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Article(id=1208154039947215199, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208154038609228128, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2022.02.0128, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1625328000000, receivedDateStr=2021-07-04, revisedDate=null, revisedDateStr=null, acceptedDate=1632844800000, acceptedDateStr=2021-09-29, onlineDate=1765975969540, onlineDateStr=2025-12-17, pubDate=1645977600000, pubDateStr=2022-02-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1765975969540, onlineIssueDateStr=2025-12-17, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1765975969540, creator=13701087609, updateTime=1765975969540, updator=13701087609, issue=Issue{id=1208154038609228128, tenantId=1146029695717560320, journalId=1189873630562394117, year='2022', volume='47', issue='2', pageStart='107', pageEnd='212', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1765975969218, creator=13701087609, updateTime=1765976148463, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1208154790459192257, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208154038609228128, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208154790459192258, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208154038609228128, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=128, endPage=135, ext={EN=ArticleExt(id=1208154040656052577, articleId=1208154039947215199, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect and mechanism of miR-125b-5p targeting ΔNp63α in keratinocyte differentiation, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

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.

, correspAuthors=Shu-Mei Feng, authorNote=null, correspAuthorsNote=
*E-mail:
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目的 探讨miR-125b-5p靶向ΔNp63α对角质形成细胞分化的影响及其机制。方法 用1.8 mmol/L氯化钙诱导角质形成细胞HaCaT，于氯化钙处理0、1、5、7 d，采用qRT-PCR检测miR-125b-5p、ΔNp63α、细胞角蛋白10(CK10)、外皮蛋白(Inv)、谷氨酰胺转移酶1(TG1)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)的mRNA表达水平。于氯化钙处理0、5 d采用细胞免疫荧光分析ΔNp63α的表达情况。利用bibiserv网站预测miR-125b-5p与ΔNp63α的结合位点。用miR-125b-5p模拟物/抑制剂和阴性对照模拟物/抑制剂转染HaCaT细胞，培养5 d后，采用qRT-PCR和Western blotting检测ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR的mRNA和蛋白相对表达水平。结果 与氯化钙处理0 d时相比，处理1、5、7 d时miR-125b-5p相对表达水平明显下降(0.17±0.02、0.08±0.01、0.07±0.02 vs. 1.00±0.02，P<0.001)，而ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR mRNA相对表达水平逐渐升高(P<0.05)。细胞免疫荧光分析结果显示，与氯化钙处理0 d时相比，氯化钙处理5 d时角质形成细胞中ΔNp63α阳性细胞率增高(96.9%±0.9% vs. 43.2%±8.2%，P<0.001)。bibiserv网站预测结果显示，miR-125b-5p可与ΔNp63α的3'-UTR位点相结合。与阴性对照模拟物组相比，miR-125b-5p模拟物组ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR mRNA相对表达水平明显降低，且ΔNp63α、CK10、Inv、TG1、PI3K、Akt、p-Akt、mTOR和p-mTOR蛋白表达水平也明显降低(P<0.05)，而抑制miR-125b-5p的表达可逆转上述效应(P<0.05)。结论 miR-125b-5p靶向ΔNp63α可通过下调PI3K/Akt/mTOR信号通路抑制角质形成细胞的分化。

, correspAuthors=冯树梅, authorNote=null, correspAuthorsNote=
冯树梅，E-mail：
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丁毅，硕士研究生，主要从事皮肤发育方面的研究

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丁毅，硕士研究生，主要从事皮肤发育方面的研究

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丁毅，硕士研究生，主要从事皮肤发育方面的研究

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Cells, 2019, 8(9): 1089., articleTitle=Fisetin, a 3, 7, 3', 4'-tetrahydroxyflavone inhibits the PI3K/Akt/mTOR and MAPK pathways and ameliorates psoriasis pathology in 2D and 3D organotypic human inflammatory skin models, refAbstract=null), Reference(id=1208154050651079398, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, doi=null, pmid=null, pmcid=null, year=2015, volume=230, issue=9, pageStart=2067, pageEnd=2074, url=null, language=null, rfNumber=[30], rfOrder=31, authorNames=Troiano A, Lomoriello IS, di Martino O, journalName=J Cell Physiol, refType=null, unstructuredReference=Troiano A, Lomoriello IS, di Martino O, et al. Y-box binding protein-1 is part of a complex molecular network linking deltaNp63alpha to the PI3K/Akt pathway in cutaneous squamous cell carcinoma[J]. 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tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, awardId=81660521, language=CN, fundingSource=国家自然科学基金(81660521), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1208154042438631801, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, xref=1, ext=[AuthorCompanyExt(id=1208154042463797627, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, companyId=1208154042438631801, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1Department of Histology and Embryology, School of Basic Medical Sciences, Urumqi 830011, China), AuthorCompanyExt(id=1208154042484769148, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, companyId=1208154042438631801, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, 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journalId=1189873630562394117, articleId=1208154039947215199, companyId=1208154042920976778, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4Xinjiang Urumqi City Central Blood Station, Urumqi 830011, China), AuthorCompanyExt(id=1208154043034222988, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, companyId=1208154042920976778, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4新疆乌鲁木齐市中心血站，乌鲁木齐 830011)])], figs=[ArticleFig(id=1208154045819241000, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig.1, caption=Expression of skin differentiation-related markers and miR-125b-5p in keratinocyte differentiation model, figureFileSmall=TDrPN0d7jNneeI9+h7BZQA==, figureFileBig=AsWKoywR7BCiE8O3cB+qvQ==, tableContent=null), ArticleFig(id=1208154045915709998, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图1, caption=皮肤分化相关标志物和miR-125b-5p在角质形成细胞分化模型中的表达情况

