探讨STE20家族基因在肺鳞癌(lung squamous cell carcinoma, LUSC)中的表达特征、临床意义及与免疫浸润关系，分析预后标志物和治疗靶点潜在价值。

整合TCGA、GTEx及GEO数据库(GSE229509、GSE268175)中85例正常肺组织和559例LUSC组织的转录组数据，分析STE20家族成员在LUSC中表达差异。采用ESTIMATE和TIMER数据库判断STE20s与免疫细胞浸润的相关性。通过LASSO回归构建预后风险模型，利用随机森林模型判断MST4的预后。采用GO和KEGG富集分析探讨MST4潜在功能。通过qRT-PCR和Western blotting在LUSC细胞系(NCIH520、H-1703、SK MES 1)中验证关键基因表达。

STE20家族中SPAK、PAK1、PAK6及MST4在LUSC组织中表达上调(P<0.05)，MST1、OSR1、TAO2、MINK、TNIK、LOK表达下调(P<0.05)。免疫浸润分析显示，MST3、MYO3B、TNIK与免疫评分呈正相关，MST1、MST4、PAK1、PAK6与免疫评分呈负相关。TIMER分析表明，MST4表达与CD8^＋ T细胞(r＝－0.23，P<0.001)、CD4^＋ T细胞(r＝－0.19，P<0.01)及巨噬细胞浸润(r＝－0.21，P<0.001)呈负相关。LASSO回归筛选出5个预后相关基因(PAK1、PAK6、MST1、OSR1、MST4)，风险评分模型显示高风险组总生存期显著短于低风险组(HR＝2.34，95%CI：1.78~3.08，P<0.001)。随机森林模型预测1年、3年、5年生存率的受试者工作特征工作(receiver operating characteristic, ROC)曲线下面积分别为0.71、0.75、0.75。MST4高表达与不良预后相关(HR＝1.89，95%CI：1.42~2.51，P<0.001)。GO/KEGG富集分析显示MST4参与细胞周期、ECM受体互作及EMT相关通路。qRT-PCR和Western blotting验证结果显示，LUSC细胞系中PAK1、PAK6、MST4 mRNA及蛋白表达水平高于正常肺上皮细胞(BEAS-2B)(P<0.01)，MST1和OSR1表达水平降低(P<0.01)。

STE20家族基因在LUSC中表达异常，与免疫浸润及预后密切相关。MST4可能通过调控细胞周期和ECM重塑促进LUSC进展，可作为预后标志物和治疗靶点。

To investigate the expression characteristics and clinical significance of STE20 family genes in lung squamous cell carcinoma (LUSC), and to explore their association with immune infiltration and their potential as prognostic biomarkers and therapeutic targets.

Transcriptomic data from TCGA, GTEx, and GEO databases (GSE229509, GSE268175) comprising 85 normal lung tissues and 559 LUSC tissues were integrated to analyze the differential expression of STE20 family members. ESTIMATE and TIMER databases were used to evaluate the correlation between STE20s and immune cell infiltration. LASSO regression was employed to construct a prognostic risk model, and random forest model was used to assess the prognostic value of MST4. GO and KEGG enrichment analyses were performed to explore the potential functions of MST4. qRT-PCR and Western blotting were used to validate the expression of key genes in LUSC cell lines (NCIH520, H-1703, SK MES 1).

Among STE20 family members, SPAK, PAK1, PAK6, and MST4 were significantly upregulated in LUSC tissues (P<0.05), while MST1, OSR1, TAO2, MINK, TNIK, and LOK were significantly downregulated (P<0.05). Immune infiltration analysis revealed that MST3, MYO3B, and TNIK were positively correlated with immune scores, whereas MST1, MST4, PAK1, and PAK6 were negatively correlated. TIMER analysis showed that MST4 expression was significantly negatively correlated with infiltration of CD8⁺ T cells (r=-0.23, P<0.001), CD4⁺ T cells (r=-0.19, P<0.01), and macrophages (r=-0.21, P<0.001). LASSO regression identified five prognosis-related genes (PAK1, PAK6, MST1, OSR1, MST4). The risk score model indicated that the high-risk group had significantly shorter overall survival than the low-risk group (HR=2.34, 95%CI: 1.78~3.08, P<0.001). The random forest model predicting 1-, 3-, and 5-year survival achieved AUC values of 0.71, 0.75, and 0.75, respectively. High MST4 expression was associated with poor prognosis (HR=1.89, 95%CI: 1.42~2.51, P<0.001). GO/KEGG enrichment analysis revealed that MST4 is involved in cell cycle, ECM-receptor interaction, and EMT-related pathways. qRT-PCR and Western blotting confirmed that mRNA and protein levels of PAK1, PAK6, and MST4 were significantly higher in LUSC cell lines than in normal lung epithelial cells (BEAS-2B) (P<0.01), while MST1 and OSR1 were significantly lower (P<0.01).

STE20 family genes are aberrantly expressed in LUSC and are closely associated with immune infiltration and prognosis. MST4, as a key member, may promote LUSC progression by regulating cell cycle and ECM remodeling, highlighting its potential as a prognostic biomarker and therapeutic target.