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Objective@#MicroRNA-221/222 is involved in the pathogenesis of intrahepatic cholestasis of pregnancy (ICP) to promote the apoptosis of placental bile acids through human trophoblastic cells. This study investigates the effects of miR-221/222 on proliferation, apoptosis and apoptosis-related proteins of human trophoblast HTR-8/SVneo (HTR-8 cells) to understand its role in promoting trophoblastic apoptosis.@*Methods@#The experiment was divided into transfection group and negative control group. Transient transfection method was used in both groups. The transfection efficiency was detected by RT-QPCR after 48 h transfection. CCK-8 was used to detect the proliferation of HTR-8 cells and the apoptosis of HTR-8 cells were analyzed by flow cytometry. Western blot was used to detect the expression of B-cell Lymphoma 2 (Bcl-2) in HTR-8 cells. Data were compared with t-test.@*Results@#The expression of miR-221/222 transfected group (25.43±0.80, 22.70±0.95) was increased significantly in the HTR-8 cells than that to negative control group (1.14±0.14, 1.58±0.14), and P value was < 0.01, the difference was statistically significant. The expression of Bcl-2 protein in mir-221/222 transfection group was (0.56 ± 0.03, 0.53 ± 0.03), and the protein expression was decreased compared with negative control group (0.72 ± 0.003, 0.76 ± 0.04). P value was < 0.05, the difference was statistically significant, and compared with the mir-221/222 negative control group (8.827 ± 0.48, 11.80 ± 0.45), cell apoptosis of mir-221/222 transfection group (42.53 ± 4.47, 24.09 ± 2.53) increased significantly, P value was < 0.01, and the difference was statistically significant. Proliferation rate in mir-221/222 transfection group was (0.82 ± 0.02, 0.74±0.01), and proliferation was inhibited, when compared with control group (1.15 ± 0.08, 1.06 ± 0.08), P value was < 0.05, and the difference was statistically significant.@*Conclusion@#miR-221/222 may promote the apoptosis of human trophoblastic cells by down regulating the expression of apoptosis inhibitory protein bcl-2, leading to placental dysfunction and impairing the normal bile acid transport function of placenta. This mechanism may be involved in the occurrence and development of ICP.
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OBJECTIVE: Recent studies have indicated the possibility that genistein may improve depression via regulating the expression of miR-221/222. This study is to explore whether genistein could improve depression by altering miR-221/222 levels and investigate the possible mechanisms involved in the improvement effect of genistein. METHODS: The animal model of depression was established through unpredictable chronic mild stress. Nest building test and splash test were adapted to evaluate the effects of genistein on depressive symptoms in mice. qRT-PCR and western blot analysis were used to detect the expression of miR-221/222 and connexin 43 (Cx43) in the prefrontal cortex of the mice. In vitro, U87-MG astrocytes were treated with genistein and the expression of miR-221/222 and Cx43 was measured. The dual-luciferase reporter assay was used to verify whether Cx43 was a direct target of miR-221/222. RESULTS: The behavioral tests showed that genistein could significantly reduce depression symptoms of mice, and this remission was not affected by gender. Genistein in vivo and in vitro could reduce increased levels of miR-221 and miR-222 in the prefrontal cortex of depressed mice, while upregulate Cx43 expression. Dual-luciferase reporter assay suggested Cx43 was directly regulated by miR-221/222 in astrocytes. CONCLUSION: Genistein can play its antidepressant effect through down-regulating miR-221/222 by targeting Cx43.
Subject(s)
Animals , Mice , Astrocytes , Behavior Rating Scale , Blotting, Western , Connexin 43 , Depression , Genistein , In Vitro Techniques , Models, Animal , Prefrontal CortexABSTRACT
Objective To study the pathway of miR-221/222 in enhancing radiation resistance of glioblastoma.Methods After 2 Gy of X-ray irradiation,the expressions of miR-221/222 in U251,U87 and LN229 glioblastma cells were detected with real-time PCR.Clonogenic assay was used to measure the radiosensitivity of glioblastoma after knocking down miR-221/222.ChIP assay was used to identify the combination situation of c-jun and miR-221/222.Luciferase assay was applied to check whether PTEN was a target of miR-221/222.Western blot was used to detect the expression of relevant proteins in the glioblastoma cells after knocking down miR-221/222.The effect of miR-221/222 and irradiation on growth of glioblastoma in nude mice was also observed.Results The expression of miR-221/222 was increased by irradiation(t =5.48 ～29.21,P ＜ 0.05) and the radiosensitivity of anti-miR-221/222-transfected cells was alsoincreased(F=1 202.22,1 789.12,1 012.32,P＜0.05).MiR-221/ 222 was transcriptionally regulated by c-jun with a target of PTEN (t =13.16,P ＜ 0.05).When miR-221/222 was knocked-down,the expression of pAkt and DNA-PKcs were down-regulated while PTEN and GSK-3β were up-regulated,and the expression of Akt were not changed.Moreover,the growth of xenograft tumor was significantly inhibited by the combination treatment of anti-miR-221/222 and irradiation(F =56.36,P ＜ 0.05).Conclusions The expression of miR-221/222 in glioblastoma cells can be increased by irradiation,and the activation of Akt pathway downstream miR-221/222 could enhance the radiation resistance of glioblastoma.