CXC型趋化因子配体10与12在胆囊癌组织中的表达水平及在肿瘤侵袭中的作用机制

张博华; 徐艳; 张松; 胡华

doi:10.12449/JCH241120

CXC型趋化因子配体10与12在胆囊癌组织中的表达水平及在肿瘤侵袭中的作用机制

DOI: 10.12449/JCH241120

张博华^a,
徐艳^a,
张松^b,
胡华^a, , ,

a.
中国人民解放军中部战区总医院药剂科，武汉 430012
b.
中国人民解放军中部战区总医院消化内科，武汉 430012

基金项目:

湖北省自然科学基金 (2021CFB001)

伦理学声明：本研究方案于2020年3月10日经由中国人民解放军中部战区总医院伦理委员会审批，批号：伦（审）-20200315，所纳入患者均签署知情同意书。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：张博华负责论文的选题和设计，参与资料分析、整理文献和撰写论文；胡华负责拟定写作思路，提供指导性意见并最后定稿；徐艳负责论文核修，对学术问题进行解答；张松参与采数据集、分析与解释。

详细信息

通信作者:
胡华， ydiyaya@163.com （ORCID： 0009-0000-0904-2694）

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出版历程
- 收稿日期: 2024-02-28
- 录用日期: 2024-05-10
- 出版日期: 2024-11-25

Expression of CXCL10 and CXCL12 in gallbladder carcinoma and their mechanism of action in tumor invasion

Bohua ZHANG^a,
Yan XU^a,
Song ZHANG^b,
Hua HU^{a
, ,
,}

a.
Department of Pharmacy，General Hospital of Central Theater Command，Wuhan 430012，China）
b.
Department of Gastroenterology，General Hospital of Central Theater Command，Wuhan 430012，China

Research funding:

Hubei Provincial Natural Science Foundation (2021CFB001)

