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Objective To explore the effect and potential mechanism of miR-125b-5p targecting ΔNp63α in keratinocyte differentiation. Methods To induce keratinocytes (HaCaT) differentiation, 1.8 mmol/L calcium chloride was added in the culture media. The mRNA level of miR-125b-5p, ΔNp63α, cytokeratin 10 (CK10), involucrin (Inv), transglutaminase 1(TG1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR) during keratinocyte differentiation were quantified by qRT-PCR (at day 0, 1, 5 and 7). The expression of ΔNp63α was detected by cellular immunofluorescence after treatment for 0 and 5 days. The binding site of miR-125b-5p to ΔNp63α was identified by the bibiserv website. The mimics/inhibitors of miR-125b-5p and the mimics/inhibitors of the negative control were transfected into keratinocytes. After five days of culture, the mRNA and protein expression of ΔNp63α, CK10, Inv, TG1, PI3K, Akt and mTOR were determined by qRT-PCR and Western blotting analysis. Results Compared with calcium chloride treatment for 0 days(1.00±0.02), the relative expression level of miR-125b-5p decreased significantly at 1 (0.17±0.02), 5 (0.08±0.01) and 7 days(0.07±0.02) (P<0.001). Compared with calcium chloride treatment for 0 days, the expression of ΔNp63α, CK10, Inv, TG1, PI3K,Akt and mTOR mRNA increased gradually after calcium chloride treatment for 1, 5 and 7 days (P<0.05). The results of cellular immunofluorescence assay showed that the positive rate of ΔNp63α increased after calcium chloride treatment for five days(96.9%±0.9% vs. 43.2%±8.2%, P<0.001). miR-125b-5p can bind to the 3'-UTR site of ΔNp63α. Compared with negative control mimic group, the mRNA expression levels of ΔNp63α, CK10, Inv, TG1, PI3K, Akt, and mTOR decreased significantly in miR-125b-5p mimic group. In addition, the protein expression levels of ΔNp63α, CK10, Inv, TG1, PI3K, Akt, p-Akt, mTOR, and p-mTOR also decreased significantly (P<0.05). Interestingly, the inhibition of miR-125b-5p could reverse the above effects (P<0.05). Conclusion miR-125b-5p targeting ΔNp63α inhibits keratinocyte differentiation by PI3K/Akt/mTOR signal pathway.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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