Effects of CircCDR1as on the biological behavior of NSCLC cells by regulating the miR-671-5p/CBX4 axis

doi:10.11958/20221405

Abstract

Abstract:

Objective To investigate effects of CircRNA antisense cerebellar degeneration-related protein 1 transcript (CircCDR1as) on the proliferation, apoptosis, migration and invasion of non-small cell lung cancer (NSCLC) cells by regulating miR-671-5p/chromobox 4 (CBX4) axis. Methods Human NSCLC cell lines (NCI-H524, NCI-H1734, Calu-3 and A549) and human bronchial epithelioid cells (HBE) were cultured in vitro. A549 cells were randomly separated into the control group, the si-NC group, the si-CircCDR1as group, the si-CircCDR1as+anti-miR-NC group, the si-CircCDR1as+anti-miR-671-5p group, the pcDNA group, the CircCDR1as group, the miR-NC group, the miR-671-5p group, the miR-671-5p+pcDNA group, the miR-671-5p+CBX4 group, the anti-miR-NC group and the anti-miR-671-5p group. The expression levels of CircCDR1as, miR-671-5p and CBX4 mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of CBX4 was detected by Western blot assay. The cell viability, proliferation, apoptosis, migration and invasion were detected by tetramethylazolium salt (MTT), clonogenic assay, flow cytometry and Transwell, respectively. The targeting relationship between miR-671-5p and CircCDR1as or CBX4 was verified by dual-luciferase reporter assay. Results Compared with HBE cells, expression levels of CircCDR1as and CBX4 were increased and the expression of miR-671-5p was decreased in the four NSCLC cell groups (P<0.05). Compared with the si-NC group, the viability and the number of colonies of A549 cells were obviously decreased in the si-CircCDR1as group, the numbers of cell migration and invasion were decreased, and the apoptosis rate was increased (P<0.05). The down regulation of miR-671-5p, a target of CircCDR1as, attenuated the regulatory effect of CircCDR1as silencing on NSCLC progression (P<0.05). miR-671-5p targeting CBX4 inhibited the malignant progression of NSCLC in vitro (P<0.05). The down regulation of CircCDR1as could reduce the expression of CBX4 by up-regulating the level of miR-671-5p (P<0.05). Conclusion CircCDR1as silencing can inhibit cell proliferation, migration and invasion and induce cell apoptosis by up-regulating miR-671-5p and down-regulating CBX4.

Key words: carcinoma, non-small-cell lung, A549 cells, CircRNA antisense cerebellar degeneration-related protein 1 transcript, miR-671-5p, chromobox 4

CLC Number:

R734.2

Figures/Tables 13

References 28

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细胞系	CircCDR1as	miR-671-5p	CBX4 mRNA	CBX4蛋白
HBE	1.00±0.01	1.00±0.02	1.00±0.02	1.00±0.03
NCI-H524	3.92±0.14^a	0.59±0.05^a	2.86±0.14^a	2.54±0.11^a
NCI-H1734	4.09±0.21^a	0.52±0.06^a	2.95±0.18^a	2.63±0.14^a
Calu-3	4.15±0.19^a	0.41±0.04^a	3.18±0.23^a	2.75±0.21^a
A549	4.29±0.28^a	0.32±0.02^a	3.32±0.25^a	2.89±0.16^a
F	328.965^＊＊	243.424^＊＊	160.266^＊＊	175.065^＊＊

细胞系	CircCDR1as	miR-671-5p	CBX4 mRNA	CBX4蛋白
HBE	1.00±0.01	1.00±0.02	1.00±0.02	1.00±0.03
NCI-H524	3.92±0.14^a	0.59±0.05^a	2.86±0.14^a	2.54±0.11^a
NCI-H1734	4.09±0.21^a	0.52±0.06^a	2.95±0.18^a	2.63±0.14^a
Calu-3	4.15±0.19^a	0.41±0.04^a	3.18±0.23^a	2.75±0.21^a
A549	4.29±0.28^a	0.32±0.02^a	3.32±0.25^a	2.89±0.16^a
F	328.965^＊＊	243.424^＊＊	160.266^＊＊	175.065^＊＊

组别	CircCDR1as	miR-671-5p	细胞活力（OD₄₉₀值）	克隆形成数（个）	凋亡率（%）	迁移细胞数（个）	侵袭细胞数（个）
Control组	1.00±0.03	1.00±0.02	1.18±0.21	91.45±7.62	10.02±0.91	94.38±8.12	97.56±6.89
si-NC组	1.00±0.02	0.99±0.03	1.17±0.19	91.38±7.49	10.01±0.89	94.29±7.91	97.54±6.85
si-CircCDR1as组	0.38±0.03^ab	1.78±0.06^ab	0.49±0.07^ab	38.62±3.51^ab	33.45±2.62^ab	40.15±10.03^ab	41.32±8.74^ab
si-CircCDR1as+anti-miR-NC组	0.39±0.02	1.75±0.08	0.47±0.06	38.67±3.48	33.48±2.14	40.18±10.06	41.35±6.95
si-CircCDR1as+anti-miR-671-5p组	0.91±0.05^cd	1.26±0.05^cd	0.99±0.12^cd	80.59±6.72^cd	14.13±1.06^cd	79.84±9.21^cd	80.69±7.24^cd
F	611.941^＊＊	328.761^＊＊	36.693^＊＊	121.564^＊＊	315.182^＊＊	55.305^＊＊	90.091^＊＊

组别	CircCDR1as	miR-671-5p	细胞活力（OD₄₉₀值）	克隆形成数（个）	凋亡率（%）	迁移细胞数（个）	侵袭细胞数（个）
Control组	1.00±0.03	1.00±0.02	1.18±0.21	91.45±7.62	10.02±0.91	94.38±8.12	97.56±6.89
si-NC组	1.00±0.02	0.99±0.03	1.17±0.19	91.38±7.49	10.01±0.89	94.29±7.91	97.54±6.85
si-CircCDR1as组	0.38±0.03^ab	1.78±0.06^ab	0.49±0.07^ab	38.62±3.51^ab	33.45±2.62^ab	40.15±10.03^ab	41.32±8.74^ab
si-CircCDR1as+anti-miR-NC组	0.39±0.02	1.75±0.08	0.47±0.06	38.67±3.48	33.48±2.14	40.18±10.06	41.35±6.95
si-CircCDR1as+anti-miR-671-5p组	0.91±0.05^cd	1.26±0.05^cd	0.99±0.12^cd	80.59±6.72^cd	14.13±1.06^cd	79.84±9.21^cd	80.69±7.24^cd
F	611.941^＊＊	328.761^＊＊	36.693^＊＊	121.564^＊＊	315.182^＊＊	55.305^＊＊	90.091^＊＊

组别	CBX4 mRNA	CBX4蛋白
miR-NC组	1.00±0.01	1.00±0.03
miR-671-5p组	0.38±0.04^a	0.42±0.04^a
anti-miR-NC组	1.00±0.02	1.00±0.02
anti-miR-671-5p组	2.12±0.06^b	1.71±0.08^b
F	2 212.351^＊＊	719.376^＊＊