探讨胱抑素4(cystatin 4, CST4)在肺腺癌组织及细胞中表达水平及对肺腺癌H1650细胞恶性行为的影响与机制。

利用TCGA数据库分析CST4在肺腺癌中表达及诊断价值；通过qRT-PCR和Western blot检测CST4在正常肺上皮细胞BEAS-2B及肺腺癌细胞系A549、PC9、H1650中表达。在H1650细胞中构建CST4敲低及过表达模型，采用CCK-8法、流式细胞术、划痕愈合实验和Transwell侵袭实验评估细胞增殖、凋亡、迁移和侵袭能力；Western blot检测上皮-间质转化(epithelial-mesenchymal transition, EMT)相关蛋白表达。通过共敲低E-钙黏蛋白(E-cadherin)进行挽救实验。

CST4在肺腺癌组织中高表达，ROC曲线显示诊断效能高。与BEAS-2B细胞相比，A549、PC9和H1650细胞中CST4蛋白(P＝0.011、0.009、0.001)及mRNA(P＝0.005、0.002、0.001)水平上调。功能实验显示，敲低CST4后H1650细胞增殖受抑制，凋亡率升高[(22.68±1.89)%比(38.23±3.62)%](P＝0.007)，划痕愈合度降低[(59.16±4.31)%比(42.45±3.87)%](P＝0.022)，侵袭细胞数减少[(51.93±4.76)个比(31.28±3.07)]个(P＝0.007)；过表达CST4呈相反效应，凋亡率降低[(24.51±1.89)%比(14.26±1.29)%](P＝0.004)，愈合度升高[(56.61±5.03)%比(75.47±6.28)%](P＝0.034)，侵袭细胞数增加[(46.39±4.23)个比(67.11±6.36)个](P＝0.016)。敲低CST4上调E-cadherin并下调N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)(P＝0.003、0.027、0.016)，过表达相反(P＝0.006、0.039、0.011)。挽救实验显示，敲低CST4基础上同时敲低E-cadherin，可逆转CST4缺失所致侵袭抑制和凋亡促进。

CST4在肺腺癌中高表达，调控EMT通路促进H1650细胞增殖、迁移和侵袭，抑制凋亡，可成为肺腺癌诊断和治疗潜在靶点。

To investigate the expression level of cystatin 4 (CST4) in lung adenocarcinoma tissues and cells and its effect on the malignant behaviors of H1650 cells, as well as the underlying mechanism.

The TCGA database was used to analyze CST4 expression in lung adenocarcinoma and its diagnostic value. qRT-PCR and Western blot were performed to detect CST4 expression in normal lung epithelial cells BEAS2B and lung adenocarcinoma cell lines A549, PC9, and H1650. CST4 knockdown and overexpression models were established in H1650 cells. Cell proliferation, apoptosis, migration, and invasion were assessed by CCK8 assay, flow cytometry, wound healing assay, and Transwell invasion assay, respectively. epithelial-mesenchymal transition (EMT)related protein expression was detected by Western blot. Rescue experiments were conducted by coknockdown of Ecadherin.

CST4 was highly expressed in lung adenocarcinoma tissues, and ROC curve analysis showed high diagnostic efficacy. Compared with BEAS2B cells, the protein (P=0.011, 0.009, 0.001) and mRNA (P=0.005, 0.002, 0.001) levels of CST4 were significantly upregulated in A549, PC9, and H1650 cells. Functional assays revealed that CST4 knockdown inhibited cell proliferation, increased the apoptosis rate [(22.68±1.89)% vs. (38.23±3.62)%, P=0.007], decreased wound healing ability [(59.16±4.31)% vs. (42.45±3.87)%, P=0.022], and reduced the number of invading cells [(51.93±4.76) vs. (31.28±3.07), P=0.007]. Conversely, CST4 overexpression showed opposite effects: apoptosis rate decreased [(24.51±1.89)% vs. (14.26±1.29)%, P=0.004], wound healing increased [(56.61±5.03)% vs. (75.47±6.28)%, P=0.034], and invading cells increased [(46.39±4.23) vs. (67.11±6.36), P=0.016]. Mechanistically, CST4 knockdown upregulated Ecadherin and downregulated Ncadherin and Vimentin (P=0.003, 0.027, 0.016), while overexpression produced the opposite effects (P=0.006, 0.039, 0.011). Rescue experiments showed that simultaneous knockdown of Ecadherin reversed the CST4deficiencyinduced inhibition of invasion and promotion of apoptosis.

CST4 is highly expressed in lung adenocarcinoma and promotes proliferation, migration, and invasion while inhibiting apoptosis in H1650 cells through regulation of the EMT pathway. CST4 may serve as a potential diagnostic and therapeutic target for lung adenocarcinoma.