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.