CK10. 细胞角蛋白10；Inv. 外皮蛋白；TG1. 谷氨酰胺转移酶1；A. qRT-PCR检测角质细胞分化模型中CK10、Inv、TG1 mRNA相对表达水平；B. qRT-PCR检测角质细胞分化模型中miR-125b-5p相对表达水平；与0 d比较，(1)P<0.05，(2)P<0.01，(3)P<0.0001；与1 d比较，(4)P<0.05，(5)P<0.01；与5 d比较，(6)P<0.01

, figureFileSmall=TDrPN0d7jNneeI9+h7BZQA==, figureFileBig=AsWKoywR7BCiE8O3cB+qvQ==, tableContent=null), ArticleFig(id=1208154046083482170, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig.2, caption=Calcium chloride promotes the expression of ΔNp63α and activates PI3K/Akt/mTOR pathway in keratinocyte, figureFileSmall=43BAemjaF3HQtAS3xQavZQ==, figureFileBig=t6O/L2b2uI1hf9GurLtYCg==, tableContent=null), ArticleFig(id=1208154046150591037, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图2, caption=氯化钙促进皮肤角质形成细胞中ΔNp63α的表达且激活PI3K/Akt/mTOR通路

PI3K. 磷脂酰肌醇3-激酶；Akt. 蛋白激酶B；mTOR. 哺乳动物雷帕霉素靶蛋白；A. qRT-PCR检测角质形成细胞中ΔNp63α mRNA相对表达水平；B. 激光共聚焦显微镜观察ΔNp63α在角质形成细胞中的表达情况；C. qRT-PCR检测PI3K/Akt/mTOR通路的mRNA相对表达水平。与0 d比较，(1)P<0.05，(2)P<0.01，(3)P<0.001，(4)P<0.0001；与1 d比较，(5)P<0.05，(6)P<0.01，(7)P<0.001，(8)P<0.0001；与5 d比较，(9)P<0.01，(10)P<0.0001

, figureFileSmall=43BAemjaF3HQtAS3xQavZQ==, figureFileBig=t6O/L2b2uI1hf9GurLtYCg==, tableContent=null), ArticleFig(id=1208154046238671427, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig.3, caption=Prediction of 3'-UTR binding sites between miR-125b-5p and ΔNp63α by bibiserv website in keratinocyte, figureFileSmall=L3VZks/qNLFF8SPrvcq/bg==, figureFileBig=Z+egaZLT/Wn+3H0XGTfr+A==, tableContent=null), ArticleFig(id=1208154046326751817, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图3, caption=bibiserv网站预测皮肤角质形成细胞中miR-125b-5p与ΔNp63α的3'-UTR结合位点信息, figureFileSmall=L3VZks/qNLFF8SPrvcq/bg==, figureFileBig=Z+egaZLT/Wn+3H0XGTfr+A==, tableContent=null), ArticleFig(id=1208154046402249292, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig.4, caption=Relationship of ΔNp63α with miR-125b-5p in keratinocyte, figureFileSmall=jMgPATBJqZblOUpFtK/QiQ==, figureFileBig=8gNqZSH3xkrRH+zi4Z0g6Q==, tableContent=null), ArticleFig(id=1208154046473552463, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图4, caption=皮肤角质形成细胞中ΔNp63α与miR-125b-5p的关系

A、B. qRT-PCR和Western blotting检测miR-125b-5p模拟物组中ΔNp63α的表达水平；C、D. qRT-PCR和Western blotting检测miR-125b-5p抑制剂组中ΔNp63α的表达水平。与阴性对照模拟物组比较，(1)P<0.05，(2)P<0.0001；与阴性对照抑制剂组比较，(3)P<0.05，(4)P<0.001

