REG1A通过调控Wnt/β-catenin信号通路对肺腺癌细胞增殖和迁移的促进作用

doi:10.13481/j.1671-587X.20220222

摘要/Abstract

摘要： 目的

探讨再生基因1A（REG1A）对肺腺癌细胞增殖和迁移的促进作用及其对Wnt/β-catenin信号通路的调控，并阐明可能的作用机制。

方法

利用肿瘤基因组图谱（TCGA）数据库下载符合本研究要求的515例肺腺癌组织和59例癌旁正常组织的基因表达谱，根据REG1A表达水平的中位值，分为低和高表达REG1A组，采用R 3.6.1软件对2组基因进行差异基因分析，根据差异基因进行基因本体（GO）功能注释，采用GSEA 4.1.0软件筛查差异基因富集的京都基因与基因组百科全书（KEGG）信号通路。筛查出得分最高的信号通路［无翅型MMTV整合位点（Wnt）信号通路］，采用实时荧光定量PCR（RT-qPCR）及Western blotting法检测人正常肺上皮细胞（BEAS-2B）和肺腺癌细胞A549、LC-2、HCC515和PC14中REG1A mRNA及蛋白表达水平。采用脂质体转染法将shREG1A和shNC质粒转染A549细胞，将REG1A敲减后的细胞，采用氯化锂（LiCl）处理，将细胞分为shNC组、shREG1A组、shREG1A+PBS组和shREG1A+LiCl组，采用细胞计数试剂盒（CCK-8）检测各组细胞增殖活性，采用Transwell实验检测各组细胞的迁移情况，RT-qPCR和Western blotting法检测各组细胞中无翅型MMTV整合位点家族成员3a（Wnt-3a）、β-连环素（β-catenin）和原癌基因（c-Myc）mRNA及蛋白表达水平。

结果

生物信息学分析，低和高表达REG1A组共有 78个差异基因，其中上调的基因50个，下调的基因28个，上述基因主要富集在脂质转运体活性、内切核糖核酸酶活性和胃肠道上皮结构稳态的维持等生物学过程和分子功能上，富集的通路有Wnt信号通路、Janus激酶-信号转导与转录激活因子（JAK-STAT）信号通路和癌症信号通路，其中Wnt信号通路富集得分最高。RT-qPCR法和Western blotting检测，肺腺癌A549、LC-2、HCC515和PC14细胞中REG1A mRNA及蛋白表达水平均明显高于BEAS-2B细胞（P<0.05）。脂质体转染法敲减REG1A后，与shNC组比较，shREG1A组A549细胞中REG1A mRNA和蛋白表达水平明显降低（P<0.05）；shNC组、shREG1A组、shREG1A+PBS组和shREG1A+LiCl组细胞增殖活性及迁移细胞数差异有统计学意义（P<0.05），shREG1A组细胞增殖活性低于shNC组和shREG1A+LiCl组（P<0.05），shREG1A组迁移细胞数少于shNC组和shREG1A+LiCl组（P<0.05），shREG1A组与shREG1A+PBS组细胞增殖活性和迁移细胞数比较差异无统计学意义（P>0.05）。

结论

REG1A在肺腺癌细胞中呈现高表达，可以通过调控Wnt/β-catenin信号通路，促进A549细胞的增殖和迁移；敲减REG1A后，A549细胞增殖和迁移能力也受到抑制。

关键词: 肺腺癌, 再生基因1A, 细胞增殖, 细胞迁移, 生物信息学

Abstract: Objective

To investigate the promotion effect of regenerating gene 1A（REG1A） on the proliferation and migration of lung adenocarcinoma cells and its regulation effect on Wnt/β-catenin signaling pathway，and to clarify its possible mechanism.

Methods

The gene expresson profiles of 515 patients with lung adenocarcinoma and 59 paracancerous normal lung tissue met the requirement of this study were obtained from The Cancer Genome Atlas （TCGA） database. According to the median value of the REG1A distribution level， they were divided into high and low expression groups.R 3.6.1 software was employed to analyze the differentially expressed genes between two groups.The Gene Ontology （GO） function annotation was carried out for the differential genes. Meanwhile， GSEA4.1.0 was used for Kyoto Encyclopedia of Genes and Genomes（KEEF） enrichment analysis of the differential genes. Wnt signaling pathway， the signaling pathway that scored the best，was screened， and the mRNA and the protein expression levels of REG1A in the human normal lung epithelial cells （BEAS-2B） and lung adenocarcinoma cells （A549， LC-2， HCC515 and PC14） were tested by real-time fluorescence quantitative PCR（RT-qPCR） and Western blotting methods. The liposome transfection method was used to transfect shREG1A and shNC into the A549 cells， and the A549 cells with REG1A expression knockdown were treated by lithium chloride. The A549 cells were divided into shNC group，shREG1A group，shREG1A+PBS group， and shREG1A+LiCl group.The proliferation activities of the cells in various groups were detected by CCK-8 method.The migration of cells in various groups was detected by Transwell assay.The mRNA and proein expression levels of Wnt-3a，β-catenin，and c-Myc in the cells in various groups were detected with RT-qPCR and Western blotting methods.

Results

The 78 differential genes in low and high expression REG1A groups were screened out， including 50 up-regulated genes and 28 down-regulated genes； these genes were mainly enriched in proton antiporter activity，endoribonuclease activity，maintenance of gastrointestinal epithelium and so on， and the enriched pathways included Wnt signaling pathway，JAK-STAT signaling pathway，and cancer signaling pathway， etc； among which the Wnt signaling pathway had the highest enrichment score. The RT-qPCR and Western blotting results indicated that the expression levels of REG1A mRNA and protein in the lung adenocarcinoma A549，LC-2，HCC515，and PC14 cells were obviously higher than those in human normal lung epithelial cells BEAS-2B（P<0.05）.The REG1A mRNA and protein expression levels in the A549 cells in shREG1A group were obviously decreased compared with shNC group （P<0.05） after REG1A knockdown. There were statistically significant differences in the cell proliferation activities and the number migration cells among shNC group，shREG1A group，shREG1A+PBS group，and shREG1A+LiCl group（P<0.05）；the cell proliferation activities in shREG1A group were significantly lower than those in shNC group and shREG1A+LiCl group（P<0.05），and the number of migration cells in shREG1A group was lower than those in shNC group and shREG1A+LiCl group（P<0.05），but there were no significant differences in the cell proliferation activities and the number of migration cells between shREG1A group and shREG1A+PBS group（P>0.05）.

Conclusion

REG1A is highly expressed in the lung adenocarcinoma cells. It could promote the proliferation and migration of A549 cells through regulating the Wnt/β-catenin signaling pathway. After knockdown REG1A， the proliferation and migration abilities of the A549 cells are significantly inhibited.

Key words: Lung adenocarcinoma, Regenerating gene 1A, Cell proliferation, Cell migration, Bioinformatics

中图分类号:

R734.2

李小燕,张维,何杰. REG1A通过调控Wnt/β-catenin信号通路对肺腺癌细胞增殖和迁移的促进作用[J]. 吉林大学学报(医学版), 2022, 48(2): 444-453.

Xiaoyan LI,Wei ZHANG,Jie HE. Promotion effect of REG1A on proliferation and migration of lung adenocarcinoma cells by regulating Wnt/β-catenin signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 444-453.

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参考文献 27

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Gene		Sequence (5'－3')
REG1A	Forward	GGCCACCTCTTGACAGTGATG
	Reverse	AAGGTCCATGGGAAAGACAC
Wnt-3a	Forward	CAGGGTCCACCACGCTCTTC
	Reverse	TTTGTAAGTGTGAGGGTCTGG
β-catenin	Forward	GGCGTTGCTCGCTTTGTA
	Reverse	CGAATGTCTGACGTATTGA
c-Myc	Forward	GAGCCAAACGGGTCATCATCT
	Reverse	GAGGGGCCATCCACAGTCTT
GAPDH	Forward	GAGTCAACGGATTTGGTCGT
	Reverse	TTGATTTTGGAGGGATCTCG

KEGG pathway enrichment	Enrichment score	NES	P	FDR
Wnt signaling pathway	0.680	2.248	<0.001	0.003
JAK-STAT signaling pathway	0.630	2.210	<0.001	0.002
Cytokine-cytokine receptor interaction pathway	0.610	2.125	<0.001	0.002
Regulation of actin cytoskeleton pathway	0.560	2.050	<0.001	0.002
MAPK signaling pathway	0.540	1.910	<0.001	0.001
Cancer signaling pathway	0.430	1.860	<0.001	0.001

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