METHODS: GC-2 and TM4 cells were treated with TCS (0.5-80 μM) for 24 or 48 hours. Effect of TCS on proliferation of GC-2 and TM4 cells was detected using a cell counting kit-8 (CCK8) assay. Expression of miR-17 family and autophagy genes were detected. The interaction between miR-20a-5 P and PTEN was determined by a dual-luciferase reporter assay.
RESULTS: TCS decreased cell proliferation of GC-2 and TM4 cells. Expression of autophagy-related genes and miR-17 family was altered by TCS. PTEN expression was significantly increased, whereas the expression of miR-20a-5 P was significantly decreased in GC-2 and TM4 cells. As predicted in relevant databases, there is a binding site of miR-20a-5 P in PTEN. The expression of PTEN was significantly down-regulated by the miR-20a-5 P mimic.
CONCLUSIONS: As a downstream target of miR-20a-5 P, PTEN functioned in the autophagy process of which TCS inhibited the proliferation of spermatocyte cells. Our results provided new ideas for revealing the molecular mechanism and protective strategy on male infertility.
方法:GC-2和TM4细胞用TCS(0.5-80μM)处理24或48小时。使用细胞计数试剂盒-8(CCK8)测定法检测TCS对GC-2和TM4细胞增殖的影响。检测miR-17家族和自噬基因的表达。miR-20a-5P和PTEN之间的相互作用通过双荧光素酶报告基因测定来确定。
结果:TCS降低了GC-2和TM4细胞的增殖。TCS改变了自噬相关基因和miR-17家族的表达。PTEN表达显著增加,而miR-20a-5P在GC-2和TM4细胞中的表达显著降低。正如在相关数据库中预测的那样,PTEN中存在miR-20a-5P的结合位点。PTEN的表达被miR-20a-5P模拟物显著下调。
结论:作为miR-20a-5P的下游靶标,PTEN在自噬过程中起作用,TCS抑制精母细胞的增殖。本研究结果为揭示男性不育的分子机制和保护策略提供了新思路。