The impact of doublecortin-like kinase 1 on the clinical characteristics of endometrioid adenocarcinoma patients

doi:10.3877/cma.j.issn.1673-5250.2024.05.016

Objective

To investigate the expression of doublecortin-like kinase （DCLK）1 in endometrioid adenocarcinoma（EA）patients and evaluate its influence on the clinical characteristics of EA patients.

Methods

A total of 95 EA patients，25 patients with atypical hyperplasia（AH），and 30 with normal endometrial tissue who underwent hysterectomy for benign uterine diseases，recruited from the Department of Gynecology，West China Second University Hospital，Sichuan University，from January to June 2019 as study subjects.According to types of endometrial diseases，they were divided into EA group （n = 95），AH group （n= 25），and control group （n = 30）.Immunohistochemistry was used to measure DCLK1 protein expression in tissues of above three groups.To further assess the role of DCLK1，EA cell lines （HEC-1-A and Ishikawa）were transfected with DCLK1 overexpression plasmids or control vectors.These cells were grouped as the DCLK1 overexpression subgroup，and vector subgroup.And HEC-1-A and Ishikawa cell lines that were not transfected with plasmids were included into blank subgroup.The expression levels of DCLK1 and key proteins in AKT/m TOR signaling pathways of HEC-1-A and Ishikawa cells in the above three subgroups were detected.Ethical approval was granted by the Ethics Committee of West China Second University Hospital，Sichuan University （Approval No.2022-038），and all participants provided clinical research informed consent.

Results

①The proportion of abnormal uterine bleeding in patients of EA group，AH group，and control group were compared，and the difference was statistically significant （χ² =45.90，P ＜0.001）.The positive expression rates of DCLK1 in endometrial glands of these three groups of patients were 83.2%（79/95），76.0%（19/25），and 40.0%（12/30），respectively，and the difference was also statistically significant （χ²=21.83，P＜0.001）.②In the EA group，there was a significant difference in the positive expression rate of DCLK1 in patients with different pathological grades of EA tissue （χ²=14.70，P=0.003），and the expression level of DCLK1 was positively correlated with EA pathological grade （r_s=0.503，P ＜0.001）.③In HEC-1-A and Ishikawa cells of DCLK1 overexpression subgroup，DCLK1 protein expression levels were significantly higher than those in vector subgroup and blank subgroup，and protein kinase B （AKT），phosphorylated protein kinase B （p-AKT）and mammalian target of rapamycin （m TOR）protein expression levels were significantly higher than those in vector subgroup，and all the differences were statistically significant （P＜0.05）.

Conclusions

The expression level of DCLK1 is closely related to the malignant phenotype of EA，and it may also be related to the AKT/m TOR signaling pathway.DCLK1 may serve as a potential biomarker for the clinical diagnosis of EA patients and a therapeutic target.

Key words: Endometrial neoplasms, Doublecortin-like kinase 1, Biomarker，tumor, AKT/m TOR signaling pathway, Female

表1 3组患者相关临床资料比较

组别	例数	年龄（岁， $\overset{ˉ}{x}$ ±s）	BMI（kg/m²， $\overset{ˉ}{x}$ ±s）	初次性生活年龄（岁， $\overset{ˉ}{x}$ ±s）	绝经［例数（%）］	肿瘤家族史［例数（%）］	妊娠史［例数（%）］	异常子宫出血［例数（%）］
EA组	95	51.3±7.0	24.6±3.5	22.7±3.0	31（32.6）	17（17.9）	93（97.9）	73（76.8）
AH组	25	48.2±6.3	24.0±3.2	23.1±2.6	10（40.0）	1（4.0）	22（88.0）	5（20.0）
对照组	30	49.9±6.0	24.2±4.7	23.2±2.7	12（40.0）	7（23.3）	30（100.0）	4（13.3）
统计量		F=2.27	F=0.31	F=0.44	χ²=0.89	χ²=3.99	χ²=4.47	χ²=45.90
P值		0.106	0.350	0.322	0.685	0.126	0.057	＜0.001

