Klotho 基因超甲基化在硫酸吲哚酚诱导的血管平滑肌细胞成骨转化中的作用机制

doi:10.3877/cma.j.issn.2095-3216.2014.05.007

目的

从细胞层面探讨Klotho 基因超甲基化在硫酸吲哚酚(IS)诱导的血管平滑肌细胞成骨转化中的作用和相关机制。

方法

体外培养人主动脉血管平滑肌细胞(HASMC),以IS 按0、200、500、1 000 μ mol /L 的浓度梯度干预6 d,采用茜素红染色观察钙盐沉积,Real-time 聚合酶链式反应(PCR)和Western 印迹检测平滑肌细胞肌动蛋白α(α-SMA)、smoothin、骨桥蛋白(OPN)、碱性磷酸酶(ALP)、核结合因子α1(Cbfα1)、Klotho 和不同DNA 甲基化转移酶(DNMT)的mRNA 和蛋白表达水平,同时利用焦磷酸测序方法检测HASMC 的Klotho 基因甲基化率;在IS 1 000 μmol/L 组培养基中加入不同浓度梯度的5-氮杂脱氧胞苷(5Aza-2dc)进行干预,观察茜素红染色、各基因表达水平和Klotho 基因甲基化率的改变。组间均数比较采用独立样本t 检验,组间率的比较采用卡方检验。

结果

IS 1 000 μmol/L组HASMC 细胞外基质呈红色,细胞结节处浓染,为钙盐沉积染色阳性。 IS 可以下调HASMC 的α-SMAmRNA 和蛋白及smoothin 表达水平,上调OPN、ALP 的mRNA 和蛋白表达水平,使HASMC 逐渐丧失平滑肌细胞表型而发生成骨转化。与对照组比较,IS 200、500、1 000 μ mol /L 刺激6 d 可显著升高HASMC 的Klotho 基因甲基化率(9 ±1% 与4 ±0.7%,t=5.38, P ＜0.01;18 ±1.3 与4 ±0.7%, t=6.92,P ＜0.01;41 ±2% 与4 ±0.7%, t=9.26,P ＜0.001)。 IS 500、1 000μ mol /L 可显著下调HASMC 的Klotho mRNA 和蛋白表达水平。同时IS 1 000 μ mol /L 可显著上调HASMC 的DNMT1 mRNA 和蛋白表达水平。 5Aza-2dc 10 μmol /L 干预可减轻IS 诱导的细胞外钙盐沉积,上调α-SMA 蛋白、smoothin 表达水平并下调Cbfα1 蛋白表达水平,同时减低HASMC 的Klotho 基因甲基化率(20 ±1%与41 ±2%, t=6.98,P ＜0.01)、上调HASMC 的Klotho 蛋白表达水平。

结论

IS 可通过上调血管平滑肌细胞DNMT 表达,启动Klotho 基因超甲基化,进而下调Klotho 基因转录和表达,诱导血管平滑肌细胞成骨转化。

关键词: Klotho 基因, 脱氧核糖核酸甲基化, 硫酸吲哚酚

Objective

This study aimed to investigate the effect and mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate (IS).

Methods

Human aortic vascular smooth muscle cells (HASMC) were cultured. IS concentrations of 0, 200, 500,1 000 μmol/L were used for 6 days. Alizarin red staining was used for observation of calcium salt deposits.Real-time polymerase chain reaction (PCR) and Western blotting were applied to detect mRNA and protein expressions of α-smooth muscle actin (α-SMA), smoothin, osteopontin (OPN), alkaline phosphatase(ALP), nuclear binding factor alpha 1 (Cbf α1), Klotho, and different DNA methylation transferases(DNMT). Klotho gene methylation level in HASMC was assessed by pyrosequencing assay. In IS 1 000 μmol/L group, different concentrations of 5-Aza-2-deoxycytidine (5Aza-2dc) were added into the culture medium for interference experiment. Mean comparison between groups was done with independent sample t-test, while rate comparison between groups was done with chi-square test.

