ABSTRACT
Objective:To explore the mechanism underlying microRNA (miR) -125a-mediated inhibition of proliferation of keratinocytes.Methods:After 24-hour pretreatment with interleukin (IL) -23, human HaCaT keratinocytes were divided into miR-125a group and miR-NC group transfected with a miR-125a overexpression plasmid and a control plasmid, respectively. Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative ability of HaCaT cells in the two groups at 0, 24, 48 and 72 hours after transfection, real-time fluorescence-based quantitative PCR to determine the mRNA expression of miR-125a and IL-23 receptors (IL-23R) in the two groups 24 hours after transfection, and Western blot analysis to determine the protein expression of IL-23R, Janus kinase 2 (JAK2) , protein kinase B (AKT) and phosphorylated AKT (p-AKT) in the two groups 48 hours after transfection. Dual-luciferase reporter assay was performed to verify the targeting relationship between miR-125a and IL-23R. Comparison of means between two groups was carried out by using t test, and changes in the proliferative ability of HaCaT cells over time were evaluated by using repeated measures analysis of variance. Results:After plasmid transfection, the relative expression of miR-125a was significantly higher in the miR-125a group (6.377 ± 0.745) than in the miR-NC group (0.700 ± 0.222; t=7.305, P=0.002) . At 0, 24 and 48 hours after transfection, there was no significant difference in cellular proliferative ability between the miR-125a group and the miR-NC group ( t=0.663, 0.623 and 1.930, respectively, all P > 0.05) ; at 72 hours after transfection, the cellular proliferative ability was significantly lower in the miR-125a group than in the miR-NC group ( t=4.407, P < 0.05) . The IL-23R mRNA expression was significantly lower in the miR-125a group than in the miR-NC group ( t=3.082, P < 0.05) . Compared with the miR-NC group, the miR-125a group showed significantly decreased protein expression of IL-23R, JAK2 and p-AKT ( t=11.715, 6.996, 12.424, P < 0.001,=0.002, < 0.001, respectively) . Dual-luciferase reporter assay showed targeted binding of miR-125a to IL-23R. Conclusion:MiR-125a may inhibit the proliferation of keratinocytes by negatively regulating the IL-23R/JAK2/AKT signaling pathway.
ABSTRACT
Objective:To investigate the correlation between microRNA-125a (miR-125a) expression and inflammatory cytokine levels in skin lesions of patients with psoriasis vulgaris, and to evaluate the effect of miR-125a on the proliferation of a human immortalized keratinocyte cell line HaCaT.Methods:Totally, lesional and adjacent non-lesional skin tissues were collected from 40 patients with psoriasis vulgaris in the Seventh People′s Hospital of Shenyang from 2017 to 2018, and real-time fluorescence-based quantitative reverse transcription PCR was performed to determine the expression of miR-125a in the skin tissues, as well as the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-17 in the lesional skin tissues. HaCaT cells were divided into 4 groups to be transfected with a miR-125a overexpression plasmid (miR-125a overexpression group), an overexpression control plasmid (overexpression control group), a miR-125a interference plasmid (miR-125a interference group) and an interference control plasmid (interference control group), respectively. Cell counting kit-8 (CCK8) assay was performed to assess the proliferative ability of HaCaT cells in the groups at 0, 24, 48, 72 hours after transfection, and double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the levels of TNF-α, IL-1β, IL-6 and IL-17 in the culture supernatant of HaCaT cells. Spearman rank correlation test was used for correlation analysis, and t test for the comparison of means between two groups. Results:The relative expression of miR-125a was significantly lower in the lesional skin tissues (expressed as 2 -ΔΔCt, 0.389 ± 0.354) than in the non-lesional skin tissues (1.106 ± 0.396, t = 7.717, P < 0.001) in patients with psoriasis vulgaris. The expression of miR-125a was negatively correlated with the mRNA expression of TNF-α, IL-1β and IL-17 in psoriatic lesions ( r = -0.447, -0.424, -0.436, all P < 0.01). Immediately and 24 hours after transfection with the plasmids, there was no significant difference in the cell proliferative ability between the miR-125a overexpression group and overexpression control group ( t = 0.282, 1.445, respectively, both P > 0.05), or between the miR-125a interference group and interference control group ( t = 0.120, 1.543, respectively, both P > 0.05). Forty-eight and 72 hours after the transfection, the cell proliferative ability was significantly lower in the miR-125a overexpression group than in the overexpression control group ( t = 3.222, 4.563, respectively, both P < 0.05), but significantly higher in the miR-125a interference group than in the interference control group ( t = 3.036, 3.269, respectively, both P < 0.05). In addition, the miR-125a overexpression group showed significantly decreased levels of TNF-α and IL-1β compared with the overexpression control group ( t = 4.318, 3.813, respectively, both P < 0.05) . Conclusions:MiR-125a is lowly expressed in skin lesions of patients with psoriasis vulgaris. MiR-125a can inhibit the proliferation of keratinocytes, and may play a protective role in the occurrence and development of psoriasis.
ABSTRACT
BACKGROUND:At present,the problems such as serious shortage of donor liver organs for transplantation,surgical injury,high incidence of surgical complications,as well as the high costs limit the development of liver transplantation,while the hepatic stem cell(HSC)transplantation provides a new pathway for the treatment of end-stage liver disease.OBJECTIVE:To introduce the source and classification of HSCs,research progress and problems of HSC transplantation for treatment of end-stage liver disease,and the clinical application prospects of HSC transplantation.METHODS:Articles were collected from CNKI and Medline database with the keywords of "hepatic stem cells,liver disease,transplantation" in both Chinese and English from 1999 to 2009.Among 87 articles,30 were included according to inclusion and exclusion criteria.Following reading titles and abstracts,original articles,and articles closely related to HSC transplantation with reliable argument and evidence and general analysis were included.Articles of repetitive studies and poor quality were excluded.RESULTS AND CONCLUSION:The HSC can be divided into liver-derived stem cells and non-liver-derived stem cells.Liver-derived stem cells include hepatic oval cells,mature liver cells and small hepatocyte-like progenitor cell.Non-liver-derived stem cells were mainly derived from embryonic stem cells,bone marrow hematopoietic stem cells and pancreatic stem cells.Currently,the research for the treatment of liver disease by HSC is still in its early stages.There are many difficult issues to be studied and solved in the discovery,separation,purification,comprehensive identification,cultivation,directed differentiation as well as clinical trials.However,as a new source of seed cells,HSC can not only replace the damaged tissue but can stimulate the receptor in tissue regeneration.Hence,compared with the clinical liver transplantation and bio-artificial liver,there are very bright future for the treatment of liver diseases by transplating HSC.