LncRNA GNAS-AS1通过调节miR-449a/Notch1轴参与胃癌细胞的增殖和迁移

doi:10.3969/j.issn.1006-5725.2024.04.008

Abstract

Abstract:

Objective To explore the impacts of long non-coding RNA （LncRNA） GNAS antisense RNA1 （GNAS-AS1） on the proliferation and migration of gastric cancer （GC） cells by regulating the miR-449a/Notch1 axis. Method Tumor tissue and adjacent tissue samples were collected from 30 patients diagnosed with GC at Sichuan Provincial People's Hospital from September 2013 to September 2017； GC cells AGS were randomly divided into Control group， si-NC group， si-GNAS-AS1 group， si-GNAS-AS1+inhibitor NC group， and si-GNAS-AS1+miR-449a inhibitor group. Real-time fluorescence quantitative PCR method was applied to detect the expression of GNAS-AS1， miR-449a， and Notch1 mRNA； MTT experiments and plate cloning experiments were applied to detect the proliferation； wound healing test was applied to detect cell migration； Transwell experiment was applied to detect cell invasion. Western Blot was applied to detect the expression of Notch1， E-cadherin， Vimentin， and N-cadherin proteins. Double Luciferase reporter gene experiment was applied to verify the relationship between GNAS-AS1 and miR-449a， between miR-449a and Notch1， respectively. Results Compared with adjacent tissues， the expression of GNAS-AS1 and Notch1 mRNA in tumor tissue was increased， the expression of miR-449a was reduced （P < 0.05）. Compared with the Control group and si-NC group， the expression of GNAS-AS1， OD₄₉₀ value， number of clones formed， scratch healing rate， number of cell invasions， and the expression of Notch1， Vimentin， and N-cadherin proteins in AGS cells in the si-GNAS-AS1 group reduced， the expression of miR-449a and E-cadherin protein increased （P < 0.05）. Compared with the si-GNAS-AS1 group and the si-GNAS-AS1+inhibitor NC group， the OD₄₉₀ value， scratch healing rate， number of cell invasions， Notch1， Vimentin， and N-cadherin expression in the si-GNAS-AS1+miR-449a inhibitor group increased， the expression of miR-449a and E-cadherin protein reduced （P < 0.05）. GNAS-AS1 targeted and negatively regulated miR-449a expression， while miR-449a targeted and negatively regulated Notch1 expression. Conclusion Silencing GNAS-AS1 may inhibit the expression of Notch1 protein by up-regulating miR-449a， thereby inhibiting the proliferation， migration， and invasion processes of GC cells.

Key words: long non-coding RNA GNAS antisense RNA1, miR-449a, Notch1, gastric cancer, migration, proliferation

CLC Number:

R735.2

Li XU,Shanshan HU,Haiming. ZHAO. LncRNA GNAS⁃AS1 participates in the proliferation and migration of gastric cancer cells by regulating the miR⁃449a/Notch1 axis[J]. The Journal of Practical Medicine, 2024, 40(4): 483-489.

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References 18

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基因名称	引物序列（5′→3′）
GNAS-AS1	上游：GACGCCTTTCCTACGG
GNAS-AS1	下游：TGGTAACGCACCTTCG
miR-449a	上游：GTCGTATCCAGTGCAGGGTCCGAG
miR-449a	下游：AACACGCTGGCAGTGTATTG
Notch1	上游：AGGTGCAGCCACAGAACTTA
Notch1	下游：TCGGACCAATCAGAGATGTT
U6	上游：GCTTCGGCAGCACATA
U6	下游：ATGGAACGCTTCACGA
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG

组别	GNAS-AS1	miR-449a	Notch1
癌旁组织	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00
肿瘤组织	2.58 ± 0.28^*	0.41 ± 0.05^*	2.17 ± 0.24^*

组别	GNAS-AS1	miR-449a	Notch1
Control组	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00
si-NC组	1.01 ± 0.12	1.02 ± 0.11	1.02 ± 0.12
si-GNAS-AS1组	0.36 ± 0.04^*#	2.25 ± 0.24^*#	0.45 ± 0.05^*#
si-GNAS-AS1+inhibitor NC组	0.34 ± 0.04	2.23 ± 0.24	0.43 ± 0.04
si-GNAS-AS1+miR-449a inhibitor组	0.35 ± 0.04	1.62 ± 0.18^{&^}	0.86 ± 0.09^{&^}

组别	OD₄₉₀	克隆形成数（个）	划痕愈合率（%）	细胞侵袭数目（个）
Control组	0.70 ± 0.08	108.42 ± 7.30	42.66 ± 5.18	157.69 ± 8.48
si-NC组	0.68 ± 0.07	106.22 ± 6.47	43.45 ± 4.42	159.44 ± 10.08
si-GNAS-AS1组	0.29 ± 0.03^*#	52.69 ± 3.38^*#	17.69 ± 2.14^*#	93.21 ± 8.34^*#
si-GNAS-AS1+inhibitor NC组	0.27 ± 0.03	55.38 ± 5.02	18.32 ± 1.97	90.89 ± 10.25
si-GNAS-AS1+miR-449a inhibitor组	0.61 ± 0.06^{&^}	86.79 ± 7.08^{&^}	38.85 ± 3.56^{&^}	138.54 ± 11.55^{&^}

组别	Notch1	E-cadherin	Vimentin	N-cadherin
Control组	0.79 ± 0.08	0.28 ± 0.03	0.85 ± 0.09	0.87 ± 0.09
si-NC组	0.81 ± 0.08	0.26 ± 0.03	0.83 ± 0.08	0.89 ± 0.09
si-GNAS-AS1组	0.32 ± 0.03^*#	0.70 ± 0.07^*#	0.40 ± 0.04^*#	0.35 ± 0.03^*#
si-GNAS-AS1+inhibitor NC组	0.30 ± 0.03	0.72 ± 0.07	0.42 ± 0.04	0.33 ± 0.03
si-GNAS-AS1+miR-449a inhibitor组	0.68 ± 0.07^{&^}	0.42 ± 0.04^{&^}	0.75 ± 0.07^{&^}	0.76 ± 0.08^{&^}

LncRNA GNAS⁃AS1 participates in the proliferation and migration of gastric cancer cells by regulating the miR⁃449a/Notch1 axis

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组别	荧光素酶活性
miR-NC与GNAS-AS1-WT共转染组	1.07 ± 0.13
miR-NC与GNAS-AS1-MUT共转染组	1.04 ± 0.13
miR-449a mimic与GNAS-AS1-WT共转染组	0.34 ± 0.04^*
miR-449a mimic与GNAS-AS1-MUT共转染组	1.06 ± 0.13

组别	荧光素酶活性
miR-NC与Notch1-WT共转染组	1.03 ± 0.13
miR-NC与Notch1-MUT共转染组	1.07 ± 0.13
miR-449a mimic与Notch1-WT共转染组	0.46 ± 0.05^*
miR-449a mimic与Notch1-MUT共转染组	1.01 ± 0.11

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