miR-128-3p靶向沉默瘦素抑制银屑病角质形成细胞增殖及炎症反应

doi:10.3969/j.issn.1674-8115.2024.10.005

摘要/Abstract

摘要：

目的·探究miR-128-3p/瘦素（leptin，LEP）轴对银屑病中角质形成细胞过度增殖及炎症反应的调控作用。方法·选取BALB/c小鼠，分为对照组（n=10）及模型组（n=10）。模型组小鼠背部行咪喹莫特软膏连续涂抹以构建银屑病小鼠模型。构建miR-128-3p过表达和干扰质粒、LEP干扰质粒分别转染人永生化角质形成细胞HaCaT细胞。经实时定量聚合酶链反应检测miR-128-3p及LEP的mRNA，Western blotting检测LEP蛋白表达；酶联免疫吸附实验检测培养液中肿瘤坏死因子α（tumor necrosis factor α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、IL-1β的含量；MTT实验检测细胞相对活力；EdU实验检测细胞增殖；采用双荧光素酶报告基因实验验证miR-128-3p与LEP的靶向关系。结果·与对照组小鼠比，模型组小鼠miR-128-3p表达下调，Lep的基因表达及蛋白水平升高（均P<0.05）。双荧光素酶报告基因实验证实LEP是miR-128-3p下游靶点。与模拟物阴性对照（negative control of mimic，NC mimic）组相比，miR-128-3p模拟物（miR-128-3p mimic）组miR-128-3p表达上调，LEP的基因表达及蛋白降低；miR-128-3p mimic组TNF-α、IL-6、IL-1β显著低于NC mimic；miR-128-3p上调后细胞相对活力、EdU阳性细胞率均降低（均P<0.05）。与抑制物阴性对照（negative control of inhibitor，NC inhibitor）组相比，miR-128-3p抑制物（miR-128-3p inhibitor）组miR-128-3p表达下调，LEP的基因表达及蛋白水平增加；miR-128-3p下调后细胞TNF-α、IL-6、IL-1β升高；miR-128-3p表达下调导致细胞相对活力和EdU阳性细胞率增加（均P<0.05）。进一步实验结果显示：与LEP抑制物（LEP inhibitor）组相比，miR-128-3pinhibitor+ LEP inhibitor组LEP表达上调，而TNF-α、IL-6、IL-1β水平升高，细胞相对活力及EdU阳性细胞率增加（均P<0.05）。结论·miR-128-3p靶向沉默LEP抑制角质形成细胞增殖及炎症反应，抑制银屑病发生发展。

关键词: 银屑病, 瘦素, 微小RNA-128-3p, 角质形成细胞

Abstract:

Objective ·To explore the role of miR-128-3p/leptin (LEP) axis in the proliferation and inflammation of keratinocytes in psoriasis. Methods ·BALB/c mice were randomly divided into a control group (n=10) and a model group (n=10). Mice in the model group were given imiquimod on the back. miR-128-3p overexpression and interference plasmids, as well as LEP interference plasmids, were constructed and transfected into HaCaT cells, respectively. miR-128-3p and LEP mRNA were quantified by real-time quantitative polymerase chain reaction, and LEPprotein levels were detected by using Western blotting. Enzyme-linked immunosorbent assay was used to measure the content of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the culture medium. MTT assay was used to evaluate cell activity and EdU assay was to used to test cell proliferation. The binding site between miR-128-3p and LEP was determined by using a dual luciferase reporter gene assay. Results ·Compared with mice in the control group, mice in the model group showed downregulated expression of miR-128-3p and upregulated expression of LEP at both RNA and protein levels (all P<0.05). The dual luciferase reporter gene assay confirmed that LEP was a downstream target of miR-128-3p. Compared with the negative control mimic (NC mimic) group, expression of miR-128-3p was up-regulated in the miR-128-3p mimic group, and expression of LEP was reduced. The levles of TNF-α, IL-6, and IL-1β were significantly lower in the miR-128-3p mimic group than in the NC mimic group. The relative cell viability and EdU-positive cell rate were also reduced after miR-128-3p up-regulation (all P<0.05). Compared with the negative control inhibitor (NC inhibitor) group, expression of miR-128-3p was down-regulated in the miR-128-3p inhibitor group, and expression of LEP was increased. The levles of TNF-α, IL-1β and IL-6 were increased after miR-128-3pdownregulation. miR-128-3p down-regulation led to an increase in relative cell viability and EdU-positive cell rate (all P<0.05). Further experimental results showed that LEP expression was up-regulated in the miR-128-3p inhibitor+LEP inhibitor group compared with that in the LEP inhibitor group, whereas the levels of TNF-α, IL-6, and IL-1β were elevated, and the relative viability of the cells and the rate of EdU-positive cells were increased (all P<0.05). Conclusion ·miR-128-3p downregulates LEP to inhibit the proliferation and inflammatory response of keratinocytes, thereby inhibiting the occurrence and development of psoriasis.

