miR-542-5p对1-磷酸鞘氨醇诱导的IEC-6细胞增殖的抑制作用

miR-542-5p down-regulates IEC-6 cell proliferation induced by sphingosine-1-phosphate

目的:观察miR-542-5p对1-磷酸鞘氨醇(S1P)诱导的大鼠小肠隐窝上皮IEC-6细胞增殖的影响。方法:建立稳定表达鞘氨醇激酶1(SphK1)的IEC-6细胞系(SphK1-IEC-C1和SphK1-IEC-C2),Western blot检测SphK1蛋白表达,放射性同位素示踪法测定SphK1酶活性和S1P分泌,细胞计数绘制生长曲线观察细胞增殖,流式细胞术分析细胞周期变化,实时荧光定量PCR检测miR-542-5p表达。结果:与对照细胞相比,细胞系SphK1-IECC1和SphK1-IEC-C2中SphK1蛋白表达显著升高,SphK1酶活性升高,细胞内外S1P浓度升高,细胞生长速度加快,细胞周期中S期细胞所占比例升高,miR-542-5p的表达量降低;在IEC-6细胞的培养液中加入S1P(0.5~10μmol/L),能显著抑制miR-542-5p表达;采用siRNA干扰降低IEC-6细胞的SphK1,可明显升高miR-542-5p表达。升高SphK1-IEC-C1和SphK1-IEC-C2细胞中miR-542-5p水平,则可降低S期细胞比例,抑制细胞增殖。结论:S1P通过降低IEC-6细胞中miR-542-5p水平,引起细胞周期由G1期向S期转换,促进IEC-6细胞增殖。

AIM： T o o b s e r v e t he e f f e c t s o f miR- 542- 5p o n th e p r o l i f e r a t io n o f r a t sma l l int e s t i ne c r y p t e pi t he-lial IEC-6 cells induced by sphingosine-1-phosphate （SIP）. METHODS： T w o IE C - 6 c e l l l i n e s （ S p h K l - I E C - C l a n d SphKl-IEC-C2） were established, which expressed sphingosine kinase-1 （SphKl） stably. Radioactive tracer was used to detect SphKl activity and SIP secretion. The cell proliferation was observed by cell counting and described by drawing growth curve, and the cell cycle analysis was carried out by flow cytometry. The level of miR-542-5p was evaluated by RT- qPCR. RESULTS： Compar ed wi th cont r ol vector cel l s wi thout SphKl cDNA, both SphKl - IEC-Cl and SphKl - I EC-C2 cel l lines showed that Sphkl was elevated, both intracellular and extracellular SIP increased dramatically, the rate of cell growth was faster, the percentage of the cells in S phase increased, and miR-542-5p expression decreased. SIP （0. 5 ？10 jjimol/L） led to the decrease in miR-542-5p expression. On the contrary, SphKl s i l e n c in g r e s u l t e d i n t h e i n c r e a s e i n m iR - 542-5p expression in the IEC-6 cells. The miR-542-5p was elevated in SphKl-IEC-Cl cells and SphKl-IEC-C2 cells, which caused the decrease in the percentage of the cells in S phase. The cell growth rate in the above-mentioned 2 cell lines decreased compared with negative control group. CONCLUSION： I n IE C - 6 c e l l s , S I P p r om o t e s p r o l i f e r a t io n b y i n h ib i t in g miR-542-5p expression, which induces the cell cycle transferring from Gl p h a s e to S p h a s e .