Article(id=1198196212982710916, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1198196207379120715, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.1576.2024.0328, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1701100800000, receivedDateStr=2023-11-28, revisedDate=null, revisedDateStr=null, acceptedDate=1705852800000, acceptedDateStr=2024-01-22, onlineDate=1763601838583, onlineDateStr=2025-11-20, pubDate=1735315200000, pubDateStr=2024-12-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763601838583, onlineIssueDateStr=2025-11-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763601838583, creator=13701087609, updateTime=1763601838583, updator=13701087609, issue=Issue{id=1198196207379120715, tenantId=1146029695717560320, journalId=1189873630562394117, year='2024', volume='49', issue='12', pageStart='1343', pageEnd='1459', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1763601837248, creator=13701087609, updateTime=1763603898104, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1198204851306988030, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1198196207379120715, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198204851306988031, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1198196207379120715, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1426, endPage=1436, ext={EN=ArticleExt(id=1198196213246952071, articleId=1198196212982710916, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Mechanism of DNA methyltransferase 3a mediating progression of lung squamous cell carcinoma and prognostic correlation analysis, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the correlation between DNA methyltransferase 3a (DNMT3a) expression and prognosis of lung squamous cell carcinoma (LSCC), as well as to elucidate the potential molecular mechanisms of DNMT3a in LSCC progression. Methods A retrospective analysis was conducted on a cohort of 47 LSCC patients who underwent surgery in the Department of Thoracic Surgery, the Second Affiliated Hospital of Air Force Medical University between May 2009 and January 2014. DNMT3a expression in LSCC tissues and paired adjacent non-cancerous tissues was assessed using immunohistochemical (IHC) staining. Patients were categorized into two groups based on the median IHC score of DNMT3a in LSCC tissues: high DNMT3a expression group (n=25) and low DNMT3a expression group (n=22). Prognostic correlation was analyzed in combination with clinicopathological data and public biological databases. To explore the molecular mechanisms of DNMT3a in LSCC progression, H1703 LSCC cell lines with overexpressed DNMT3a were established using a lentiviral infection method, with the creation of DNMT3a overexpression group and control group. Functional phenotype experiments were then conducted to test the differences in cell proliferation and migration between the two groups. DNMT3a overexpression tumor xenograft models were also established in nude mice, with the creation of DNMT3a overexpression group and control group (3 mice per group), to observe the growth of subcutaneous xenograft tumors. Western blotting analysis was employed to detect the expression of related proteins in the two groups of cells and subcutaneous xenograft tumors. Functional rescue experiments involved treating DNMT3a overexpression cells with c-Myc inhibitor (10058-F4) and assessing cell proliferation using EdU proliferation staining. Subsequently, DNMT3a overexpression cells were infected with RNAi-Zinc finger E-box binding homeobox 1 (ZEB1) lentivirus to knock down ZEB1 expression, and a Transwell migration assay was utilized to detect cell migration. Finally, DNMT3a overexpression group and control group were treated with DNMT specific inhibitor (SGI-1027), and the effects of DNMT3a inhibition on cell proliferation and migration were observed in both overexpression and control groups. Results IHC analysis revealed significantly higher DNMT3a level in LSCC tissues compared to adjacent non-cancerous tissues (P<0.0001). High DNMT3a expression was closely associated with N stage, clinical stage and tumor differentiation degree (P<0.05 or P<0.01), and it was identified as an independent risk factor for poor prognosis in LSCC patients (P<0.05). Functional phenotype experiments indicated that DNMT3a overexpression group exhibited significantly higher colony formation number, proportion of EdU-positive cells, wound healing migration rate, and Transwell cell migration number compared with control group (P<0.05). The volume and weight of subcutaneous xenograft tumors in DNMT3a overexpression group were significantly higher than those in control group (P<0.001). Western blotting showed that the protein expression levels of c-Myc and ZEB1 in DNMT3a overexpression group were significantly higher than those in control group. Functional rescue experiments demonstrated a significant reduction in the proportion of EdU-positive cells after 10058-F4 treatment in DNMT3a overexpression group (P<0.05). Knockdown of ZEB1 led to a significant decrease in the number of Transwell cell migration in DNMT3a overexpression group (P<0.05), with no significant change in DNMT3a protein expression. Additionally, inhibition of DNMT3a with SGI-1027 resulted in a significant decrease in colony formation number and migration rate in both DNMT3a overexpression group and control group (P<0.05). Conclusions High expression of DNMT3a is a significant independent risk factor for poor prognosis of LSCC patients. DNMT3a is likely to promote the proliferation of LSCC by upregulating c-Myc expression and to enhance the migration of LSCC by increasing ZEB1 expression.