, figureFileSmall=jMgPATBJqZblOUpFtK/QiQ==, figureFileBig=8gNqZSH3xkrRH+zi4Z0g6Q==, tableContent=null), ArticleFig(id=1208154046553244245, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig.5, caption=Effect of miR-125b-5p transfection on the expression of skin differentiation-related markers in keratinocytes, figureFileSmall=NC1iL4PJuvSwYZifl3elUQ==, figureFileBig=pzVx81KEYbBwcHK+ntNT7g==, tableContent=null), ArticleFig(id=1208154046624547421, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图5, caption=miR-125b-5p转染对皮肤角质形成细胞中分化相关标志物表达的影响

CK10. 细胞角蛋白10；Inv. 外皮蛋白；TG1. 谷氨酰胺转移酶1；A. 转染miR-125b-5p模拟物5 d后，qRT-PCR和Western blotting检测角质细胞CK10、Inv、TG1 mRNA和蛋白表达水平；B. 转染miR-125b-5p抑制剂5 d后，qRT-PCR和Western blotting检测角质细胞CK10、Inv、TG1 mRNA和蛋白表达水平；与阴性对照模拟物组比较，(1)P<0.05，(2)P<0.01，(3)P<0.001，(4)P<0.0001；与阴性对照抑制剂组比较，(5)P<0.05，(6)P<0.01，(7)P<0.001，(8)P<0.0001

, figureFileSmall=NC1iL4PJuvSwYZifl3elUQ==, figureFileBig=pzVx81KEYbBwcHK+ntNT7g==, tableContent=null), ArticleFig(id=1208154046716822110, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Fig. 6, caption=Effects of miR-125b-5p expression on PI3K/Akt/mTOR pathway in keratinocytes, figureFileSmall=M+DJUoZHHniZIOH+nRm2Xg==, figureFileBig=4HndtpUaj3SnFCKmY8ljzg==, tableContent=null), ArticleFig(id=1208154046809096804, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=图6, caption=miR-125b-5p表达对皮肤角质形成细胞中PI3K/Akt/mTOR通路的影响

PI3K. 磷脂酰肌醇3-激酶；Akt. 蛋白激酶B；mTOR. 哺乳动物雷帕霉素靶蛋白；A. 转染miR-125b-5p模拟物5 d后，qRT-PCR和Western blotting检测PI3K/Akt/mTOR通路mRNA及总蛋白、磷酸化蛋白相对表达水平；B. 转染miR-125b-5p抑制剂5 d后，qRT-PCR和Western blotting检测PI3K/Akt/mTOR通路mRNA及总蛋白、磷酸化蛋白相对表达水平；与阴性对照模拟物组比较，(1)P<0.05，(2)P<0.01，(3)P<0.001，(4)P<0.0001；与阴性对照抑制剂组比较，(5)P<0.05，(6)P<0.01，(7)P<0.001，(8)P<0.0001

, figureFileSmall=M+DJUoZHHniZIOH+nRm2Xg==, figureFileBig=4HndtpUaj3SnFCKmY8ljzg==, tableContent=null), ArticleFig(id=1208154046897177193, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=EN, label=Tab.1, caption=

Primer sequence of qRT-PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
基因引物
ΔNp63α正向：5'-ATTGTAAGGGTCTCGGGGTG-3'
反向：5'-GGGCATTGTTTTCCAGGTACA-3'
CK10正向：5'-GGAGGAGGAGGAGGAGTGTCATC-3'
反向：5'-AAGCCAGGCGGTCATTCAGATTC-3'
Inv正向：5'-AGCTCCTCAAGACTGTTCCTCCTC-3'
反向：5'-TCCTGCTCCTGTGGCTCCTTC-3'
TG1正向：5'-GTGGAACGACTGCTGGATGAAGAG-3'
反向：5'-TGTGACAATGAGTGTGCCGATGG-3'
PI3K正向：5'-GAGATTGCAAGCAGTGATAGTG-3'
反向：5'-TAATTTTGGCAGTGATTGTGGG-3'
Akt正向：5'-TGACCATGAACGAGTTTGAGTA-3'
反向：5'-GAGGATCTTCATGGCGTAGTAG-3'
mTOR正向：5'-GAGATACGCTGTCATCCCTTTTA-3'
反向：5'-CTGTATTATTGACGGCATGCT-3'
β-actin正向：5'-CAACTTGATGTATGAAGGCTTTGGT-3'
反向：5'-ACTTTTATTGGTCTCAAGTCAGTGTACAG-3'
miR-125b-5p正向：5'-TCCCTGAGACCCTAACTTGTGA-3'
反向：5'-CCAGTGCAGG-GTCCGAGGTATT-3'
U6正向：5'-AGCTAGCTAGCTAGCTAGCTAA-3'
反向：5'-ACGTAGCTAGCCAGCTAGCTCA-3'
), ArticleFig(id=1208154047010423405, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208154039947215199, language=CN, label=表1, caption=

