LncRNA NEAT1靶向miR-185-5p调控骨肉瘤的机制研究

doi:10.3877/cma.j.issn.2096-0263.2023.04.006

目的

探究LncRNA NEAT1对骨肉瘤细胞的调控机制。

方法

采用qRT-PCR检测正常人成骨细胞和骨肉瘤细胞中LncRNA NEAT1的差异表达，构建siRNA-NEAT1#1、siRNA-NEAT1#2、siRNA-NEAT1#3转染至骨肉瘤细胞并验证其转染效率。通过CCK-8法、流式细胞术、Transwell实验分别检测细胞活力、凋亡、迁移和侵袭能力。使用核质分离实验验证NEAT1定位。利用生物信息学预测网站预测LncRNA NEAT1的靶基因，继而采取双荧光素酶精准靶点验证实验。miR-185-5p inhibitor和siRNA-NEAT1共同转染至骨肉瘤细胞中验证LncRNA NEAT1通过海绵结合靶基因miR-185-5p对骨肉瘤细胞生物学行为进行调控。

结果

LncRNA NEAT1在骨肉瘤组织及细胞中高表达，在骨肉瘤细胞中敲低LncRNA NEAT1表达抑制细胞活力、迁移、侵袭，促进细胞凋亡，LncRNA NEAT1竞争性结合miR-185-5p，同时敲低骨肉瘤细胞LncRNA NEAT1与miR-185-5p的表达部分逆转了以上细胞行为。

结论

敲低miR-185-5p的表达水平可部分逆转siRNA-NEAT1对骨肉瘤细胞活力的抑制作用

关键词: 骨肉瘤, LncRNA NEAT1, miR-185-5p, 细胞活力

Objective

To investigate the regulatory mechanism of LncRNA NEAT1 on osteosarcoma cells.

Methods

qRT-PCR was used to detect the differential expression of LncRNA NEAT1 in normal human osteoblasts and osteosarcoma cells. siRNA-NEAT1#1, siRNA-NEAT1#2 and siRNA-NEAT1#3 were constructed and transfected into osteosarcoma cells and their transfection efficiency was verified. Cell viability, apoptosis, migration and invasion ability were detected by CCK-8 assay, flow cytometry and transwell assay, respectively. NEAT1 localization was verified using nucleoplasmic isolation experiments. Predicted target genes of LncRNA NEAT1 was detected by bioinformatics prediction website, followed by dual luciferase assay to verify. Co-transfection of miR-185-5p inhibitor and siRNA-NEAT1 into osteosarcoma cells was applied to verify that LncRNA NEAT1 regulated the biological behavior of osteosarcoma cells through sponge binding of the target gene miR-185-5p.

Results

LncRNA NEAT1 was highly expressed in osteosarcoma cells. Knockdown of LncRNA NEAT1 expression in osteosarcoma cells inhibited cell viability, migration, invasion and promoted apoptosis. LncRNA NEAT1 sponged binding to miR-185-5p, knockdown of LncRNA NEAT1 expression with miR-185-5p in osteosarcoma cells partially reversed the above cellular behaviors.

Conclusion

Knockdown of miR-185-5p expression level could partially reverse the inhibitory effect of siRNA-NEAT1 on osteosarcoma cell viability.

Key words: Osteosarcoma, LncRNA NEAT1, MiR-185-5p, Cell viability

表1 qRT-PCR引物序列

Gene	Forward primer（5’-3’）	Reverse primer（5’-3’）
NEAT1	GCGAGGTGCCTTTACTACAT		TGGAACCCAGAAGACAGA
miR-185-5p	CGCTGGAGAGAAAGGCAGT	GTGCAGGGTCCGAGGT
GAPDH	ACGGCAAGTTCAACGGCACAG	GACGCCAGTAGACTCCACGACA
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

表1 qRT-PCR引物序列

Gene	Forward primer（5’-3’）	Reverse primer（5’-3’）
NEAT1	GCGAGGTGCCTTTACTACAT		TGGAACCCAGAAGACAGA
miR-185-5p	CGCTGGAGAGAAAGGCAGT	GTGCAGGGTCCGAGGT
GAPDH	ACGGCAAGTTCAACGGCACAG	GACGCCAGTAGACTCCACGACA
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

图1 LncRNA NEAT1表达水平在骨肉瘤细胞中上调注：qRT-PCR检测LncRNA NEAT1在骨肉瘤细胞系中的表达。**代表与正常人成骨细胞系进行比较P<0.01，***代表与正常人成骨细胞系进行比较P<0.001

图2 qRT-PCR检测siRNA-NEAT1的转染效率

图2~5 LncRNA NEAT1提升骨肉瘤细胞活力并抑制凋亡。图3　CCK-8检测细胞活力；图4　流式细胞术检测细胞凋亡；图5　transwell迁移实验检测细胞迁移、侵袭能力注：OS：正常培养的U2OS细胞，siRNA-NEAT1/NC：siRNA-NEAT1（或其阴性对照NC）转染至U2OS细胞，**：P<0.01，***：P<0.001

图12~15 敲低miR-185-5p的表达水平可部分逆转siRNA-NEAT1对骨肉瘤细胞活力的抑制作用。图12　qRT-PCR检测miR-185-5p在不同处理组U2OS细胞中的表达；图13　CCK-8检测细胞活力；图14　流式细胞术检测细胞凋亡；图15　transwell迁移实验检测细胞迁移、侵袭能力

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Expression of LncRNA NEAT1 in osteosarcoma cells and study of the mechanism of cellular activity via miR-185-5p

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Expression of LncRNA NEAT1 in osteosarcoma cells and study of the mechanism of cellular activity via miR-185-5p