口腔鳞癌来源的游离DNA 通过诱导巨噬细胞极化调控口腔癌细胞系干性和迁移能力

郑适泽; 孟琳; 任飞龙; 任春霞; 李杏; 王丹丹; 孙宏晨

doi:10.12182/20230560206

口腔鳞癌来源的游离DNA 通过诱导巨噬细胞极化调控口腔癌细胞系干性和迁移能力

doi:

郑适泽^{1, 2,},
孟琳^{1, 2},
任飞龙^{1, 2},
任春霞^{2, 3},
李杏^{1, 2},
王丹丹^{1, 2},
孙宏晨^{1, 2, ,}

1. 辽宁大学本科齿科医生方面的问题科（成都 130021）
2. 辽源市省牙骨骼发育及颌骨重朔与再生利用要点实验操作室（哈尔滨市 130021）
3. 辽源市大家嘴巴门诊牙体科（沈阳 130021）

基金项目: 国家自然科学基金重点国际（地区）合作研究项目（No. 81920108012）和吉林大学研究生创新研究计划项目（No. 101832020CX304）资助

详细信息

作者简介: 孙宏晨，吉林市社会“唐敖庆學者”有远见教受A岗，主管专门医师，博士生生任课老师，中国级领退伍军人物人材，“长白山學者”聘请教受，中国国耳鼻喉科临床会耳鼻喉科病症学专门分委会会主管分委会。首要开展牙和骨胸部发育怪物学与修整复苏理论研究。对于首席总裁物理学者节目主持中国重心生产制造工作方案1项，对于开展人需承担收录重心項目和重心国外公司合作項目内的中国那自然物理学债卷項目9项。刊登国外杂志期刊论文提纲140余篇，H系数45。获中国专利申请技术专利技术代理权10件，得到省部级科技开发进步英语头等奖1项，一级奖2项。讲授中国《耳鼻喉科企业病症学》上品必修课程、上品信息分享课和全渠道金课，获省级重点教学课优秀成果头等奖2项

通讯作者:
E-mail：hcsun@mail.jlu.edu.cn

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出版历程
- 收稿日期: 2023-10-13
- 修回日期: 2024-04-25
- 网络出版日期: 2024-05-20
- 刊出日期: 2024-05-20

Oral Squamous Cell Carcinoma-Derived Cell-Free DNA Modulates Stemness and Migration of Oral Squamous Cell Carcinoma Cell Line by Inducing M2 Macrophage Polarization

koko体育app:ZHENG Shi-ze^{1, 2
,},
MENG Lin^{1, 2},
REN Fei-long^{1, 2},
koko体育app:REN Chun-xia^{2, 3},
LI Xing^{1, 2},
WANG Dan-dan^{1, 2},
SUN Hong-chen^{1, 2
, ,}

1. Department of Pathology, Hospital of Stomatology, Jilin University, Changchun 130021, China
2. Jilin Provincial Key Laboratory of Tooth Development, Jaw Bone Remodeling and Regeneration, Changchun 130021, China
3. Department of Periodontology, Hospital of Stomatology, Jilin University, Changchun 130021, China

More Information

Corresponding author: E-mail: hcsun@mail.jlu.edu.cn

摘要

摘要: 目的研究口腔鳞状细胞癌细胞来源的游离DNA对巨噬细胞极化作用，以及极化的巨噬细胞对口腔鳞癌细胞系的干性和迁移能力调控作用。方法取病理确诊为口腔鳞状细胞癌的组织标本30例，异常增生的组织标本10例，正常口腔上皮组织标本10例。通过免疫组化染色、免疫荧光染色检测M2型巨噬细胞在不同口腔组织中数量及位置。收集人舌鳞状细胞癌细胞系Cal-27细胞的条件培养基，纯化并提取游离DNA（cell free DNA, cfDNA）并进行鉴定。用cfDNA处理巨噬细胞，观察细胞形态学变化，RT-qPCR检测极化相关指标表达水平。用cfDNA 诱导后的巨噬细胞条件培养基处理 CAL-27 细胞，RT-qPCR检测其干性基因变化水平；并且通过划痕实验验证cfDNA诱导的巨噬细胞调控肿瘤细胞迁移的能力。结果与正常口腔上皮组织相比，异常增生的口腔上皮深层结缔组织和口腔鳞癌间质中M2型巨噬细胞数量较多（P<0.05）。CAL-27细胞分泌长度在 10000～15000 bp的cfDNA。CAL-27细胞分泌的cfDNA可诱导巨噬细胞高表达 M2 型巨噬细胞标记（P<0.05）。cfDNA 处理的巨噬细胞诱导肿瘤细胞高表达肿瘤干性基因（P<0.05），同时促进了肿瘤细胞迁移能力（P<0.05）。结论口腔鳞状细胞癌细胞来源的cfDNA通过诱导巨噬细胞向 M2 型极化促进口腔癌细胞系干性和迁移。
- koko体育app: 肿瘤相关巨噬细胞 /
- koko体育app: 口腔鳞状细胞癌 /
- koko体育app: 循环游离DNA /
- koko体育app: 极化 /
- koko体育app: 干性
Abstract: Objective To investigate the effect of oral squamous cell carcinoma (OSCC)-derived cell-free DNA (cfDNA) on the polarization of macrophages and the regulatory effect of polarized macrophages on the stemness and migration of OSCC cells. Methods A total of 30 OSCC tissue samples, 10 dysplastic oral tissue samples, and 10 normal oral tissue samples were collected. The status of all tissue samples was confirmed by pathology analysis. Immunohistochemical (IHC) staining and immunofluorescence (IF) staining were performed to examine the cell count and location of M2 macrophages in different types of oral tissue samples. The conditioned medium (CM) of OSCC cell line CAL-27 from the human tongue was collected and the cfDNA was concentrated and isolated for identification. The macrophages were treated by cfDNA and their morphological characteristics were observed under microscope. The expression levels of polarization-related indicators were determined by RT-qPCR. CAL-27 cell line was treated with macrophage CM induced by cfDNA and the expression levels of stemness-related genes were determined by RT-qPCR. Scratch-wound assay was conducted to verify that the migration ability of CAL-27 was modulated by macrophages induced by cfDNA. Results There were more M2 macrophages in the deep connective tissue of dysplastic oral epithelium and the stroma of OSCC compared with those in the normal oral tissues (P<0.05). OSCC cell line CAL-27 could secret cfDNA of 10000-15000 bp in length. cfDNA secreted by CAL-27 could induced in macrophages significantly higher expression of M2-macrophage-related genes (P<0.05). cfDNA-treated macrophages induced significantly increased expression of stemness-related genes in CAL-27 cell line (P<0.05) and promoted the migration ability of CAL-27 cell line (P<0.05). Conclusion OSCC-derived cfDNA promotes stemness and migration of OSCC cell line by inducing M2 macrophage polarization.
- Tumor-associated macrophage /
- koko体育app: 🦂 Oral squam🔯ous cell carcinoma /
- koko体育app: Cell-free DNA /
- koko体育app: Polarization /
- koko体育app: Stemness