qRT-PCR引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因引物
ΔNp63α正向：5'-ATTGTAAGGGTCTCGGGGTG-3'
反向：5'-GGGCATTGTTTTCCAGGTACA-3'
CK10正向：5'-GGAGGAGGAGGAGGAGTGTCATC-3'
反向：5'-AAGCCAGGCGGTCATTCAGATTC-3'
Inv正向：5'-AGCTCCTCAAGACTGTTCCTCCTC-3'
反向：5'-TCCTGCTCCTGTGGCTCCTTC-3'
TG1正向：5'-GTGGAACGACTGCTGGATGAAGAG-3'
反向：5'-TGTGACAATGAGTGTGCCGATGG-3'
PI3K正向：5'-GAGATTGCAAGCAGTGATAGTG-3'
反向：5'-TAATTTTGGCAGTGATTGTGGG-3'
Akt正向：5'-TGACCATGAACGAGTTTGAGTA-3'
反向：5'-GAGGATCTTCATGGCGTAGTAG-3'
mTOR正向：5'-GAGATACGCTGTCATCCCTTTTA-3'
反向：5'-CTGTATTATTGACGGCATGCT-3'
β-actin正向：5'-CAACTTGATGTATGAAGGCTTTGGT-3'
反向：5'-ACTTTTATTGGTCTCAAGTCAGTGTACAG-3'
miR-125b-5p正向：5'-TCCCTGAGACCCTAACTTGTGA-3'
反向：5'-CCAGTGCAGG-GTCCGAGGTATT-3'
U6正向：5'-AGCTAGCTAGCTAGCTAGCTAA-3'
反向：5'-ACGTAGCTAGCCAGCTAGCTCA-3'
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miR-125b-5p靶向ΔNp63α对角质形成细胞分化的影响及其机制
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丁毅 1 , 李敏 2 , 陈龙 3 , 张美林 4 , 冯树梅 1, *
解放军医学杂志 | 基础研究 2022,47(2): 128-135
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解放军医学杂志 | 基础研究 2022, 47(2): 128-135
miR-125b-5p靶向ΔNp63α对角质形成细胞分化的影响及其机制
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丁毅1, 李敏2, 陈龙3, 张美林4, 冯树梅1, *
作者信息
  • 1新疆医科大学基础医学院组织学与胚胎学教研室，乌鲁木齐 830011
  • 2新疆第二医学院基础医学院解剖组胚教研室，乌鲁木齐 830011
  • 3新疆医科大学基础医学院技能中心，乌鲁木齐 830011
  • 4新疆乌鲁木齐市中心血站，乌鲁木齐 830011

    • 丁毅，硕士研究生，主要从事皮肤发育方面的研究

通讯作者:

冯树梅，E-mail：
Effect and mechanism of miR-125b-5p targeting ΔNp63α in keratinocyte differentiation
Yi Ding1, Min Li2, Long Chen3, Mei-Lin Zhang4, Shu-Mei Feng1, *
Affiliations
  • 1Department of Histology and Embryology, School of Basic Medical Sciences, Urumqi 830011, China
  • 2Department of Anatomy and Histoembryology, School of Basic Medical Sciences, Xinjiang Second Medical College, Urumqi 830011, China
  • 3Functional Center, Institute of Basic Medicine, Xinjiang Medical University, Urumqi 830011, China
  • 4Xinjiang Urumqi City Central Blood Station, Urumqi 830011, China
出版时间: 2022-02-28 doi: 10.11855/j.issn.0577-7402.2022.02.0128
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摘要
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目的 探讨miR-125b-5p靶向ΔNp63α对角质形成细胞分化的影响及其机制。方法 用1.8 mmol/L氯化钙诱导角质形成细胞HaCaT，于氯化钙处理0、1、5、7 d，采用qRT-PCR检测miR-125b-5p、ΔNp63α、细胞角蛋白10(CK10)、外皮蛋白(Inv)、谷氨酰胺转移酶1(TG1)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)的mRNA表达水平。于氯化钙处理0、5 d采用细胞免疫荧光分析ΔNp63α的表达情况。利用bibiserv网站预测miR-125b-5p与ΔNp63α的结合位点。用miR-125b-5p模拟物/抑制剂和阴性对照模拟物/抑制剂转染HaCaT细胞，培养5 d后，采用qRT-PCR和Western blotting检测ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR的mRNA和蛋白相对表达水平。结果 与氯化钙处理0 d时相比，处理1、5、7 d时miR-125b-5p相对表达水平明显下降(0.17±0.02、0.08±0.01、0.07±0.02 vs. 1.00±0.02，P<0.001)，而ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR mRNA相对表达水平逐渐升高(P<0.05)。细胞免疫荧光分析结果显示，与氯化钙处理0 d时相比，氯化钙处理5 d时角质形成细胞中ΔNp63α阳性细胞率增高(96.9%±0.9% vs. 43.2%±8.2%，P<0.001)。bibiserv网站预测结果显示，miR-125b-5p可与ΔNp63α的3'-UTR位点相结合。与阴性对照模拟物组相比，miR-125b-5p模拟物组ΔNp63α、CK10、Inv、TG1、PI3K、Akt和mTOR mRNA相对表达水平明显降低，且ΔNp63α、CK10、Inv、TG1、PI3K、Akt、p-Akt、mTOR和p-mTOR蛋白表达水平也明显降低(P<0.05)，而抑制miR-125b-5p的表达可逆转上述效应(P<0.05)。结论 miR-125b-5p靶向ΔNp63α可通过下调PI3K/Akt/mTOR信号通路抑制角质形成细胞的分化。

