长链非编码RNA-MALAT1在高糖诱导的人腹膜间皮细胞纤维化过程中的作用

doi:10.3877/cma.j.issn.2095-3216.2016.03.008

目的

探讨长链非编码RNA(lncRNA)- MALAT1在人腹膜间皮细胞(HPMCs)高糖损伤导致的腹膜纤维化中的作用。

方法

将永生化的HPMCs分为对照组及高糖(50 mmol/L葡萄糖)刺激1、3、5、7 d组，使用实时荧光定量PCR及Western印迹方法，检测MALAT1、表型转换及纤维化相关分子E-钙黏素(E-cad)、ɑ-平滑肌肌动蛋白(ɑ-SMA)、胶原蛋白I(ColⅠ)、胶原蛋白Ⅲ(Col Ⅲ)、纤连蛋白(Fn)、结缔组织生长因子(CTGF)在mRNA及蛋白水平的表达变化；给予HPMCs转染MALAT1的过表达慢病毒，检测MALAT1、表型转换及纤维化相关分子E-cad、ɑ-SMA、ColⅠ、ColⅢ、Fn、CTGF在mRNA及蛋白水平的表达变化；给予高糖刺激72 h后的HPMCs转染MALAT1siRNA，再检测以上成分的mRNA及蛋白水平的表达变化。采用SPSS 18.0统计软件进行数据分析。

结果

高糖刺激HPMCs后，随时间的延长，MALAT1及间质标志分子ɑ-SMA、纤维化相关分子ColⅠ、ColⅢ、Fn、CTGF的表达明显增加与对照组比较差异有统计学意义(mRNA水平MALAT1、α-SMA、ColⅠ、ColⅢ、Fn、CTGF第7天时分别为t＝ 7.080、t＝6.325、t＝7.205、t＝11.70、t＝5.739、t＝9.221，P<0.05；蛋白水平α-SMA、ColⅠ、ColⅢ、Fn、CTGF第7天时分别为t＝3.429、t＝3.846、t＝5.274、t＝3.751、t＝4.093，P<0.05)，上皮标志分子E-cad mRNA及蛋白水平表达逐渐下降(第7天时mRNA t＝7.270、P＝0.0019，蛋白水平t＝5.658、P＝0.0048)；慢病毒转染使MALAT1上调后，MALAT1、ɑ-SMA、ColⅠ、ColⅢ、Fn、CTGF的表达增加(mRNA水平MALAT1 α-SMA、ColⅠ、ColⅢ、Fn、CTGF分别为t＝7.151、t＝6.495、t＝6.068、t＝8.660、t＝5.283、t＝3.230，P<0.05；蛋白水平α-SMA、ColⅠ、ColⅢ、Fn、CTGF分别为t＝3.980、t＝3.623、t＝3.351、t＝4.965、t＝5.804，P<0.05)，E-cad表达下降(mRNA水平t＝5.511，P＝0.0053；蛋白水平t＝6.397, P＝0.0031)；使用siRNA下调MALAT1后，MALAT1、ɑ-SMA、ColⅠ、ColⅢ、Fn、CTGF的表达下降(mRNA水平MALAT1、ɑ-SMA、ColⅠ、ColⅢ、Fn、CTGF分别为t＝2.854、t＝5.511、t＝3.442、t＝2.442、t＝4.407、t＝6.556，P< 0.05；蛋白水平ɑ-SMA、ColⅠ、ColⅢ、Fn、CTGF分别为t＝3.241、t＝7.589、t＝7.427、t＝4.319、t＝3.976，P< 0.05), E-cad表达增加(mRNA水平t＝2.865、P＝ 0.0457；蛋白水平t＝2.823、P＝0.0477)。

结论

MALAT1参与高糖刺激所引起的HPMCs的纤维化过程，并且可以促进腹膜纤维化的发生。

关键词: 腹膜纤维化, 上皮-间充质转分化, 长链非编码RNA, MALAT1

Objective

To study the role of long noncoding RNA-MALAT1 in human peritoneal mesothelial cells (HPMCs) fibrosis induced with high glucose (HG).

Methods

The expression of MALAT1, E-cadherin, alpha-smooth muscle actin (ɑ-SMA), collagenⅠ, collagen Ⅲ, fibronectin, and connective tissue growth factor (CTGF) were tested by real-time PCR and Western blot in HPMCs incubated with HG medium (50 mmol/L glucose) or in control medium for different time (for 0, 1, 3, 5, and 7 days). The expression of MALAT1, E-cadherin, ɑ-SMA, collagenⅠ, collagenⅢ, fibronectin, and CTGF were also assayed by real-time PCR and Western blot in HPMCs after transfection with MALAT1 lentivirus, or control lentivirus. MALAT1 small interfering RNA (siRNA), or control siRNA was transfected into HPMCs that had been cultured with HG medium for 72 hours.