, correspAuthors=Yi Hu, authorNote=null, correspAuthorsNote=, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Xin Zhou, Hao Fan, An Wang, Jia-Pei Qin, Yi-Bing Bai, Zhi-Qiang Ma, Yi Hu), CN=ArticleExt(id=1198196448153141451, articleId=1198196212982710916, tenantId=1146029695717560320, journalId=1189873630562394117, language=CN, title=DNA甲基转移酶3a与肺鳞癌预后的相关性及其介导肺鳞癌进展的分子机制, columnId=1190310110472798614, journalTitle=解放军医学杂志, columnName=基础研究, runingTitle=null, highlight=null, articleAbstract=

目的 探讨DNA甲基转移酶3a(DNMT3a)表达与肺鳞癌预后的相关性及其介导肺鳞癌进展的潜在分子机制。方法 收集2009年5月－2014年1月于空军军医大学第二附属医院胸外科行手术治疗的47例肺鳞癌患者进行回顾性分析。采用免疫组织化学染色检测DNMT3a在肺鳞癌组织与癌旁组织中的表达，根据肺鳞癌中DNMT3a免疫组化评分中位数分为DNMT3a高表达组(n=25)与DNMT3a低表达组(n=22)，结合临床病理信息与公共生物信息学数据库进行预后相关性分析。为探索DNMT3a介导肺鳞癌进展的分子机制，采用慢病毒感染法构建DNMT3a过表达的肺鳞癌H1703细胞系，设置DNMT3a过表达组与对照组，采用功能表型实验检测两组细胞增殖、迁移的差异。构建DNMT3a过表达的裸鼠皮下肿瘤异种移植模型，设置DNMT3a过表达组与对照组(n=3)，观察两组皮下移植瘤的生长。Western blotting检测两组细胞与两组皮下移植瘤中相关蛋白的表达。功能回复实验先采用c-Myc抑制剂(10058-F4)处理DNMT3a过表达细胞，EdU增殖染色实验检测细胞增殖；再采用RNAi-锌指E盒结合同源框1(ZEB1)慢病毒感染DNMT3a过表达细胞敲低ZEB1表达，Transwell迁移实验检测细胞迁移。采用DNMT特异性抑制剂(SGI-1027)处理DNMT3a过表达组与对照组细胞，观察抑制DNMT3a后的细胞增殖及迁移。结果 免疫组化染色结果显示，肺鳞癌中DNMT3a免疫组化评分明显高于癌旁组织(P<0.0001)。DNMT3a高表达与N分期、TNM分期、肿瘤分化程度等密切相关(P<0.05或P<0.01)，DNMT3a高表达是肺鳞癌患者预后不良的独立危险因素(P<0.05)。功能表型实验表明，DNMT3a过表达组的克隆形成数、Edu阳性细胞比例、细胞迁移率、细胞迁移数均高于对照组(P<0.05)。DNMT3a过表达组裸鼠皮下移植瘤体积与重量明显高于对照组(P<0.001)。Western blotting检测结果显示，DNMT3a过表达组细胞与皮下移植瘤的c-Myc、ZEB1蛋白表达量明显高于对照组(P<0.05)。功能回复实验表明，10058-F4处理DNMT3a过表达组细胞EdU阳性细胞比例明显降低(P<0.05)；慢病毒敲低ZEB1后，DNMT3a过表达组细胞迁移数明显减少(P<0.05)，而DNMT3a蛋白的表达则无明显变化(P>0.05)。此外，SGI-1027抑制DNMT3a表达后，DNMT3a过表达组与对照组克隆形成数及细胞迁移率均明显降低(P<0.05)。结论 DNMT3a高表达是肺鳞癌患者预后不良的独立危险因素；DNMT3a可能通过上调c-Myc表达增强肺鳞癌的增殖能力，上调ZEB1表达增强肺鳞癌的迁移能力。

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²Department of Oncology, the Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071, China
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²解放军总医院第五医学中心肿瘤医学部肿瘤内科，北京 100071
³解放军总医院第二医学中心心血管内科，北京 100853, bio={"content":"