miR-125b-5p  /  ΔNp63α  /  细胞分化  /  角质形成细胞

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.

miR-125b-5p  /  ΔNp63α  /  cell differentiation  /  keratinocytes
丁毅, 李敏, 陈龙, 张美林, 冯树梅. miR-125b-5p靶向ΔNp63α对角质形成细胞分化的影响及其机制. 解放军医学杂志, 2022 , 47 (2) : 128 -135 . DOI: 10.11855/j.issn.0577-7402.2022.02.0128
Yi Ding, Min Li, Long Chen, Mei-Lin Zhang, Shu-Mei Feng. Effect and mechanism of miR-125b-5p targeting ΔNp63α in keratinocyte differentiation[J]. Medical Journal of Chinese People’s Liberation Army, 2022 , 47 (2) : 128 -135 . DOI: 10.11855/j.issn.0577-7402.2022.02.0128
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皮肤是生物体远离外界环境影响的第一道屏障，位于皮肤最外层的表皮层主要由不断经历自我更新、分化和退化过程的角质形成细胞组成[1-2]。角质形成细胞的异常分化与银屑病、特应性皮炎及皮肤鳞癌等的发生关系密切[3-4]。因此，阐明角质形成细胞的分化机制对于了解皮肤疾病的发生非常重要。MicroRNAs(miRNAs)是一类内源性、长度为22~23个核苷酸的非编码小RNA，通过与靶基因的3'非翻译区(3'-UTR)配对来调节基因的表达[5]。多项研究发现，miRNA可调控分化、增殖和凋亡等多种重要的细胞功能，并且其表达异常与皮肤疾病密切相关[4-6]。miR-125b-5p作为一种重要的抑癌分子，在银屑病、皮肤鳞癌和黑色素瘤中低表达，并可调节细胞凋亡，参与肿瘤的发生发展[7-8]；过表达miR-125b-5p可抑制角质形成细胞和黑色素瘤细胞的增殖[9]。ΔNp63α是皮肤发育的重要调控因子，在银屑病和皮肤鳞癌中表达上调[10]，过表达ΔNp63α可促进角质形成细胞增殖[11]。靶基因预测发现，miR-125b-5p与ΔNp63α密切相关，但miR-125b-5p能否通过调控ΔNp63α的表达影响角质形成细胞的分化鲜见报道。本研究旨在分析miR-125b-5p和ΔNp63α对角质形成细胞分化的影响及可能机制，以期为了解皮肤病的发病机制提供新的理论依据。
1 材料与方法
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1.1 主要试剂及仪器
DMEM培养基、胎牛血清、青霉素-链霉素溶液购自美国Gibco公司；转染试剂riboFECTTM CP、miR-125b-5p的模拟物及抑制剂、阴性对照模拟物/抑制剂购自广州锐博生物科技有限公司；ΔNp63α单抗(ab203826)、β-actin单抗(ab75186)、蛋白激酶B(protein kinase B，Akt；ab179463)、p-Akt(Ser473；ab192623)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR；ab32028)、p-mTOR(Ser2448；ab109268)购自英国Abcam公司；磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase，PI3K；sc-365290)、细胞角蛋白10(cytokeratin 10，CK10；sc-53252)、外皮蛋白(involucrin，Inv；sc-21748)、谷氨酰胺转移酶1(transglutaminase，TG1；sc-166767)单抗购自美国Santa Cruz公司；皂素(saponin)购自美国Sigma公司；辣根过氧化物酶标记的二抗购自上海爱必信生物科技有限公司；反转录试剂盒、实时荧光定量PCR(qRT-PCR)检测试剂盒购自北京全式金生物技术有限公司；BCA蛋白分析试剂盒和RIPA裂解液购自北京索莱宝科技有限公司；ECL化学发光底物试剂盒购自中国Biosharp公司。生物安全柜和CO2培养箱购自美国Thermo Fisher公司；激光共聚焦显微镜(Nikon C2)购自日本Nikon公司。
