miR-490-3p Suppresses EMT of SW1990 Pancreatic Cancer Cells
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摘要:
目的 研究miR-490-3p在分子水平上通过HMGA2蛋白对SW1990细胞上皮间充质转化的影响。 方法 通过实时荧光定量PCR法（RT-qPCR）检测正常胰腺导管上皮细胞HPDE和人胰腺癌细胞SW1990中miR-490-3p的表达。通过转染质粒[miR-490-3p阻遏物（inhibitor）和miR-490-3p模拟物（mimic）以及各自的阴性对照质粒]调控miR-490-3p在SW1990细胞中的表达并通过RT-qPCR法验证转染效率。转染48 h后，通过CCK8检测、划痕法、Transwell小室检测，流式细胞仪检测等分别检测细胞增殖、凋亡、迁移和侵袭等恶性生物学特征的影响。通过Western blot检测细胞中上皮间充质转化相关蛋白E-cadherin和N-cadherin的蛋白表达水平。同时，用数据库预测miR-490-3p及其靶基因HMGA2的靶向关系，并通过双荧光素酶报告基因实验进行验证，通过Western blot检测细胞中HMGA2的蛋白表达水平。 结果 （1）与HPDE正常细胞相比，SW1990癌细胞中miR-490-3p表达水平明显降低（P = 0.215）；（2）miR-490-3p 上调后，SW1990细胞的凋亡率显著高于对照组（P < 0.0001），而细胞增殖、迁移和侵袭能力显著低于对照组（P < 0.0001）。miR-490-3p 下调后，SW1990细胞的凋亡率显著低于对照组（P < 0.0001），而细胞增殖、迁移和侵袭能力显著高于对照组（P < 0.0001）；（3）miR-490-3p 上调后，SW1990细胞中的N-cadherin表达量显著高于对照组（P < 0.0001），而E-cadherin的表达水平显著低于对照组（P < 0.0001）；miR-490-3p 下调后，SW1990细胞中的N-cadherin表达量显著低于对照组（P < 0.0001），而E-cadherin的表达水平显著高于对照组（P < 0.0001）；（4）HMGA2是miR-490-3p的靶向基因。 结论 miR-490-3p可通过靶向HMGA2抑制SW1990胰腺癌细胞EMT，从而影响胰腺癌的的发生发展进程，揭示HMGA2和miR-490-3p可能为胰腺癌诊断和治疗的靶点。 -
关键词:
- 胰腺癌 /
- 上皮间充质转化 /
- miR-490-3p /
- HMGA2
Abstract:Objective To elucidate the role of miR-490-3p in regulating HMGA2 expression and affecting epithelial-mesenchymal transition （EMT） of SW1990 cells. Methods miR-490-3p expression was measured in HPDE and SW1990 cell lines by Real-time PCR. SW1990 cell lines were cultured in vitro and divided into four groups, including inhibitor-Negative control （inhibitor-NC）, miR-490-3p inhibitor, mimic- Negative control （mimic-NC） and miR-490-3p mimic group, and the transfection efficiency was verified by RT-qPCR. After 48 h of transfection, CCK8 assay, wound healing assay, Transwell invasion assay andflow cytometric were used to detect the effects of malignant biological characteristics such as proliferation, migration, invasion and apoptosis of SW1990 cell linerespectively. Western blot was used to detect the expression of E-cadherin and N-cadherin. Meanwhile, database predicted the target genes of miR-490-3p, and dual luciferase assay confirmed the targeted relationship between miR-490-3p and HMGA2, western blot was used to detect the expression of HMGA2. Results 1） The expression of miR-490-3p was downregulated in SW1990 cells （P = 0.215）. 2） Overexpression of miR-490-3p significantly inhibited the proliferation （P < 0.0001）, migration （P < 0.0001） and invasion （P < 0.0001） of SW1990 cell line and prompted apoptosis （P < 0.0001）; In contrast, downregulation of miR-490-3p significantly promoted the proliferation （P < 0.0001）, migration （P < 0.0001） and invasion （P < 0.0001） of SW1990 cell line and inhibited apoptosis （P < 0.0001）. 3） Upregulation of miR-490-3p significantly decreased the expression of N-cadherin （P < 0.0001） and increased E-cadherin （P < 0.0001） expression in SW1990 cell line. In contrast, downregulation of miR-490-3p significantly upregulated （P < 0.0001） the expression of N-cadherin, decreased E-cadherin （P < 0.0001） expression. 4） HMGA2 was a target gene of miR-490-3p. Conclusion These findings indicate that miR-490-3p acts as a tumor suppressor during the process of EMT through targeting HMGA2, suggesting miR-490-3p is a potential new diagnostic and therapeutic target for the treatment of pancreatic cancer. -
Key words:
- Pancreatic cancer /
- EMT /
- miR-490-3p /
- HMGA2
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图 1 miR-490-3p在SW1990细胞中表达降低
A：miR-490-3p mRNA在不同细胞系中的表达；B：miR-490-3p转染水平验证。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. The expression of miR-490-3p significantly decreased in SW1990
图 2 miR-490-3p促进SW1990细胞凋亡
A：细胞凋亡实验；B：细胞凋亡率；C:细胞活力。***P < 0.001；****P < 0.000 1。
Figure 2. miR-490-3p promoted SW1990 cell apoptosis
图 3 miR-490-3p抑制SW1990细胞迁移和侵袭
A：细胞划痕实验；B：细胞侵袭实验；C：细胞迁移率；D：侵袭细胞数。****P < 0.000 1。
Figure 3. miR-490-3p inhibited the invasion and migration of SW1990 cell
图 4 miR-490-3p抑制SW1990细胞的EMT
A：E-cadherin和N-cadherin蛋白的表达；B：E-cadherin蛋白表达水平分析；C：N-cadherin蛋白表达水平分析。***P < 0.001；****P < 0.000 1。
Figure 4. miR-490-3p inhibited the EMT of SW1990 cell
图 5 HMGA2和miR-490-3p的靶向关系验证
A：HMGA2蛋白表达水平与胰腺癌预后分析；B：miR-490-3p与HMGA2相关性分析；C：miR-490-3p与HMGA2基因3′UTR结合位点示意图；D：293T细胞荧光素酶活性；E:HMGA2蛋白表达水平与表达水平分析。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 5. Validation of targeting relationship between HMGA2 and miR-490-3p
表 1 RT-qPCR扩增引物序列表
Table 1. Nucleotide sequences of the primers used for real-time quantitative PCR
引物名称 引物序列 miR-490-3p forward 5′-CGTGGATCCTTCTTCAACCAACGGTGGTG-3′ miR-490-3p reverse 5′-CCAGAATTCAAAGCAGGAAGAGTAAGACTTCC-3′ U6 forward 5′-GCTTCGGCAGCACATATACTAA-3′ U6 reverse 5′-CGAATTTGCGTGTCATCCTT-3′ 
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