koko体育app: HTML全文

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图 1 M2型巨噬细胞在不同口腔组织中的分布

Figure 1. Distribut♈ion of M2 macrophages in different types of🎶 oral tissues

A: IHC staining of CD163 (scale bar=200 μm [top], 50 μm [bottom]); B: IF staining of CD163 (scale bar=20 μm).

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图 2 鉴定cfDNA

Figure 2. Determination of cfDNA

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图 3 cfDNA对巨噬细胞活性作用

Figure 3. Effect of cfDNA on macrophage activity

^** P <0.01,^*** P <0.001, vs. control group.

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图 4 cfDNA 对巨噬细胞的极化作用

Figure 4. E💖ffect of cfDNA on gene expression of related cytokines in M1 and M2 macrophages

A: microscopic image (the yellow arrows are pointed at polarized macrophages); B: the mRNA levels were determined by qPCR. ^** P<0.01, ^*** P<0.001, vs. CTR-CM group, n=3. CTR-CM: control-ꦓconditioned medium; cfDNA-CM: conditioned medium of macrophages after induction by cfDNA.

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图 5 cfDNA诱导的☂巨噬细胞对CAL-27细胞干性基因表达𒐪（A）和CAL-27细胞迁移能力（B）的影响

Figure 5. Effect of cfDNA-induced macrophages on the expression of stemness-related genes in CAL-27 cells (A) and on the migration ability of🦩 CAL-27 cells (B)

^* P <0.05, ^{* * *} P <0.001, vs. CTR-CM group, n=3. CTR-CM: control-conditioned medium; cfDNA-CM: conditioned medium of m💞acrophages after induction by cfDNA.

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表 1 引物合成序列

Table 1. Gene primer sequences

Gene (human)	Primer sequence (5′-3′)	Primer length/bp
β-actin	F: GGAGATTACTGCCCTGGCTCCTA	23
	R GACTCATCGTACTCCTGCTTGCTG	22
CD86	F: TGCTCATCTATACACGGTTACC	22
	R: TGCATAACACCATCATACTCGA	22
TNF-α	F: TGGCGTGGAGCTGAGAGATAACC	23
	R: CGATGCGGCTGATGGTGTGG	20
IL-6	F: CACTGGTCTTTTGGAGTTTGAG	22
	R: GGACTTTTGTACTCATCTGCAC	22
CD163	F: ATCAACCCTGCATCTTTAGACA	22
	R: CTTGTTGTCACATGTGATCCAG	22
CD204	F: GGACACTGATAGCTGCTCCGAATC	24
	R: CACGAGGAGGTAAAGGGCAATCAG	24
Arg-1	F: GGACCTGCCCTTTGCTGACATC	22
	R: TCTTCTTGACTTCTGCCACCTTGC	24
TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; Arg-1: arginase 1.

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表 2 RT-qPCR引物合成序列

Table 2. RT-qPCR primer sequences

Gene (human)	Primer sequence (5′-3′)	Primer length/bp
β-actin	F: GGAGATTACTGCCCTGGCTCCTA	23
	R: GACTCATCGTACTCCTGCTTGCTG	24
SOX2	F: GTGAGCGCCCTGCAGTACAA	20
	R: GCGAGTAGGACATGCTGTAGGTG	23
OCT4	F: GCTGGATGTCAGGGCTCTTTG	21
	R: TTCAAGAGATTTATCGAGCACCTTC	25
SOX2: SRY-box transcription factor 2; OCT4: organic cation/carnitine transporter 4.

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