临床肿瘤学杂志

目的 探讨生长抑制因子4(ING4)在子宫内膜癌（EC）中的表达及其对癌细胞生物学功能的影响。方法 收集138例子宫内膜组织标本，包括30例正常子宫内膜（NE）、31例不伴非典型性子宫内膜增生（EHA）、30例非典型性子宫内膜增生（EAH）及47例EC组织，通过免疫组化检测ING4、肿瘤蛋白53(p53)、细胞周期蛋白依赖性激酶抑制剂1A(p21)及细胞周期蛋白依赖性激酶抑制剂2A(p16)的蛋白表达。通过慢病毒转染构建稳定过表达ING4(Ad-ING4组)及阴性对照（NC组）的Ishikawa和HEC-1B细胞株。通过实时荧光定量PCR检测ING4、p53、含半胱氨酸的天冬氨酸蛋白水解酶-3(CASP3)、B细胞淋巴瘤-2(Bcl-2)及Bcl-2相关X蛋白（Bax）的mRNA表达水平，并采用蛋白质印迹检测ING4、p53、p21、p16、CASP3、Bcl-2、Bax、信号转导与转录激活因子3(STAT3)及其磷酸化形式（p-STAT3）的蛋白表达水平。利用细胞计数试剂盒-8(CCK-8)实验、克隆形成实验、划痕愈合实验、Transwell侵袭实验评估细胞增殖、迁移及侵袭能力；通过流式细胞术定量检测细胞凋亡与周期分布。采用过表达ING4的HEC-1B细胞构建裸鼠皮下移植瘤模型，观察ING4过表达对体内肿瘤生长的影响。结果 在EC组织中，ING4蛋白表达水平（平均光密度值：0.288±0.056）显著低于NE(0.480±0.077)、EHA(0.506±0.084)及EAH(0.406±0.096)组织（P<0.001），并与p21(r=0.353,P=0.015)和p16(r=0.451,P=0.002)的表达呈显著正相关。在细胞实验中，与NC组相比，Ad-ING4组的Ishikawa和HEC-1B细胞表现出增殖、迁移和侵袭能力受到抑制。具体表现为：Ad-ING4组的EC细胞增殖活力（72 h、96 h）、克隆形成能力、划痕愈合率及Transwell侵袭细胞数均显著降低，同时G1期细胞比例与早期凋亡比例升高。Ad-ING4组细胞中p53/p21/p16信号通路被激活，CASP3表达上调且Bax/Bcl-2比值增加，而p-STAT3的蛋白表达则受到抑制。在体内动物实验中，Ad-ING4组裸鼠的皮下移植瘤体积显著小于NC组[（89.00±7.55）mm3 vs.(206.70±8.51)mm3,P<0.01]，且瘤体内ING4、p53、p16的蛋白表达水平更高。结论 ING4在EC中低表达，其过表达可通过激活p53/p21/p16通路、促进凋亡并抑制STAT3磷酸化，有效抑制肿瘤细胞的增殖、侵袭及体内成瘤，提示ING4具有肿瘤抑制功能。

Objective To investigate the expression of inhibitor of growth family member 4(ING4) in endometrial cancer(EC) and its effect on the biological functions of cancer cells. Methods A total of 138 endometrial tissue specimens were collected, including 30 normal endometrial(NE), 31 endometrial hyperplasia without atypia(EHA), 30 atypical endometrial hyperplasia(EAH), and 47 EC tissues. The protein expression of ING4, tumor protein p53(p53), cyclin-dependent kinase inhibitor 1A(p21), and cyclindependent kinase inhibitor 2A(p16) was detected by immunohistochemistry. Stable ING4-overexpressing cell lines(Ad-ING4 group) and negative control cell lines(NC group) were established in Ishikawa and HEC-1B cells via lentiviral transfection. The mRNA expression levels of ING4, p53, cysteine-containing aspartate-specific protease-3(CASP3), B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax) were measured by quantitative real-time PCR, while the protein expression levels of ING4, p53, p21, p16, CASP3, Bcl-2, Bax, signal transducer and activator of transcription 3(STAT3), and its phosphorylated form(p-STAT3) were evaluated by Western blot. Cell proliferation, migration, and invasion were assessed using the Cell Counting Kit-8(CCK-8) assay, colony formation assay, wound healing assay, and Transwell invasion assay. Apoptosis and cell cycle distribution were analyzed by flow cytometry. A subcutaneous xenograft tumor model was established in nude mice using ING4-overexpressing HEC-1B cells to observe the effect of ING4 overexpression on in vivo tumor growth. Results In EC tissues, the expression level of ING4 protein(mean optical density: 0.288±0.056) was significantly lower than that in NE(0.480±0.077), EHA(0.506±0.084), and EAH(0.406±0.096) tissues(P<0.001). Furthermore, ING4 expression showed a significant positive correlation with the expression of p21(r=0.353, P=0.015) and p16(r=0.451, P=0.002). In cellular experiments, compared with the NC group, the Ad-ING4 group of Ishikawa and HEC-1B cells exhibited inhibited proliferation, migration, and invasion capabilities. Specifically, EC cells in the Ad-ING4 group showed significantly reduced proliferative activity(72 h, 96 h), colony formation ability, wound healing rate, and the number of Transwell invading cells, along with an increased proportion of G1-phase cells and early apoptotic cells. In the Ad-ING4 group, the p53/p21/p16 signaling pathway was activated, CASP3 expression was upregulated, the Bax/Bcl-2 ratio increased, while p-STAT3 protein expression was inhibited. In vivo animal experiments demonstrated that the subcutaneous xenograft tumor volume in the Ad-ING4 group was significantly smaller than that in the NC group [(89.00±7.55) mm3 vs.(206.70±8.51) mm3, P<0.01], and the protein expression levels of ING4, p53, and p16 in the tumor tissues were higher. Conclusion ING4 is downregulated in EC. Its overexpression effectively inhibits tumor cell proliferation, invasion, and in vivo tumorigenesis by activating the p53/p21/p16 pathway, promoting apoptosis, and inhibiting STAT3 phosphorylation, suggesting that ING4 possesses tumor-suppressive functions.