Key words: psoriasis, leptin, miR-128-3p, keratinocyte

中图分类号:

R758.63

彭静, 尹婧, 夏萍, 陈柳青. miR-128-3p靶向沉默瘦素抑制银屑病角质形成细胞增殖及炎症反应[J]. 上海交通大学学报（医学版）, 2024, 44(10): 1241-1248.

PENG Jing, YIN Jing, XIA Ping, CHEN Liuqing. miR-128-3p inhibits the proliferation of keratinocytes in psoriasis via repressing leptin[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(10): 1241-1248.

图/表 6

表1 qPCR引物序列

Tab 1 Sequences of primers for qPCR

Primer name	Sequence (5'→3')
Human miR-128-3p-F	CGG TCA CAG TGA ACC GGT
Human miR-128-3p-R	GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA AAG AG
Mouse miR-128-3p-F	TCA CAG TGA ACC GGT CTC
Mouse miR-128-3p-R	GAA CAT GTC TGC GTA TCT C
LEP-F	GCT GTG CCC ATC CAA AAA GTC C
LEP-R	CCC AGG AAT GAA GTC CAA ACC G
Lep-F	GCA GTG CCT ATC CAG AAA GTC C
Lep-R	GGA ATG AAG TCC AAG CCA GTG AC
GAPDH-F	GTC TCC TCT GAC TTC AAC AGC G
GAPDH-R	ACC ACC CTG TTG CTG TAG CCA A
Gapdh-F	CAT CAC TGC CAC CCA GAA GAC TG
Gapdh-R	ATG CCA GTG AGC TTC CCG TTC AG

表2 RNA干扰序列及miRNA模拟物序列

Tab 2 Sequences of RNA interference and miRNA mimic

Name	Sequence (5'→3')
miR-128-3p inhibitor	AAA GAG ACC GGU UCA CUG UGA
miR-128-3p mimic sense	UCA CAG UGA ACC GGU CUC UUU
miR-128-3p mimic antisense	AGA GAC CGG UUC ACU GUG AUU
LEP siRNA sense	GCA GAT AGC TCA TGA CCT GTT
LEP siRNA antisense	AAC AGG TCA TGA GCT ATC TGC

图1 miR-128-3p及LEP在银屑病皮肤组织中的表达Note： A. Expressions of miR-128-3pand Lep in skin tissues from mice with psoriasisdetected by qPCR. B. Expressions of LEPprotein in skin tissues from mice with psoriasis detected by Western blotting.

Fig 1 miR-128-3p and LEPexpression in psoriatic skin tissues

图2 miR-128-3p与 LEP 的靶向关系验证Note： A. The potential binding site between LEP and miR-128-3p predicted by Starbase. B. Validation of the binding site between miR-128-3p and LEP by usingdual luciferase reporter genes assay. C. Expression of LEP detected byqPCR. D. Expression of LEP protein detected by Western blotting.

Fig 2 Validation of the binding site between miR-128-3pand LEP

图3 miR-128-3p对HaCaT细胞增殖及炎症反应的影响Note： A. Expression of miR-128-3pdetected by qPCR. B. The levels of TNF-α, IL-6, and IL-1β in the cell supernatant detected by ELISA. C. Cell viability detected by MTT assay. D. Cell proliferation studied by EdU assay. Scale bar =50 μm.

Fig 3 Effect of miR-128-3p on the proliferation and inflammatory response of HaCaT cells

图4 miR-128-3p/LEP轴对HaCaT细胞增殖及炎症反应的影响Note： A. Expression of LEP protein detected by Western blotting. B. The levels of TNF-α, IL-1β, and IL-6 in the cell supernatant measured by ELISA. C. Cell viability measured by MTT assay. D. Cell proliferation studied by EdU assay. Scale bar =50 μm.

Fig 4 Effect of miR-128-3p/LEP axis on the proliferation and inflammatory response of HaCaT cells

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