长链非编码RNA-ATB对瘢痕疙瘩成纤维细胞增殖和凋亡的影响及其机制研究

Effects of lncRNA-ATB on the proliferation and apoptosis of keloid fibroblasts and its mechanism

摘要:

目的 探讨被转化生长因子b激活的长链非编码RNA（lncRNA-ATB）对瘢痕疙瘩成纤维细胞增 殖和凋亡的影响，并分析其作用机制。方法 检测瘢痕疙瘩和正常皮肤组织中ATB、microRNA-200c（miR200c）和DNA甲基转移酶DNMT3B的表达水平，并分析其相关性。在成纤维细胞中分别转染sh-ATB、o/eATB、miR-200c mimics、miR-200c inhibitors和sh-DNMT3B，采用CCK-8法检测细胞增殖情况；流式细胞分 析仪检测细胞凋亡情况；双荧光素酶测定法检测 ATB 和 miR-200c、miR-200c 和 DNMT3B 靶向结合情况； Western blotting和qRT-PCR检测增殖相关分子周期素依赖性激酶6（CDK6）和凋亡相关分子半胱氨酸天冬氨 酸蛋白酶-3（Caspase-3）相对表达量。结果 ATB和DNMT3B在瘢痕疙瘩组织中的表达水平均高于正常皮肤 （P <0.05），miR-200c在瘢痕疙瘩组织中的表达水平低于正常皮肤（P <0.05），同时ATB与miR-200c在瘢痕疙 瘩组织中呈负相关（rs =-3.429，P <0.05），miR-200c 与 DNMT3B 在瘢痕疙瘩组织中呈负相关（rs=-2.011，P < 0.05）。双荧光素酶结果显示ATB能够靶向结合miR-200c，miR-200c能够靶向结合DNMT3B。在瘢痕疙瘩 成纤维细胞中转染sh-ATB或miR-200c mimics后，细胞的增殖能力和增殖相关分子CDK6的表达水平均降低 （P <0.05），细胞的凋亡率和凋亡相关分子Caspase-3的表达水平均升高（P <0.05）。在回复实验中，瘢痕疙瘩成 纤维细胞中共转染sh-ATB和miR-200c inhibitors能够逆转单独转染sh-ATB对细胞增殖和凋亡的影响（P > 0.05），共转染o/e-ATB和miR-200c inhibitors能够进一步增强单独转染o/e-ATB对细胞增殖和凋亡的影响 （P <0.05）；瘢痕疙瘩成纤维细胞中共转染 miR-200c inhibitors 和 sh-DNMT3B 能够逆转单独转染 miR-200c inhibitors对细胞增殖和凋亡的影响（P <0.05），共转染miR-200c mimics和sh-DNMT3B能够进一步增强单独 转染 miR-200c mimics 对细胞增殖和凋亡的影响（P <0.05）。结论 ATB 在瘢痕疙瘩组织中高表达，低表达 ATB能够通过调控miR-200c/DNMT3B通路，抑制瘢痕疙瘩成纤维细胞增殖，并促进细胞凋亡。

Abstract:

Objective To investigate the effects of long non-coding RNA activated by transforming growth factor-β (lncRNA-ATB) on proliferation and apoptosis of keloid fibroblasts and to analyze its mechanism. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of ATB, microRNA-200c (miR-200c) and DNA methyltranserase 3 beta (DNMT3B) in keloids and normal skin tissues, and the correlation among the three was analyzed. The fibroblasts were transfected with sh-ATB, o/e-ATB, miR-200c mimics, miR-200c inhibitors and sh-DNMT3B. The cell proliferation was detected by cell counting kit-8 (CCK-8) assay, and the cell apoptosis was detected by flow cytometry. The dual luciferase assay was used to detect the binding of ATB and miR-200c, and that of miR-200c and DNMT3B. The expression levels of proliferation-related molecule cyclin-dependent kinase 6 (CDK-6) and apoptosis-related molecule caspase-3 were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). Results The expression levels of ATB and DNMT3B in keloid tissues were higher than those in normal skin (P < 0.05). The expression level of miR-200c in keloid tissues was lower than that in normal skin (P < 0.05). The expression levels of ATB and miR-200c (rs = - 3.429), and those of miR-200c and DNMT3B (rs = -2.011) were negatively correlated in keloid tissues (P < 0.05). The dual luciferase assay showed that ATB was able to target miR-200c, and that miR-200c was able to target DNMT3B. After transfection of sh-ATB or miR-200c mimics into keloid fibroblasts, the proliferation of cells and the expression of CDK-6 were decreased (P < 0.05), while apoptosis rate and the expression of caspase-3 were increased (P < 0.05). In the recovery experiment, co-transfection of sh-ATB and miR-200c inhibitors into keloid fibroblasts reversed the effects of single transfection of sh-ATB on cell proliferation and apoptosis (P > 0.05). Co-transfection of o/e- ATB and miR-200c inhibitors could further enhance the effects of single transfection of o/e-ATB on cell proliferation and apoptosis (P < 0.05). Co-transfection of miR-200c inhibitors and sh-DNMT3B in keloid fibroblasts could reverse the effects of single transfection of miR-200c inhibitors on cell proliferation and apoptosis (P > 0.05). Co-transfection of miR-200c mimics and sh-DNMT3B can further enhance the effects of single transfection of miR200c mimics on cell proliferation and apoptosis (P < 0.05). Conclusions ATB was highly expressed in keloid tissues, and low expression of ATB could inhibit proliferation and promote apoptosis of fibroblasts by regulating miR-200c/DNMT3B pathway.