周鑫，硕士研究生，主要从事肺癌表观遗传学相关分子机制方面的研究

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周鑫，硕士研究生，主要从事肺癌表观遗传学相关分子机制方面的研究

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tableContent=null), ArticleFig(id=1198318898429915346, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1198196212982710916, language=CN, label=图1, caption=DNMT3a在肺鳞癌中高表达并与患者预后不良相关

DNMT3a. DNA甲基转移酶3a；A. 免疫组织化学染色检测DNMT3a在肺鳞癌患者肿瘤组织与癌旁组织中的表达；B. 肺鳞癌组织及癌旁组织中DNMT3a水平比较(n=47)；C. Kaplan-Meier生存分析DNMT3a不同表达对肺鳞癌患者累积总生存时间(OS)的影响；D. 基于GEPIA2数据库中482例肺鳞癌不同DNMT3a水平与患者生存的Kaplan-Meier生存分析；E. 基于Kaplan-Meier plotter数据库中300例肺鳞癌不同DNMT3a水平与患者生存的Kaplan-Meier生存分析

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DNMT3a. DNA甲基转移酶3a；ZEB1. 锌指E盒结合同源框1；A. Western blotting检测DNMT3a过表达组和对照组肺鳞癌细胞中DNMT3a、c-Myc、ZEB1蛋白的表达；B. CCK-8法检测两组肺鳞癌细胞体外增殖能力；C. 克隆形成实验检测两组肺鳞癌细胞体外增殖能力；D. 肿瘤异种移植实验模型检测两组裸鼠皮下肿瘤重量；E. Western blotting检测两组肿瘤组织中DNMT3a、c-Myc、ZEB1蛋白的表达；F. c-Myc特异性抑制剂10058-F4药物孵育肺鳞癌细胞48 h，采用EdU增殖染色实验检测各组细胞增殖能力；与对照组比较，(1)P<0.05，(2)P<0.001；与DNMT3a过表达组比较，(3)P<0.05；与DNMT3a过表达+DMSO组比较，(4)P<0.05

, figureFileSmall=cGZU8XTnNferQ8qf2Mki/w==, figureFileBig=wHDkZL1XKFn/AqsntxxAfA==, tableContent=null), ArticleFig(id=1198318898765459678, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1198196212982710916, language=EN, label=Fig.3, caption=Effect of overexpression of DNMT3a on migration of lung squamous cell carcinoma cells, figureFileSmall=GHX25RKxHJLsIphKcxp3Ug==, figureFileBig=lv9ag7rw9qnRvpwxASH5tw==, tableContent=null), ArticleFig(id=1198318898853540063, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1198196212982710916, language=CN, label=图3, caption=DNMT3a过表达对肺鳞癌细胞迁移能力的影响

DNMT3a. DNA甲基转移酶3a；ZEB1. 锌指E盒结合同源框1；A. 划痕实验检测两组肺鳞癌细胞体外迁移能力；B. Transwell迁移实验检测两组肺鳞癌细胞体外迁移能力(×100)；C. RNAi-ZEB1慢病毒转染DNMT3a过表达肺鳞癌细胞以敲低ZEB1的表达，采用Western blotting检测进行验证；D. Transwell迁移实验检测各组细胞迁移能力(×100)；与对照组比较，(1)P<0.05，(2)P<0.01；与DNMT3a过表达组比较，(3)P<0.05；与DNMT3a过表达+阴性对照组比较，(4)P<0.05

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DNMT3a. DNA甲基转移酶3a；A. 克隆形成实验检测各组肺鳞癌细胞体外迁移能力；B. 划痕实验检测各组肺鳞癌细胞体外迁移能力；与对照组比较，(1)P<0.05；与SGI-1027组比较，(2)P<0.05；与DNMT3a过表达组比较，(3)P<0.05

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Correlation of DNMT3a expression with clinicopathological characteristics of lung squamous cell carcinoma patients [n(%)]

