长链非编码RNA对牙本质基质蛋白1的调控机制

doi:10.3877/cma.j.issn.1674-1366.2017.01.002

目的

初步探究长链非编码RNA（lncRNA）对牙本质基质蛋白1（DMP1）基因表达的调控机制。

方法

在MC3T3-E1细胞中，运用Western blot以及实时荧光定量聚合酶链反应（PCR）实验观察lncRNA32865与DMP1的变化趋势。构建含有荧光素酶报告基因的DMP1启动子载体，并与lncRNA过表达质粒、si-lncRNA32865分别共转染后，观察DMP1启动子活性以及lncRNA32865对其的影响。数据进行统计学处理，以P<0.05为差异具有统计学意义。

结果

Western blot以及实时荧光定量PCR实验结果显示，在MC3T3-E1细胞中过表达lncRNA32865时，DMP1基因表达量（0.236 ± 0.022）较对照组降低（t= 59.816，P<0.001），抑制lncRNA32865时，DMP1基因表达量（1.994 ± 0.133）较对照组升高（t=-12.989，P= 0.006）。荧光素酶报告基因检测表明DMP1启动子有活性（t=-77.360，P<0.001）。与转染DMP1启动子处理组（6.018 ± 0.105）比较，DMP1启动子质粒与lncRNA32865过表达质粒共转染组荧光强度（3.877 ± 0.120）显著降低（t= 50.713，P<0.001），而DMP1启动子质粒与si-lncRNA32865共转染组荧光强度（17.296 ± 0.674）显著增加（t=-26.612，P= 0.001）。

结论

初步证明lncRNA32865与DMP1表达趋势相反，lncRNA32865通过与DMP1基因的启动子区相互作用，以此调控DMP1的基因表达。

关键词: 基因表达调控, RNA，未翻译, 长链, 牙本质基质蛋白类

Objective

To investigate the regulation mechanism of long noncoding RNA (lncRNA) on dentin matrix protein 1 (DMP1) .

Methods

The lipidosome infection protocol was used to transfect the lncRNA32865 plasmid and lncRNA32865 siRNA into the MC3T3-E1 cells. Using the methods of Western blot and RT-qPCR to detect the expression tendency of lncRNA32865 and DMP1 after transfection. The method of luciferase reporter gene was used to examine the DMP1 promoter activity and the effect of lncRNA32865 on it. For all statistical analyses, P<0.05 was considered statistically significant.

Results

Over expression of lncRNA32865 led to the decrease of DMP1 expression (0.236 ± 0.022) , there was a statistically significant difference between the groups (t= 59.816, P<0.001) . DMP1 expression was increased (1.994 ± 0.133) caused by inhibiting lncRNA32865 gene, there was a statistically significant difference between the groups (t=-12.989, P= 0.006) . The luciferase reporter gene analysis showed that DMP1 promoter was active (t=-77.360, P<0.001) . The Luciferase expression level of DMP1 promotor and lncRNA32865 plasmid cotransfection (3.877 ± 0.120) was lower than the control group (6.018 ± 0.105) with statistically significant difference (t= 50.713, P<0.001) . The Luciferase expression level of DMP1 promotor and lncRNA32865 siRNA cotransfection was increased (17.296 ± 0.674) with statistically significant difference (t=-26.612, P= 0.001) .

Conclusions

Western blot and RT-qPCR analyses demonstrated that the expression tendency of lncRNA32865 and DMP1 is opposite. LncRNA32865 regulates the expression of DMP1 gene by binding to the DMP1 promoter.

Key words: Gene expression regulation, RNA untranslated, Long chain, Dentin matrix proteins

表1 聚合酶链反应引物的碱基序列

基因	引物序列
DMP1	F:5′-TGGTATCAGGTCGGAAGAATC-3′
?	R:5′-CCCTGCTGTTGCTGTCAGTA-3′
lncRNA32865	F:5′-CTCAGCATCTCTGGTAAGGT-3′
?	R:5′-AGGTAGGGCATGGACTAGAA-3′
GAPDH	F:5′-GGCCTCCAAGGAGTAAGAAA-3′
?	R:5′GCCCCTCCTGTTATTATGG-3′
U6	F:5′CTCGCTTCGGCAGCACA-3′
?	R:5′AACGCTTCACGAATTTGCGT-3′

表1 聚合酶链反应引物的碱基序列

基因	引物序列
DMP1	F:5′-TGGTATCAGGTCGGAAGAATC-3′
?	R:5′-CCCTGCTGTTGCTGTCAGTA-3′
lncRNA32865	F:5′-CTCAGCATCTCTGGTAAGGT-3′
?	R:5′-AGGTAGGGCATGGACTAGAA-3′
GAPDH	F:5′-GGCCTCCAAGGAGTAAGAAA-3′
?	R:5′GCCCCTCCTGTTATTATGG-3′
U6	F:5′CTCGCTTCGGCAGCACA-3′
?	R:5′AACGCTTCACGAATTTGCGT-3′

图1 在MC3T3-E1细胞中过表达或沉默lncRNA32865、lncRNA32865在细胞中的表达情况

图2 在MC3T3-E1细胞中过表达或沉默lncRNA32865、DMP1基因的表达情况

图3 MC3T3-E1细胞转染lncRNA32865过表达质粒和si-lncRNA32865后对DMP1蛋白表达变化的影响

图4 MC3T3-E1细胞中lncRNA32865对DMP1基因启动子活性的影响

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Study on the regulation mechanism of long noncoding RNA on dentin matrix protein 1

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Study on the regulation mechanism of long noncoding RNA on dentin matrix protein 1