Results

In IS 1 000 μmol/L group, extracellular matrix of HASMC was stained red, which was concentrated in cell nodules, showing positive calcium salt deposition. IS decreased the expression of α-SMA mRNA and protein as well as smoothin, and increased the mRNA and protein expression of OPN and ALP, making the HASMC to gradually lose the SMC phenotype and transfer to osteoblast. Stimulation of HASMC by IS at the concentration of 200,500, and 1 000 μmol/L for 6 days significantly induced Klotho gene hypermethylation ratio compareal with the control group (9 ±1% vs 4 ±0.7%, t=5.38, P ＜0.01;18 ±1.3 vs 4 ±0.7%,t=6.92, P ＜0.01; 41 ±2% vs 4 ±0.7%, t =9.26, P ＜0.001). IS at the concentration of 500 and 1 000 μmol /L also significantly decreased Klotho mRNA and protein levels in HASMC. At the same time,IS obviously increased the expression of DNMT1 mRNA and protein level. 5Aza-2dc 10 μmol/L reduced the calcium deposition induced by IS, upregulated α-SMA protein and smoothin expression, down-regulated Cbf αl protein level, simultaneously decreased the Klotho gene methylation ratio (20 ±1% vs 41 ±2%,t=6.98, P ＜0.01), and increased the protein level of Klotho in HASMC.

Conclusion

IS induced the vascular SMC-osteoblast conversion. Through upregulating of DNMT1 activity, IS induced Klotho gene hypermethylation, and down-regulated Klotho protein expression. Block of Klotho gene hypermethylation reduced the vascular SMC-osteoblast conversion induced by IS.

Key words: Klotho gene, DNA methylation, Indoxyl sulfate

图1 Klotho 基因的测序位点图注:粗横线为Klotho 基因第1 外显子和测序位置,中间细横线上每一竖线代表一个CpG 位点

表1 亚硫酸氢盐修饰后Klotho 的引物及序列

引物	序列(5'→3')	长度(bp)
PCR引物F	GTGGGAGAAAAGTGAGAGTAG	21
PCR引物R	AAACCCTCAAATTCATTCTCTTTACCTACC	30
测序引物S	AAGTGAGAGTAGGTG	15
待分析序列	TTTTTTTAGYGGYGYGTTTYGTTAGGGTTYGGTAGGATTTYGTTTTTAAGT(焦磷酸盐变性修饰后)	51

表1 亚硫酸氢盐修饰后Klotho 的引物及序列

引物	序列(5'→3')	长度(bp)
PCR引物F	GTGGGAGAAAAGTGAGAGTAG	21
PCR引物R	AAACCCTCAAATTCATTCTCTTTACCTACC	30
测序引物S	AAGTGAGAGTAGGTG	15
待分析序列	TTTTTTTAGYGGYGYGTTTYGTTAGGGTTYGGTAGGATTTYGTTTTTAAGT(焦磷酸盐变性修饰后)	51

表2 各基因引物序列

基因	引物
Klotho	5'-CACGGCAAGGGTGCGTCCAT-3'
α-SMA	5'-TCGCGCCCACGAGATGGAGA-3'
	5'-AGTACGACGACGACGGCTA-3'
	5'-CACACTCCACGCAAAAGCAC-3'
Cbfα1	5'-TCAACGATCTGAGATTTGTGGG-3'
	5'-GGGGAGGATTTGTGAAGACGG-3'
OPN	5'-GAAGTTTCGCAGACCTGACAT-3'
	5'-GTATGCACCATTCAACTCCTCG-3'
DNMT1	5'-AGGCGGCTCAAAGATTTGGAA-3'
	5'-GCAGAAATTCGTGCAAGAGATTC-3'
DNMT3a	5'-AGTACGACGACGACGGCTA-3'
	5'-CACACTCCACGCAAAAGCAC-3'
DNMT3b	5'-ACCTCGTGTGGGGAAAGATCA-3'
	5'-CCATCGCCAAACCACTGGA-3'
β-actin	5'-TCACCCACACTGTGCCCATCTACGA-3'
	5'-CAGCGGAACCGCTCATTGCCAATGG-3'