More Information

Corresponding author: HU Hua， ydiyaya@163.com （ORCID： 0009-0000-0904-2694）

摘要

摘要: 目的探讨CXC型趋化因子配体10（CXCL10）和12在胆囊癌组织中的表达水平及其参与肿瘤侵袭的机制。方法选择2020年4月—2023年4月在中国人民解放军中部战区总医院手术切除的56例胆囊癌患者肿瘤组织及癌旁组织标本，使用RT-PCR法检测癌组织及癌旁组织CXCL10 mRNA、CXCL12 mRNA表达，分析患者癌组织中CXCL10 mRNA、CXCL12 mRNA表达与患者临床病理参数的关系。使用人胆囊癌细胞株GBC-SD，构建CXCL10、CXCL12低表达胆囊癌细胞，CCK-8法检测CXCL10、CXCL12低表达对胆囊癌细胞株增殖的影响，Transwell检测CXCL10、CXCL12低表达对胆囊癌细胞株侵袭能力的影响，并采用Western Blot法检测胆囊癌细胞PI3K/Akt通路表达情况。计量资料两组间比较采用配对t检验或成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果在胆囊癌患者中，癌组织CXCL10、CXCL12 mRNA相对表达量均显著高于癌旁组织（1.857±0.315 vs 1.024±0.203、2.038±0.374 vs 1.064±0.221，t值分别为16.342、16.778，P值均<0.05）。不同TNM分期、有无淋巴结转移、有无远处转移以及不同肿瘤直径患者的CXCL10、CXCL12 mRNA比较差异均有统计学意义（P值均<0.05）。CXCL10：si-CXCL10组细胞CXCL10 mRNA相对表达量、CXCL10蛋白表达、CCK-8吸光值、细胞迁移数量、p-PI3K及p-Akt蛋白表达均显著低于对照组和si-RNA组（P值均<0.05）；CXCL12：si-CXCL12组细胞CXCL12 mRNA相对表达量、CXCL12蛋白表达、CCK-8吸光值、细胞迁移数量、p-PI3K及p-Akt蛋白表达均显著低于对照组和si-RNA组（P值均<0.05）。结论胆囊癌组织中的CXCL10及CXCL12表达升高，抑制CXCL10、CXCL12表达后，胆囊癌细胞增殖、侵袭受到明显抑制，其机制可能与抑制PI3K/Akt通路磷酸化有关。
- 胆囊肿瘤 /
- 趋化因子CXCL10 /
- 趋化因子CXCL12 /
- 肿瘤浸润
Abstract: Objective To investigate the expression levels of CXCL10 and CXCL12 in gallbladder carcinoma and their mechanism of action in tumor invasion. Methods Tumor tissue samples and adjacent tissue samples were collected from 56 patients with gallbladder carcinoma who underwent surgical resection in General Hospital of Central Theater Command from April 2020 to April 2023. RT-PCR was used to measure the mRNA expression levels of CXCL10 and CXCL12 in cancerous tissue and adjacent tissue， and the correlation of the mRNA expression levels of CXCL10 and CXCL12 in cancerous tissue with clinicopathological parameters was analyzed. The human gallbladder carcinoma cell line GBC-SD was used to construct gallbladder carcinoma cells with low expression of CXCL10 and CXCL12. CCK8 assay was used to observe the effect of low expression of CXCL10 and CXCL12 on the proliferation of gallbladder carcinoma cells， Transwell assay was used to observe the effect of low expression of CXCL10 and CXCL12 on the invasion ability of gallbladder carcinoma cells， and Western blot was used to measure the expression of the PI3K/Akt pathway in gallbladder carcinoma cells. The paired t-test or independent-samples t-test was used for comparison of measurement data between two groups； an analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results In the patients with gallbladder carcinoma， the relative mRNA expression levels of CXCL10 and CXCL12 in cancerous tissue were significantly higher than those in adjacent tissue （CXCL10： 1.857±0.315 vs 1.024±0.203， t=16.342， P<0.05； CXCL12： 2.038±0.374 vs 1.064±0.221， t=16.778， P<0.05）. There were significant differences in the relative mRNA expression levels of CXCL10 and CXCL12 between the patients with different TNM stages， presence or absence of lymph node metastasis or distant metastasis， and tumor diameters （all P<0.05）. Compared with the control group and the si-RNA group， the si-CXCL10 group had significantly lower relative mRNA and protein expression levels of CXCL10 and CXCL12， CCK-8 absorbance values， number of cell migration， and protein expression levels of p-PI3K and p-Akt （all P<0.05）. Conclusion There are increases in the expression of CXCL10 and CXCL12 in gallbladder carcinoma tissue， and the proliferation and invasion of gallbladder carcinoma cells are significantly inhibited after inhibition of the expression of CXCL10 and CXCL12， which might be associated with the inhibition of the phosphorylation of the PI3K/Akt pathway.
- Gallbladder Neoplasms /
- Chemokine CXCL10 /
- Chemokine CXCL12 /
- Neoplasm Invasiveness

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图 1 癌组织及癌旁组织CXCL10、CXCL12蛋白表达（HE染色，×400）

注： a，癌组织CXCL10表达阳性；b，癌旁组织CXCL10表达阴性；c，癌组织CXCL12表达阳性；d，癌旁组织CXCL12表达阴性。

Figure 1. Expression of CXCL10 and CXCL12 proteins in cancer tissue and adjacent tissue （HE staining， ×400）

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图 2 低表达CXCL10、CXCL12对细胞迁移的影响（结晶紫染色，×200）

注： a、b、c分别为CXCL10的对照组、si-RNA组和si-CXCL10组；d、e、f分别为CXCL12的对照组、si-RNA组和si-CXCL12组。

Figure 2. Low expression of CXL10 RNA and CXL12 RNA affects cell migration（crystal violet staining， ×200）

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图 3 各组细胞PI3K/Akt通路表达情况

Figure 3. Expression of PI3K/Akt pathway in each group

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表 1 癌组织及癌旁组织CXCL10 mRNA、CXCL12 mRNA表达比较

Table 1. Comparison of CXCL10 mRNA and CXCL12 mRNA expression in cancer tissue and adjacent tissue

组别	例数	CXCL10 mRNA	CXCL12 mRNA
癌组织	56	1.857±0.315	2.038±0.374
癌旁组织	56	1.024±0.203	1.064±0.221
t值		18.359	20.506
P值		<0.001	<0.001

