微RNA-939靶向启动子区调控CD2相关蛋白的表达

MicroRNA- 939 regulating CD2 -associated protein expression by targeting promoter

目的验证微RNA-939（microRNA-939，miR-939）与CD2相关蛋白（CD2AP）的靶向调控关系。方法用RegRNA软件预测人CD2AP启动子区潜在的miR-939结合位点。将人CD2AP启动子荧光素酶报告基因重组质粒pGL3-2K与microRNA阴性对照物（miR—NC）或miR-939模拟物瞬时共转染HEK-293T细胞，24h后测定相对荧光素酶活性。用miR—NC或miR-939模拟物转染HEK-293T细胞48h，反转录-实时荧光定量PCR检测CD2APmRNA表达水平，Western blot检测CD2AP蛋白表达水平。结果1．CD2AP启动子区存在2个miR-939结合位点，分别位于起始密码子ATG（定位＋1）上游-468～-491和-654～-677。2．在30nmol／L、50nmol／L水平，miR—NC组和miR-939组的相对荧光素酶活性分别为6．81±0．88比6．07±2．24、5．88±1．44比3．94±0．79，2组间差异均无统计学意义（t=3．04、2．06，P均〉0．05）；达100nmol／L水平时，荧光素酶活性分别为5．58±0．58比3．29±0．64，差异有统计学意义（t=4．07，P〈0．05）。3．在30nmol／L、50nmol／L、100nmol／L水平，miR-NC组和miR-939组的CD2APmRNA相对表达量分别为1．004±0．01比0．80±0．08、1．00±0．00比0．80±0．13、1．00±0．00比0．72±0．07，CD2APmRNA水平在各浓度均下调20％～30％，组间差异有统计学意义（t=4．44、2．93、6．84，P均〈0．05）。4．在50nmol／L水平，miR—NC组和miR-939组的CD2AP蛋白相对表达量为0．48±0．09比0．19±0．12，CD2AP蛋白表达水平下调，差异有统计学意义（t=3．36，P〈0．05）。结论CD2AP是miR-939的靶基因，miR-939通过靶向启动子区下调CD2AP的转录和表达，提示miR-939可能介导足突细胞的损伤。

Objective To verify the targeting regulatory relationship between microRNA -939 （miR -939） and CD2 - associated protein （CD2AP）. Methods The online RegRNA software was used to predict the human CD2AP promoter for potential binding sites complementary to miR - 939. HEK - 293T cells were cotransfected with human CD2AP promoter plasmid pGL3 -2K and microRNA negative control （miR -NC） or miR- 939 mimics, and the relative luciferase activity （RLA） was detected at 24 h post - transfection. HEK - 293T cells were transfected with miR - NC or miR - 939 mimics for 48 h, and the CD2AP mRNA expression level was detected by adopting reverse transcript and real- time fluorescence quantification- PCR, while the CD2AP protein expression level was detected by using Western blot. Results （ 1 ） There were 2 miR - 939 binding sites at CD2AP promoter region,located at - 468 to - 491 and - 654 to - 677 upstream of initiation codon ATG （ marked as ＋ 1 ） relatively. （2） At 30 nmol/L, 50 nmol/L,the RLA in miR - NC group and miR -939 group were 6.81±0.88 vs 6.07±2.24,5.88 ± 1.44 vs 3.94± 0. 79 relatively, and there were no significant differences between the 2 groups （ t = 3.04,2.06, all P 〉 0.05 ） , while the RLA between the 2 groups were 5.58 ±0.58 vs 3.29 ±0.64 at 100 mnol/L,and the difference was significant between the 2 groups（ t = 4.07, P 〈 0. 05 ）. （3） At 30 nmol/L,50 nmol/L and 100 nmol/L, the relative CD2AP mRNA expression in miR - NC group and miR - 939 group were 1.00 ± 0.01 vs 0. 80 ± 0.08,1.00 ± 0.00 vs 0. 80 ± 0.13 and 1.00 ±0. 00 vs 0. 72 ±0.07 relatively,while the CD2AP mRNA expression was decreased by 20% -30% at each concentration level, and there were significant differences between the 2 groups （t =4.44,2.93,6.84, all P 〈 0.05 ）. （4） At 50 nmoL/L, the relative CD2AP protein expression in miR - NC group and miR - 939 group were 0.48 ± 0.09 vs 0. 19 ± 0.12, and the CD2AP protein expression was decreased,and the difference was significant （t = 3.36,P 〈 0.05）. Conclusions CD2AP is the target gene of miR -939,and miR -939 can down -regulate the expression of CD2AP both in mRNA and protein levels by targeting its promoter region ,which indicates that miR-939 may mediate the podocyte injury.