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临床病理变量	总数(n=47)	DNMT3a高表达组(n=25)	DNMT3a低表达组(n=22)	P
性别				0.926
女	2(4.3)	1(4.0)	1(4.5)
男	45(95.7)	24(96.0)	21(95.5)
年龄(岁)				0.319
≥60	25(53.2)	15(60.0)	10(45.5)
<60	22(46.8)	10(40.0)	12(54.5)
吸烟史				0.297
无	6(12.8)	2(8.0)	4(18.2)
有	41(87.2)	23(92.0)	18(81.8)
T分期				0.775
T3	33(70.2)	18(72.0)	15(68.2)
T4	14(29.8)	7(28.0)	7(31.8)
N分期				<0.010
N0	25(53.2)	10(40.0)	15(68.2)
N1–N3	22(46.8)	15(60.0)	7(31.8)
远处转移				0.281
否	46(97.9)	25(100.0)	21(95.5)
是	1(2.1)	0	1(4.5)
TNM分期				<0.050
Ⅰ–Ⅱ	19(40.4)	8(32.0)	11(50.0)
Ⅲ–Ⅳ	28(59.6)	17(68.0)	11(50.0)
肿瘤分化程度				<0.050
高中度分化	30(63.8)	12(48.0)	18(81.8)
低分化	17(36.2)	13(52.0)	4(18.2)

), ArticleFig(id=1198318900338323696, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1198196212982710916, language=CN, label=表1, caption=

DNMT3a表达与肺鳞癌患者临床病理特征的相关性[例(%)]

, figureFileSmall=null, figureFileBig=null, tableContent=

临床病理变量	总数(n=47)	DNMT3a高表达组(n=25)	DNMT3a低表达组(n=22)	P
性别				0.926
女	2(4.3)	1(4.0)	1(4.5)
男	45(95.7)	24(96.0)	21(95.5)
年龄(岁)				0.319
≥60	25(53.2)	15(60.0)	10(45.5)
<60	22(46.8)	10(40.0)	12(54.5)
吸烟史				0.297
无	6(12.8)	2(8.0)	4(18.2)
有	41(87.2)	23(92.0)	18(81.8)
T分期				0.775
T3	33(70.2)	18(72.0)	15(68.2)
T4	14(29.8)	7(28.0)	7(31.8)
N分期				<0.010
N0	25(53.2)	10(40.0)	15(68.2)
N1–N3	22(46.8)	15(60.0)	7(31.8)
远处转移				0.281
否	46(97.9)	25(100.0)	21(95.5)
是	1(2.1)	0	1(4.5)
TNM分期				<0.050
Ⅰ–Ⅱ	19(40.4)	8(32.0)	11(50.0)
Ⅲ–Ⅳ	28(59.6)	17(68.0)	11(50.0)
肿瘤分化程度				<0.050
高中度分化	30(63.8)	12(48.0)	18(81.8)
低分化	17(36.2)	13(52.0)	4(18.2)

), ArticleFig(id=1198318900430598389, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1198196212982710916, language=EN, label=Tab.2, caption=

Univariate and multivariate analysis of prognostic factors for lung squamous cell carcinoma patients' survival

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临床病理变量	单因素分析			多因素分析
	HR	95%CI	P	HR	95%CI	P
性别(女/男)	2.446	0.335~17.850	0.378
年龄(<60岁/≥60岁)	0.755	0.405~1.409	0.377
吸烟史(从未吸烟/有过吸烟)	1.073	0.419~2.752	0.883
T分期(T3/T4)	1.769	0.899~3.481	0.099
N分期(N0/N1－N3)	1.406	0.750~2.638	0.288
TNM分期(Ⅰ－Ⅱ/Ⅲ－Ⅳ)	1.367	0.716~2.609	0.343
肿瘤分化程度(高中度分化/低分化)	3.785	1.911~7.497	<0.001	3.025	1.468~6.232	<0.01
肿瘤DNMT3a表达(低表达/高表达)	2.723	1.404~5.284	<0.010	2.073	1.017~4.222	<0.05

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影响肺鳞癌患者生存的单因素和多因素分析

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临床病理变量	单因素分析			多因素分析
	HR	95%CI	P	HR	95%CI	P
性别(女/男)	2.446	0.335~17.850	0.378
年龄(<60岁/≥60岁)	0.755	0.405~1.409	0.377
吸烟史(从未吸烟/有过吸烟)	1.073	0.419~2.752	0.883
T分期(T3/T4)	1.769	0.899~3.481	0.099
N分期(N0/N1－N3)	1.406	0.750~2.638	0.288
TNM分期(Ⅰ－Ⅱ/Ⅲ－Ⅳ)	1.367	0.716~2.609	0.343
肿瘤分化程度(高中度分化/低分化)	3.785	1.911~7.497	<0.001	3.025	1.468~6.232	<0.01
肿瘤DNMT3a表达(低表达/高表达)	2.723	1.404~5.284	<0.010	2.073	1.017~4.222	<0.05

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