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表 2 癌组织CXCL10 mRNA、CXCL12 mRNA表达与患者临床病理参数的关系

Table 2. The relationship between the expression of CXCL10 mRNA and CXCL12 mRNA in cancer tissues and clinical pathological parameters of patients

临床病理特征	例数	CXCL10 mRNA	CXCL12 mRNA
性别
男	27	1.796±0.293	1.985±0.337
女	29	1.914±0.356	2.083±0.405
t值		1.359	0.980
P值		0.183	0.331
年龄
≥60岁	25	1.894±0.229	1.993±0.311
<60岁	31	1.827±0.336	2.074±0.401
t值		0.850	0.828
P值		0.399	0.411
TNM分期
Ⅰ～Ⅱ期	21	1.447±0.285	1.570±0.324
Ⅲ～Ⅳ期	35	2.103±0.407	2.319±0.452
t值		6.483	6.630
P值		<0.001	<0.001
病理类型
腺癌	49	1.844±0.394	2.015±0.416
其他	7	1.948±0.410	2.199±0.375
t值		0.650	1.106
P值		0.518	0.274
淋巴结转移
有	30	2.153±0.473	2.349±0.332
无	26	1.515±0.338	1.679±0.415
t值		5.724	6.709
P值		<0.001	<0.001
远处转移
有	19	2.203±0.374	2.412±0.423
无	37	1.679±0.310	1.846±0.325
t值		5.580	5.561
P值		<0.001	<0.001
肿瘤直径
>5 cm	21	2.115±0.263	2.414±0.462
≤5 cm	35	1.702±0.364	1.812±0.394
t值		4.531	5.187
P值		<0.001	<0.001

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表 3 各组细胞CXCL10、CXCL12 mRNA及蛋白的比较

Table 3. Comparison of CXCL10 and CXCL12 mRNA and protein expression among different groups

分组	mRNA	蛋白
CXCL10
对照组	1.029±0.204	0.937±0.152
si-RNA组	1.048±0.156^1）	1.035±0.224^1）
si-CXCL10组	0.492±0.115^1）2）	0.533±0.128^1）2）
F值	18.878	4.453
P值	<0.001	0.036
CXCL12
对照组	0.937±0.152	1.685±0.217
si-RNA组	1.035±0.224^1）	1.603±0.301^1）
si-CXCL12组	0.533±0.128^1）2）	0.997±0.205^1）2）
F值	11.845	9.186
P值	<0.001	<0.001
注：与对照组比较，1）P<0.05；与si-RNA组比较，2）P<0.05。

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表 4 各组细胞吸光值和迁移数量的比较

Table 4. Comparison of light absorption value and migration number of cells in each group

分组	吸光值	细胞迁移数量
CXCL10
对照组	1.44±0.32	159.43±24.09
si-RNA组	1.39±0.40^1）	150.35±31.54^1）
si-CXCL10组	0.74±0.15^1）2）	108.30±25.60^1）2）
F值	8.029	5.005
P值	0.006	0.026
CXCL12
对照组	1.51±0.23	162.39±39.42
si-RNA组	1.59±0.33^1）	158.23±41.39^1）
si-CXCL12组	0.86±0.20^1）2）	101.31±29.84^1）2）
F值	11.915	4.202
P值	0.001	0.041
注：与对照组比较，1）P<0.05；与si-RNA组比较，2）P<0.05。

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表 5 各组细胞PI3K/Akt通路蛋白的比较

Table 5. Comparison of PI3K/Akt pathway proteins in each group

分组	p-PI3K	p-Akt
CXCL10
对照组	0.984±0.132	1.294±0.120
si-RNA组	1.023±0.174^1）	1.317±0.219^1）
si-CXCL10组	0.485±0.097^1）2）	0.720±0.137^1）2）
F值	23.638	21.152
P值	0.006	<0.001
CXCL12
对照组	1.127±0.133	1.423±0.210
si-RNA组	1.094±0.165^1）	1.389±0.197^1）
si-CXCL12组	0.603±0.102^1）2）	0.792±0.174^1）2）
F值	23.353	16.692
P值	<0.001	<0.001
注：与对照组比较，1）P<0.05；与si-RNA组比较，2）P<0.05。

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