1.2 细胞培养
人正常皮肤永生化角质形成细胞株HaCaT购自武汉大学中国典型培养物保藏中心，用含15%灭活胎牛血清(FBS)、1%青霉素和链霉素的DMEM培养。
1.3 角质形成细胞分化模型的构建
取对数生长期HaCaT细胞，按照3×105个/孔接种于6孔板中，加入1.8 mmol/L氯化钙处理0、1、5、7 d，提取RNA行qRT-PCR检测。
1.4 细胞免疫荧光分析ΔNp63α的表达情况
取对数生长期HaCaT细胞，按照8000个/孔铺于含细胞爬片的6孔板中，次日加入1.8 mmol/L氯化钙处理0、5 d。用PBS清洗3次，4%多聚甲醛溶液固定15 min；PBS洗涤10 min×3次，将ΔNp63α一抗(1∶300)于saponin中稀释后加在玻片上孵育1 h；PBS洗涤，加入二抗(1∶300)孵育1 h，PBS洗涤后封片，利用激光共聚焦显微镜进行共聚焦成像分析。
1.5 细胞转染
取对数生长期HaCaT细胞，按照3×105个/孔接种于6孔板中。次日待细胞贴壁后按照如下分组进行转染：阴性对照模拟物组(转染阴性对照模拟物)、miR-125b-5p模拟物组(转染miR-125b-5p模拟物)、阴性对照抑制剂组(转染阴性对照抑制剂)、miR-125b-5p抑制剂组(转染miR-125b-5p抑制剂)。因瞬时转染效率仅可维持3 d左右，故在3 d后重新转染，转染5 d后收集细胞用于后续实验。
1.6 qRT-PCR检测miR-125b-5p及ΔNp63α、CK10、Inv、TG1、PI3K、Akt、mTOR的mRNA表达水平
利用Trizol法提取细胞总RNA，采用反转录试剂盒进行反转录。PCR反应条件：94 ℃ 30 s；94 ℃ 5 s，60 ℃ 30 s，40个循环；95 ℃ 15 s，60 ℃ 60 s，90 ℃15 s。以U6或β-actin为内参，采用2–ΔΔCt法计算目的基因mRNA的相对表达水平。引物由中国生工生物工程公司合成，引物序列见表1。
1.7 Western blotting检测ΔNp63α、CK10、Inv、TG1、PI3K、Akt、mTOR蛋白表达水平
取转染阴性对照模拟物/抑制剂、miR-125b-5p模拟物/抑制剂的角质形成细胞，RIPA冰上裂解20 min，用BCA蛋白分析试剂盒进行定量检测。蛋白质经SDS-PAGE凝胶电泳后，转移到硝酸纤维素膜(NC)上。1×TBST洗涤后，用5%脱脂奶粉封闭2 h，加入TBST稀释的一抗ΔNp63α(1∶1000)、CK10(1∶100)、Inv(1∶100)、TG1(1∶1000)、PI3K(1∶3000)、Akt(1∶3000)、mTOR(1∶3000)和β-actin(1∶1000)，4 ℃孵育过夜。加入辣根过氧化物酶(HRP)标记的抗兔/鼠IgG二抗(1∶5000)室温孵育1 h；洗膜后用ECL试剂盒发光显影，然后用ImageJ软件分析蛋白条带的灰度值。
1.8 miR-125b-5p与ΔNp63α结合位点预测
在NCBI(https://www.ncbi.nlm.nih.gov/)网站查询miR-125b-5p和ΔNp63α的完整RNA序列，利用bibiserv(https://bibiserv.cebitec.uni-bielefeld.de/)在线网站对miR-125b-5p和ΔNp63α的结合位点进行分析。
1.9 统计学处理
采用SPSS 28.0软件进行统计分析。符合正态分布的计量资料以$\bar{x}±s$表示，两组间比较采用t检验，多组间比较采用方差分析，进一步两两比较采用Games-Howell检验。P<0.05为差异有统计学意义。
2 结果
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2.1 皮肤分化相关标志物和miR-125b-5p在氯化钙诱导的角质形成细胞分化模型中的表达情况
qRT-PCR检测结果显示，氯化钙处理7 d时角质形成细胞中CK10、Inv和TG1 mRNA相对表达水平(分别为300.70±13.27、9.14±1.23、5.49±0.34)高于处理0 d(分别为1.10±0.11、1.02±0.23、1.00±0.08)、1 d(分别为2.13±0.45、1.21±0.21、1.33±0.04)和5 d(分别为11.90±2.10、5.86±1.03、2.61±0.11)时，且处理5 d时高于处理1 d时，差异有统计学意义(P<0.05，图1A)；氯化钙处理5 d时miR-125b-5p相对表达水平(0.08±0.01)低于处理0 d(1.00±0.02)和1 d(0.17±0.02)时，差异有统计学意义(P<0.0001或P<0.05，图1B)，但氯化钙处理5 d与7 d(0.07±0.02)时miR-125b-5p相对表达水平差异无统计学意义(P>0.05)。