表2 各基因引物序列

基因	引物
Klotho	5'-CACGGCAAGGGTGCGTCCAT-3'
α-SMA	5'-TCGCGCCCACGAGATGGAGA-3'
	5'-AGTACGACGACGACGGCTA-3'
	5'-CACACTCCACGCAAAAGCAC-3'
Cbfα1	5'-TCAACGATCTGAGATTTGTGGG-3'
	5'-GGGGAGGATTTGTGAAGACGG-3'
OPN	5'-GAAGTTTCGCAGACCTGACAT-3'
	5'-GTATGCACCATTCAACTCCTCG-3'
DNMT1	5'-AGGCGGCTCAAAGATTTGGAA-3'
	5'-GCAGAAATTCGTGCAAGAGATTC-3'
DNMT3a	5'-AGTACGACGACGACGGCTA-3'
	5'-CACACTCCACGCAAAAGCAC-3'
DNMT3b	5'-ACCTCGTGTGGGGAAAGATCA-3'
	5'-CCATCGCCAAACCACTGGA-3'
β-actin	5'-TCACCCACACTGTGCCCATCTACGA-3'
	5'-CAGCGGAACCGCTCATTGCCAATGG-3'

图2 各组人主动脉平滑肌细胞茜素红钙盐沉积染色结果( ×40) 注:A 为对照组;B 为硫酸吲哚酚1 000 μmol/L 组;C 为硫酸吲哚酚1 000 μmol/L+5-Aza-2dc 10 μmol/L 组

图3 不同浓度硫酸吲哚酚干预下Klotho 及钙化相关蛋白的表达注:IS 为硫酸吲哚酚;Cbfα1 为核结合因子α1;α-SMA 为α-平滑肌肌动蛋白; OPN 为骨桥蛋白;ALP 为碱性磷酸酶A 为Klotho 蛋白的表达;B 为Cbfa1 蛋白的表达;C 为a-SMA、smoothin 蛋白的表达;D 为ALP、OPN 蛋白的表达

图4 不同硫酸吲哚酚干预浓度Klotho 基因6 个CG 位点甲基化程度注:IS 为硫酸吲哚酚;与未加入IS 组比较:^aP ＜0.05, ^bP ＜0.001

图5 不同硫酸吲哚酚干预浓度甲基化转移酶蛋白的表达注:IS 为硫酸吲哚酚;DNMI 为甲基化转移酶A 为随硫酸吲哚酚干预浓度的增加,DNMT1 mRNA、DNMT3amRNA 的表达随之增加,DNMT3b mRNA 的表达未随硫酸吲哚酚干预浓度变化;B 为随硫酸吲哚酚干预浓度增加,DNMT1 蛋白表达增加

图6 5-氮杂胞苷降低硫酸吲哚酚诱导的Klotho 基因超甲基化注:IS 为硫酸吲哚酚;5-Aza-2-dc 为5-氮杂胞苷;与未加入5-氮杂胞苷组比较为^aP ＜0.05;^b P ＜0.01

图7 5-氮杂胞苷抑制硫酸吲哚酚诱导的血管平滑肌细胞成骨转化注:IS 为硫酸吲哚酚;5-Aza-2dc 为5-氮杂脱氧胞苷;Cbfα1 为核转录因子α1;α-SMA 为平滑肌细胞肌动蛋白α;ALP 为碱性磷酸酶;OPN 为骨桥蛋白A 为5-Aza-2dc 可抑制IS 诱导的Klotho 表达下调;B、C、D 为5-Aza-2dc 可抑制IS 诱导的血管平滑肌细胞发生成骨转化(a-SMA 及smoothelln表达较IS 组上调,Cbfa1、ALP 及OPN 表达较IS 组下调)

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Effect mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate

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Effect mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate