.324
P值	＜0.001	＜0.001	＜0.001

组别	LINC00839-WT	LINC00839-MUT	WEE1-WT	WEE1-MUT
miR-NC组	1.00±0.09	1.02±0.13	1.01±0.09	0.98±0.07
miR-17-5p mimics组	0.47±0.03^a	0.96±0.07	0.52±0.04^a	0.91±0.08
t值	13.685	0.995	12.187	1.613
P值	＜0.001	0.343	＜0.001	0.138

组别	LINC00839	miR-17-5p	WEE1 mRNA	WEE1
Control组	1.03±0.09	0.98±0.07	1.01±0.12	1.12±0.10
si-NC组	0.99±0.07	0.95±0.09	1.03±0.10	1.08±0.09
si-LINC00839组	0.45±0.03^a	1.77±0.12^a	0.32±0.02^a	0.53±0.06^a
si-LINC00839+NC inhibitor组	0.47±0.03	1.82±0.15	0.45±0.04	0.42±0.05
si-LINC00839+miR-17-5p inhibitor组	0.43±0.05	0.74±0.09^b	1.46±0.13^b	0.85±0.07^b
F值	163.838	131.674	150.852	103.247
P值	＜0.001	＜0.001	＜0.001	＜0.001

组别	细胞活力（OD值）	Edu阳性率（%）
Control组	0.89±0.07	47.41±3.06
si-NC组	0.95±0.08	44.58±2.72
si-LINC00839组	0.56±0.04^a	26.29±1.53^a
si-LINC00839+NC inhibitor组	0.53±0.03	28.37±1.14
si-LINC00839+miR-17-5p inhibitor组	0.72±0.05^b	34.65±2.07^b
F值	65.798	108.696
P值	＜0.001	＜0.001

组别	细胞凋亡率（%， $\bar{x}$ ±s）
Control组	4.13±0.68
si-NC组	6.72±1.05
si-LINC00839组	24.58±2.96^a
si-LINC00839+NC inhibitor组	22.36±2.14
si-LINC00839+miR-17-5p inhibitor组	15.49±1.73^b
F值	139.244
P值	＜0.001

组别	细胞凋亡率（%， $\bar{x}$ ±s）
Control组	4.13±0.68
si-NC组	6.72±1.05
si-LINC00839组	24.58±2.96^a
si-LINC00839+NC inhibitor组	22.36±2.14
si-LINC00839+miR-17-5p inhibitor组	15.49±1.73^b
F值	139.244
P值	＜0.001

组别	划痕愈合率（%， $\bar{x}$ ±s）
Control组	73.42±6.51
si-NC组	70.88±5.43
si-LINC00839组	31.96±2.25^a
si-LINC00839+NC inhibitor组	35.07±3.19
si-LINC00839+miR-17-5p inhibitor组	54.34±3.87^b
F值	110.394
P值	＜0.001

组别	划痕愈合率（%， $\bar{x}$ ±s）
Control组	73.42±6.51
si-NC组	70.88±5.43
si-LINC00839组	31.96±2.25^a
si-LINC00839+NC inhibitor组	35.07±3.19
si-LINC00839+miR-17-5p inhibitor组	54.34±3.87^b
F值	110.394
P值	＜0.001

组别	PCNA	Bax	Bcl-2	caspase-3	E-cadherin	N-cadherin
Control组	0.94±0.08	0.26±0.03	1.15±0.10	0.53±0.03	0.32±0.03	1.04±0.09
si-NC组	0.87±0.06	0.22±0.02	1.08±0.08	0.49±0.02	0.27±0.02	1.10±0.11
si-LINC00839组	0.35±0.02^a	0.84±0.06^a	0.56±0.04^a	1.07±0.09^a	0.61±0.05^a	0.46±0.05^a
si-LINC00839+NC inhibitor组	0.39±0.03	0.92±0.10	0.63±0.05	0.98±0.07	0.67±0.06	0.41±0.04
si-LINC00839+miR-17-5p inhibitor组	0.61±0.05^b	0.45±0.05^b	0.82±0.07^b	0.74±0.06^b	0.35±0.04^b	0.80±0.06^b
F值	143.413	182.448	81.579	113.413	110.933	109.699
P值	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001

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