2.2 氯化钙处理对角质形成细胞中ΔNp63α表达及PI3K/Akt/mTOR通路的影响
qRT-PCR检测结果显示，氯化钙处理1、5、7 d时角质形成细胞中ΔNp63α mRNA相对表达水平均高于0 d，差异有统计学意义(3.55±0.24、5.58±0.42、20.82±0.58 vs. 1.01±0.23，P<0.01，图2A)。激光共聚焦显微镜观察显示，与氯化钙处理0 d时相比，氯化钙处理5 d时角质形成细胞中ΔNp63α阳性细胞率增高，差异有统计学意义(96.9%±0.9% vs. 43.2%±8.2%，t=6.469，P<0.001，图2B)。氯化钙处理1 d(分别为2.28±0.05、1.66±0.04、1.77±0.22)、5 d(分别为4.37±0.22、2.42±0.05、2.47±2.22)和7 d(分别为12.77±0.59、2.88±0.20、4.39±0.33)时PI3K、Akt和mTOR mRNA相对表达水平高于0 d(分别为1.00±0.11、1.03±0.05、1.00±0.05)，差异有统计学意义(P<0.05，图2C)。
2.3 ΔNp63α与miR-125b-5p的关系
bibiserv网站预测结果显示，miR-125b-5p与ΔNp63α的3'-UTR区有一个结合位点(图3)。
qRT-PCR和Western blotting检测结果显示，miR-125b-5p模拟物组ΔNp63α mRNA和蛋白表达水平明显低于阴性对照模拟物组(mRNA：0.64±0.02 vs. 1.00±0.01；蛋白：0.92±0.19 vs. 1.22±0.20，P<0.05，图4A、B)；miR-125b-5p抑制剂组ΔNp63α mRNA和蛋白表达水平均高于阴性对照抑制剂组(mRNA：1.79±0.13 vs. 0.99±0.01；蛋白：0.78±0.09 vs. 0.60±0.11，P<0.05，图4C、D)。
2.4 miR-125b-5p转染对角质细胞中皮肤分化相关标志物表达的影响
qRT-PCR和Western blotting检测结果显示，miR-125b-5p模拟物组CK10、Inv、TG1 mRNA和蛋白表达水平明显低于阴性对照模拟物组(P<0.05，图5A)；miR-125b-5p抑制剂组CK10、Inv和TG1 mRNA和蛋白表达水平明显高于阴性对照抑制剂组(P<0.05，图5B)。
2.5 miR-125b-5p转染对角质形成细胞中PI3K/Akt/mTOR通路的影响
qRT-PCR和Western blotting检测结果显示，与阴性对照模拟物组相比，miR-125b-5p模拟物组PI3K、Akt、mTOR mRNA表达水平降低，PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白表达水平降低，差异均有统计学意义(P<0.05，图6A)；与阴性对照抑制剂组相比，miR-125b-5p抑制剂组PI3K、Akt、mTOR mRNA表达水平升高，PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白表达水平升高，差异均有统计学意义(P<0.05，图6B)。
3 讨论
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表皮再生和动态平衡对于维持正常皮肤功能非常重要，且表皮的形成伴随着角质形成细胞的分化、增殖和迁移。分化的基底层角质形成细胞是表皮细胞补充的主要来源[12]。角质形成细胞的异常分化与银屑病和特应性皮炎等皮肤病相关，并与皮肤肿瘤的形成有关[13]。因此，研究角质形成细胞的分化对理解皮肤疾病的发生具有重要意义。
近年来多项研究发现，miRNA与角质形成细胞的分化、增殖和凋亡等相关。Wang等[14]发现，miR-744-3p可通过靶向KLLN抑制银屑病中角质形成细胞的分化。在皮肤表皮层分布的miR-155过表达可明显抑制角质形成细胞的终末分化[15]。但miRNA调控角质形成细胞分化的相关研究较少。据报道，miR-125b-5p在部分癌症中起到癌基因或抑癌基因的作用，这可能是由于miR-125b-5p在不同组织中的靶基因不同而导致的[16]。此外，Zheng等[17]发现，miR-125b-5p在银屑病患者皮肤中呈低表达。Lu等[18]发现，miR-125b-5p在皮肤鳞癌中呈低表达。过表达miR-125b-5p可促进皮肤鳞癌细胞的凋亡，抑制肿瘤的增长。Tian等[19]也得到了相似的结果。同时，miR-125b-5p在口腔鳞癌、头颈鳞癌、肺鳞癌和食管鳞癌中也呈低表达[20-22]。由此可见，miR-125b-5p可能在角质形成细胞中作为一种抑癌基因而存在。本研究对角质形成细胞分化模型中miR-125b-5p的表达情况进行分析，发现miR-125b-5p的表达水平随着角质形成细胞的分化而降低，推测miR-125b-5p可能参与角质形成细胞的分化。本研究结果显示，miR-125b-5p模拟物组CK10、Inv、TG1的mRNA和蛋白表达水平明显降低，且转染miR-125b-5p抑制剂后可逆转这一效应，提示miR-125b-5p在角质形成细胞分化过程中发挥着重要作用。采用生物信息学技术分析发现，miR-125b-5p可以与ΔNp63α的3'-UTR区结合。过表达miR-125b-5p可降低ΔNp63α mRNA和蛋白的表达水平，进一步表明miR-125b-5p抑制角质形成细胞的分化是通过下调ΔNp63α实现的。
ΔNp63α为p53家族的成员，在上皮组织分化和组织发育中发挥重要作用，且与多种肿瘤的发生发展密切相关。ΔNp63α在皮肤鳞癌、宫颈癌、肺癌组织中高表达，且其表达水平与肿瘤的浸润、转移关系密切[23-24]。有研究发现，ΔNp63α表达水平与皮肤鳞癌患者的生存率呈负相关[23]。此外，ΔNp63α过度表达可促进皮肤鳞癌的发生和发展[25]。King等[26]发现，ΔNp63α敲除小鼠的表皮不再生长。Nylander等[27]发现，在p63敲除小鼠中过表达ΔNp63α可以恢复皮肤分化标志物的水平，并维持上皮的完整性。上述研究表明，ΔNp63α在皮肤发育中发挥着重要的调控作用。
此外，本研究证实，miR-125b-5p和ΔNp63α对角质形成细胞分化的作用可能与PI3K/Akt/mTOR通路有关。既往研究发现，激活PI3K/Akt/mTOR信号通路可促进角质形成细胞的分化[28-29]。Troiano等[30]发现，在皮肤鳞癌中ΔNp63α过表达与PI3K/Akt/mTOR信号通路异常激活有关。因此，本研究评价miR-125b-5p和ΔNp63α对角质形成细胞分化的影响时，重点关注了PI3K/Akt/mTOR信号通路的作用。结果表明，上调miR-125b-5p可降低角质形成细胞中PI3K/Akt/mTOR通路mRNA、总蛋白及磷酸化蛋白的表达水平，而下调miR-125b-5p则促进了PI3K/Akt/mTOR通路的激活，提示miR-125b-5p靶向ΔNp63α通过PI3K/Akt/mTOR信号通路调节角质形成细胞的分化。
综上所述，本研究结果表明，miR-125b-5p靶向ΔNp63α通过PI3K/Akt/mTOR通路抑制角质形成细胞的分化。该结果为理解皮肤疾病的发病机制提供了新的见解，并为表皮细胞生物学研究奠定了基础。但本研究仅在体外细胞水平证实miR-125b-5p对角质形成细胞分化的影响，且ΔNp63α调控角质形成细胞分化是否与PI3K/Akt/mTOR信号通路相关尚无法证实。因此，体内功能验证及ΔNp63α的潜在机制尚需进一步研究。
基金
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  • 国家自然科学基金(81660324)
  • 国家自然科学基金(81660521)
文献
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doi: 10.11855/j.issn.0577-7402.2022.02.0128
  • 接收时间：2021-07-04
  • 首发时间：2025-12-17
  • 出版时间：2022-02-28
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  • 收稿日期：2021-07-04
  • 录用日期：2021-09-29
基金
National Natural Science Foundation of China(81660324)
国家自然科学基金(81660324)
National Natural Science Foundation of China(81660521)
国家自然科学基金(81660521)
作者信息
    1新疆医科大学基础医学院组织学与胚胎学教研室，乌鲁木齐 830011
    2新疆第二医学院基础医学院解剖组胚教研室，乌鲁木齐 830011
    3新疆医科大学基础医学院技能中心，乌鲁木齐 830011
    4新疆乌鲁木齐市中心血站，乌鲁木齐 830011

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