表1 3组患者相关临床资料比较

组别	例数	年龄（岁， $\overset{ˉ}{x}$ ±s）	BMI（kg/m²， $\overset{ˉ}{x}$ ±s）	初次性生活年龄（岁， $\overset{ˉ}{x}$ ±s）	绝经［例数（%）］	肿瘤家族史［例数（%）］	妊娠史［例数（%）］	异常子宫出血［例数（%）］
EA组	95	51.3±7.0	24.6±3.5	22.7±3.0	31（32.6）	17（17.9）	93（97.9）	73（76.8）
AH组	25	48.2±6.3	24.0±3.2	23.1±2.6	10（40.0）	1（4.0）	22（88.0）	5（20.0）
对照组	30	49.9±6.0	24.2±4.7	23.2±2.7	12（40.0）	7（23.3）	30（100.0）	4（13.3）
统计量		F=2.27	F=0.31	F=0.44	χ²=0.89	χ²=3.99	χ²=4.47	χ²=45.90
P值		0.106	0.350	0.322	0.685	0.126	0.057	＜0.001

图1 3组患者子宫内膜组织免疫组化染色结果（图1A：EA 组；图1B：AH 组；图1C：对照组）（苏木精染色，高倍）注：对照组为因子宫良性疾病进行子宫切除术的子宫内膜组织正常患者。EA 为子宫内膜样腺癌，AH 为非典型增生

表2 不同临床特征EA 组患者DCLK1阳性表达率比较［例数（%）］

临床指标	例数	DCLK1阳性表达率	χ²值	P值
FIGO分期（期）			0.07	0.678
Ⅰ～Ⅱ	61	50（82.0）
Ⅲ～Ⅳ	34	29（85.3）
ER表达			1.51	0.476
阳性	88	72（81.8）
阴性	7	7（100.0）
组织病理学分级			14.70	0.003
高分化	31	20（64.5）
中分化	32	29（90.6）
低分化	32	30（93.8）
PR表达			3.16	0.128
阳性	80	64（80.0）
阴性	15	15（100.0）
p53表达^a			0.08	0.849
阳性	47	39（83.0）
阴性	45	38（84.4）
PTEN表达^a			0.46	0.815
阳性	10	9（90.0）
阴性	75	61（81.3）

表2 不同临床特征EA 组患者DCLK1阳性表达率比较［例数（%）］

临床指标	例数	DCLK1阳性表达率	χ²值	P值
FIGO分期（期）			0.07	0.678
Ⅰ～Ⅱ	61	50（82.0）
Ⅲ～Ⅳ	34	29（85.3）
ER表达			1.51	0.476
阳性	88	72（81.8）
阴性	7	7（100.0）
组织病理学分级			14.70	0.003
高分化	31	20（64.5）
中分化	32	29（90.6）
低分化	32	30（93.8）
PR表达			3.16	0.128
阳性	80	64（80.0）
阴性	15	15（100.0）
p53表达^a			0.08	0.849
阳性	47	39（83.0）
阴性	45	38（84.4）
PTEN表达^a			0.46	0.815
阳性	10	9（90.0）
阴性	75	61（81.3）

图2 EA 组EA 细胞系中过表达DCLK1 基因序列质粒图谱注：EA 为子宫内膜样腺癌，DCLK1为双肾上腺皮质激素样激酶1

图3 过表达DCLK1亚组、空载体亚组、空白对照亚组HEC-1-A 和Ishikawa细胞DCLK1表达水平柱状图与电泳图（图3A、3B：HEC-1-A 细胞中，DCLK1表达水平柱状图与电泳图；图3C、3D：Ishikawa细胞中，DCLK1表达水平柱状图与电泳图）注：Blank表示空白对照亚组，Vector表示空载体亚组，OE代表过表达DCLK1亚组。 ^a表示P＜0.001。DCLK1为双肾上腺皮质激素样激酶1，GADPH 为磷酸苷油酸脱氢酶

图4 过表达DCLK1亚组与空载体亚组HEC-1-A 和Ishikawa细胞AKT/m TOR 信号通路中关键蛋白表达水平（图4A：HEC-1-A 和Ishikawa细胞中，AKT/m TOR 信号通路关键蛋白表达水平电泳图；图4B：HEC-1-A 细胞中，AKT 表达水平柱状图；图4C：Ishikawa细胞中，AKT 表达水平柱状图；图4D：HEC-1-A 细胞中，p-AKT 表达水平柱状图；图4E：Ishikawa细胞中，p-AKT 表达水平柱状图；图4F：HEC-1-A 中，m TOR 表达水平柱状图；图4G：Ishikawa细胞中，m TOR 表达水平柱状图）注：Vector表示空载体亚组，OE代表过表达DCLK1亚组。 ^a表示P＜0.05； ^b表示P＜0.01。DCLK1为双肾上腺皮质激素样激酶1，AKT为蛋白激酶B，p-AKT 为磷酸化蛋白激酶B，m TOR 为哺乳动物雷帕霉素靶蛋白，GAPDH 为磷酸苷油酸脱氢酶

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