Results

The expression of MALAT1, ɑ-SMA, collagenⅠ, collagenⅢ, fibronectin, and CTGF in HPMCs cultured with HG medium for 72 hours were significantly increased (P<0.05), but E-cadherin decreased with the stimulation time of HG (P<0.05). Compared with control lentivirus, the expression of MALAT1, ɑ-SMA, collagenⅠ, collagenⅢ, fibronectin, and CTGF were increased, but E-cadherin was reduced after transfection with the MALAT1 lentivirus (P<0.01), suggesting that overexpression of MALAT1 might lead to an increase in epithelial-mesenchymal transition (EMT) and fibrosis of HPMCs. The expression of MALAT1, ɑ-SMA, collagen, collagenⅢ, fibronectin, and CTGF decreased (P<0.05), but E-cadherin increased (P<0.05) after silencing the expression of MALAT1 by siRNA compared with the control cells, demonstrating that down-regulation of MALAT1 could inhibit EMT and fibrosis in HG-induced HPMCs.

Conclusion

MALAT1 might participate in and promote the HG-induced fibrosis and damage of HPMCs.

Key words: Peritoneal fibrosis, Epithelial-mesenchymal transition, LncRNA, MALAT1

表1 实时荧光定量PCR引物序列

基因	引物序列
β-actin	正义引物：5′-CTCCATCCTGGCCTCGCTGT-3′
β-actin	反义引物：5′-GCTGTCACCTTCACCGTTCC-3′
α-SMA	正义引物：5′-TCCGGAGCGCAAATACTCTG-3′
α-SMA	反义引物：5′-CCGGCTTCATCGTATTC-3′
CTGF	正义引物：5′-CTCTGCTCTCGCAGCTTACC-3′
CTGF	反义引物：5′-CATCCCACAGGTCTTGGAAC-3′
ColⅠ	正义引物：5′-TGACCTCAAGATGTGCCACT-3′
ColⅠ	反义引物：5′-CCTCTTGTCCTTGGGGTTCT-3′
ColⅢ	正义引物：5′-CGAAACACACTGGGGAATGG-3′
ColⅢ	反义引物：5′-TTTGTCGGTCACTTGCACTG-3′
Fn	正义引物：5′-GGACATGCATTGCCTACTCG-3′
Fn	反义引物：5′-GAATCCTGGCATTGGTCGAC-3′
E-cad	正义引物：5′-CGCATTGCCACATACACTCT-3′
E-cad	反义引物：5′-TTGGCTGAGGATGGTGTAAG-3′
MALAT1	正义引物：5′- CTAGCTAGCGCTTACAATCTTAGAGTGGTAGGCA -3′
MALAT1	反义引物：5′- CCGCTCGAGTCTAACATAGTTCAACCCACCAAAG-3′

表1 实时荧光定量PCR引物序列

基因	引物序列
β-actin	正义引物：5′-CTCCATCCTGGCCTCGCTGT-3′
β-actin	反义引物：5′-GCTGTCACCTTCACCGTTCC-3′
α-SMA	正义引物：5′-TCCGGAGCGCAAATACTCTG-3′
α-SMA	反义引物：5′-CCGGCTTCATCGTATTC-3′
CTGF	正义引物：5′-CTCTGCTCTCGCAGCTTACC-3′
CTGF	反义引物：5′-CATCCCACAGGTCTTGGAAC-3′
ColⅠ	正义引物：5′-TGACCTCAAGATGTGCCACT-3′
ColⅠ	反义引物：5′-CCTCTTGTCCTTGGGGTTCT-3′
ColⅢ	正义引物：5′-CGAAACACACTGGGGAATGG-3′
ColⅢ	反义引物：5′-TTTGTCGGTCACTTGCACTG-3′
Fn	正义引物：5′-GGACATGCATTGCCTACTCG-3′
Fn	反义引物：5′-GAATCCTGGCATTGGTCGAC-3′
E-cad	正义引物：5′-CGCATTGCCACATACACTCT-3′
E-cad	反义引物：5′-TTGGCTGAGGATGGTGTAAG-3′
MALAT1	正义引物：5′- CTAGCTAGCGCTTACAATCTTAGAGTGGTAGGCA -3′
MALAT1	反义引物：5′- CCGCTCGAGTCTAACATAGTTCAACCCACCAAAG-3′

图1 高糖刺激不同时间MALAT1、细胞表型转换及纤维化相关分子的mRNA及蛋白水平表达(实时荧光定量PCR, Western印迹)

图2 MALAT1过表达慢病毒转染后MALAT1、细胞表型转换及纤维化相关分子在mRNA、蛋白水平的表达(实时荧光定量PCR，Western印迹)

图3 MALAT1 siRNA转染后MALAT1、细胞表型转换及纤维化相关分子在mRNA、蛋白水平的表达变化(实时荧光定量PCR，Western印迹)

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Role of long noncoding RNA-MALAT1 in human peritoneal mesothelial cells fibrosis induced with high glucose

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Role of long noncoding RNA-MALAT1 in human peritoneal mesothelial cells fibrosis induced with high glucose