miR-126过表达和ADAM9基因沉默对胃癌SGC-7901细胞生物学行为的影响及其机制

doi:10.13481/j.1671-587X.20240203

摘要/Abstract

摘要：

目的探讨微小RNA-126（miR-126）过表达和解整联蛋白金属蛋白酶9（ADAM9）基因沉默对胃癌细胞生物学行为的影响，并阐明其作用机制。方法体外培养人胃低分化腺癌SGC-7901细胞和人正常胃黏膜上皮NGEC细胞，提取细胞中总RNA，采用实时荧光定量PCR（RT-qPCR）法检测2种细胞中miR-126和ADAM9 mRNA表达水平。将处于对数生长期的SGC-7901细胞分为 miR-126 过表达组（miR-126-OE组）和ADAM9基因沉默组（ADAM9 siRNA组），以Lipofectamine^TM 2000为载体分别转染miR-126模拟物（miR-126 mimics）和敲低ADAM9的RNA寡核苷酸，并设置相应的阴性对照组。采用噻唑蓝（MTT）法检测各组细胞增殖活性，细胞划痕实验检测各组细胞迁移率，Transwell小室实验检测各组细胞的迁移细胞数和侵袭细胞数，Western blotting法检测各组细胞中E-钙黏蛋白、N-钙黏蛋白和波形蛋白表达水平。TargerScan网站预测miR-126的靶基因并通过双荧光素酶报告基因实验验证miR-126和ADAM9的靶向调控关系，采用RT-qPCR法和Western blotting法检测转染miR-126 mimics后SGC-7901细胞中ADAM9 mRNA和蛋白表达水平。结果 RT-qPCR法检测，与人正常胃黏膜上皮NGEC细胞比较，胃癌SGC-7901细胞中miR-126表达水平明显降低（P<0.05），而ADAM9 mRNA表达水平明显升高（P<0.05）。MTT法检测，SGC-7901细胞过表达miR-126或沉默ADAM9基因48和72 h后，与相应阴性对照组比较，miR-126 OE组和ADAM9 siRNA组细胞增殖活性均明显降低（P<0.05或P<0.01）。细胞划痕实验检测，与相应阴性对照组比较，48 h后miR-126 OE组和ADAM9 siRNA组细胞迁移率明显降低（P<0.05）。Transwell小室实验检测，与相应阴性对照组比较，miR-126 OE组和ADAM9 siRNA组迁移细胞数和侵袭细胞数明显减少（P<0.05或P<0.01）。Western blotting法检测，与相应阴性对照组比较，miR-126-OE组和ADAM9 siRNA组细胞中E-钙黏蛋白表达水平明显升高（P<0.05或P<0.01），而N-钙黏蛋白和波形蛋白表达水平明显降低（P<0.05或P<0.01）。靶基因预测，ADAM9的3'-UTR含有与miR-126-3p互补的核苷酸序列。双荧光素酶报告基因实验，ADAM9是miR-126靶向负调控的下游基因。SGC-7901细胞转染miR-126 mimics 48 h后，与mimics NC组比较，miR-126 OE组细胞中ADAM9 mRNA和蛋白表达水平均明显降低（P<0.05或P<0.01）。结论胃癌SGC-7901细胞中miR-126低表达而ADAM9基因高表达。miR-126过表达可抑制胃癌SGC-7901细胞增殖活性、迁移和侵袭能力，其机制可能与miR-126靶向负调控ADAM9并抑制胃癌细胞的上皮-间质转化（EMT）进程有关。

关键词: 胃肿瘤, 微小RNA-126, 靶基因, 解整联蛋白金属蛋白酶9, 上皮-间质转化

Abstract:

Objective To discuss the effects of microRNA-126 （miR-126） over-expression and disintegrin and metalloproteinase 9 （ADAM9） gene silencing on the biological behavior of the gastric cancer cells， and to clarify the mechanism. Methods The human poorly differentiated gastric adenocarcinoma SGC-7901 cells and human normal gastric mucosa epithelial NGEC cells were cultured in vitro. The total RNA was extracted from the cells， and the expression levels of miR-126 and ADAM9 mRNA in both types of cells were detected by real-time fluorescence quantitative PCR （RT-qPCR）method. The SGC-7901 cells at logarithmic growth phase were divided into miR-126 over-expression group （miR-126-OE group） and ADAM9 gene silencing group （ADAM9 siRNA group）. The transfection with miR-126 mimics （miR-126） mimics and ADAM9 RNA oligonucleotides were conducted by Lipofectamine^TM 2000， and the corresponding negative control group was established； MTT assay was used to detect the proliferation activities of the cells in various groups； cell wound assay was used to detect the migration rate of the cells in various groups；Transwell chamber assay was used to detect the the numbers of migration and invasion cells in various groups； Western blotting method was used to detect the expression levels of E-cadherin， N-cadherin， and vimentin proteins in the cells in various gorups. The miR-126 target genes were predicted by TargetScan website， and the targeting regulatory relationship between miR-126 and ADAM9 was confirmed by dual-luciferase reporter assay. The expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics were detected by RT-qPCR and Western blotting methods. Results The RT-qPCR results showed that compared with human normal gastric mucosa epithelial NGEC cells， the expression level of miR-126 in the gastric cancer SGC-7901 cells was significantly decreased （P<0.05）， while the expression level of ADAM9 mRNA was significantly increased （P<0.05）. The MTT assay results showed that after 48 and 72 h of over-expressing miR-126 or silencing the ADAM9 gene in the SGC-7901 cells， compared with the corresponding negative control group， the proliferation activities of the cells in both miR-126-OE and ADAM9 siRNA groups were significantly decreased （P<0.05 or P<0.01）. The cell wound assay results indicated that compared with the corresponding negative control group， the migration rates of the cells in both miR-126 OE and ADAM9 siRNA groups 48 h after transfection were significantly decreased （P<0.05）. The Transwell chamber assay results showed that the numbers of migration and invasion cells in both miR-126-OE and ADAM9 siRNA groups were significantly lower than those in corresponding negative control group （P<0.05 or P<0.01）.The Western blotting method results showed that compared with the corresponding negative control groups， the expression level of E-cadherin protein in the cells in miR-126-OE and ADAM9 siRNA groups were significantly increased （P<0.05 or P<0.01）， while the expression levels of N-cadherin and vimentin proteins were significantly decreased （P<0.05 or P<0.01）. The target prediction results showed that the 3'-UTR of ADAM9 contains nucleotide sequences complementary to miR-126-3p. The dual-luciferase reporter assay results showed that ADAM9 was a downstream target gene negatively regulated by miR-126. Compared with mimics NC group， the expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics for 48 h were decreased （P<0.05 or P<0.01）. Conclusion The gastric cancer SGC-7901 cells are characterized by low expression of miR-126 and high expression of ADAM9 gene. Over-expression of miR-126 can inhibit the proliferative activity， migration， and invasion capabilities of the gastric cancer SGC-7901 cells； the mechanism may be related to the negative regulation of ADAM9 by miR-126 and the inhibition of epithelial-mesenchymal transition （EMT） process in the gastric cancer cells.

Key words: Gastric neoplasm, MicroRNA-126, Target gene, A disintegrin and metalloprotease 9, Epithelial-mesenchymal transition

中图分类号:

R735.2

魏海峰,倪志强,魏雁虹,王起来,李首庆,马寅芙,谭岩,方艳秋. miR-126过表达和ADAM9基因沉默对胃癌SGC-7901细胞生物学行为的影响及其机制[J]. 吉林大学学报(医学版), 2024, 50(2): 310-319.

Haifeng WEI,Zhiqiang NI,Yanhong WEI,Qilai WANG,Shouqing LI,Yinfu MA,Yan TAN,Yanqiu FANG. Effects of miR-126 over-expression and ADAM9 gene silencing on biological behavior of gastric cancer SGC-7901 cells and their mechanisms[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 310-319.

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Target gene	Primer sequence
ADAM9	F: 5'-GGAAGAGTGTGACTGTGGTAC-3'
ADAM9	R: 5'-CCTCGGCATAAAGTACCTCC-3'
Hsa-miR-126 stem-loop RT primer	5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCATT-3'
MiR-126-3p	F: 5'-GTCTCGTACCGTGAGTAAT-3'
MiR-126-3p	R: 5'-GTGCAGGGTCCGAGGT-3'
U6	F: 5'-CTCGCTTCGGCAGCACA-3'
U6	R: 5'-AACGCTTCACGAATTTGCGT-3'
GAPDH	F: 5'-CAATGACCCCTTCATTGACC-3'
GAPDH	R: 5'-GACAAGCTTCCCGTTCTCAG-3'

Group	Proliferation activity of SGC-7901 cells
Group	（t/h）0	24	48	72
Mimics NC	0.15±0.03	0.34±0.08	0.57±0.11	1.26±0.12
MiR-126-OE	0.13±0.04	0.23±0.07	0.28±0.12^*	0.85±0.13^*
SiRNA NC	0.16±0.02	0.36±0.04	0.73±0.14	1.79±0.25
ADAM9 siRNA	0.17±0.06	0.31±0.05	0.40±0.09^△	0.79±0.11^△△

Group	Migration rate of SGC-7901 cells
Group	（t/h） 24	48
Mimics NC	59.2±12.4	89.6±15.4
MiR-126-OE	41.7±8.6	53.1±12.7*
SiRNA NC	60.4±9.5	91.6±15.8
ADAM9 siRNA	43.5±11.3	46.9±9.2^△

Group	Number of migration cells	Number of invasion cells
Mimics NC	163.24±21.47	125.41±18.18
MiR-126-OE	103.15±16.49*	76.64±10.29*
SiRNA NC	147.91±19.15	113.24±14.29
ADAM9 siRNA	98.64±13.28^△	62.47±8.26^△△

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