HAPLN3基因在肾透明细胞癌中的表达及其与预后、免疫的关系

目的 探讨透明质酸和蛋白多糖链接蛋白家族基因成员3（HAPLN3）在肾透明细胞癌（ccRCC）中的表达及其与预后、免疫的关系。方法 利用多个公共数据库分析HAPLN3在ccRCC中的差异表达、预后价值，以及与肿瘤微环境、免疫细胞浸润、免疫检查点及免疫检查点阻断反应评分（TIDE）等的相关性，并行富集分析HAPLN3在ccRCC中的潜在作用机制。通过免疫组织化学法检测HAPLN3蛋白在癌组织和癌旁组织中的表达，采用实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting检测ccRCC细胞株（OS-RC-2、ACHN）HAPLN3 mRNA和蛋白的表达。细胞转染siRNA沉默HAPLN3，用qRT-PCR检测不同ccRCC细胞株中HAPLN3基因沉默的有效性。采用CCK-8法检测HAPLN3基因表达降低对不同ccRCC细胞株增殖的影响。结果 生物信息学分析结果表明，ccRCC中HAPLN3 mRNA相对表达量高于正常肾脏组织（P <0.05），在不同WHO/ISUP分级的ccRCC中发现G4组HAPLN3 mRNA相对表达量高于G3组（P <0.05）。生存分析结果表明，HAPLN3高表达组总体生存率（OS）低于低表达组（P <0.05），且G4组OS也低于G3组（P <0.05）。单因素Cox回归分析结果表明，HAPLN3高表达［HR=1.548（95% CI：1.325，1.801）］、高龄［HR=1.029（95% CI：1.016，1.042）］、高pT分期［HR=1.923（95% CI：1.632，2.265）］、高pN分期［HR=3.425（95% CI：1.818，6.456）］、高pTNM分期［HR=1.867（95% CI：1.638，12.12）］、高Grade分级［HR=2.291（95% CI：1.870，2.806）］是ccRCC患者不良预后的影响因素（P <0.05）；多因素Cox回归分析结果表明，HAPLN3高表达［HR=1.430（95% CI：1.144，1.789）］、高龄［HR=1.006（95% CI：1.006，1.043）］、高pTNM分期［HR=1.661（95% CI：1.205，2.289）］、高Grade分级［HR=1.486（95% CI：1.070，2.065）］是ccRCC患者不良预后的独立影响因素（P <0.05）。HAPLN3 mRNA相对表达量与B淋巴细胞、CD4⁺ T淋巴细胞、CD8⁺ T淋巴细胞、中性粒细胞、巨噬细胞、树突状细胞浸润呈正相关（r_s =0.284、0.532、0.584、0.617、0.323和0.620，均P =0.000）。G4组CTLA4、LAG3、PDCD1、TIGIT mRNA相对表达量和TIDE评分高于G3组（P <0.05）。GESA富集分析结果表明，HAPLN3在ccRCC中与多条免疫及肿瘤通路相关。癌组织HAPLN3阳性表达率高于癌旁组织（P <0.05）。OSRC-2、ACHN细胞HAPLN3 mRNA和蛋白相对表达量高于HK-2细胞（P <0.05）。si-NC组、si-HAPLN3组不同时间点OS-RC-2、ACHN细胞的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点OS-RC-2、ACHN细胞的OD值有差异（F =481.158和292.321，均P =0.000）；②两组OS-RC-2、ACHN细胞的OD值有差异（F =27.471和255.219，均P =0.002）；③两组OS-RC-2、ACHN细胞的OD值变化趋势有差异（F =20.799和11.301，P =0.017和0.040）。结论 HAPLN3在ccRCC中表达上调，并与肿瘤细胞增殖相关，可能是ccRCC患者的独立预后标志物，并有作为免疫治疗靶点的潜在价值。

Expression of HAPLN3 gene in clear cell renal cell carcinoma and its prognostic and immunological implications

Objective To investigate the expression and prognostic value of hyaluronan and proteoglycan link protein 3 (HAPLN3) in clear cell renal cell carcinoma (ccRCC).Methods Multiple public databases were used to analyze the expression and prognostic value of HAPLN3 in ccRCC, to explore the correlations of HAPLN3 expression with tumor microenvironment, immune cell infiltration and Tumor Immune Dysfunction and Exclusion (TIDE) score, and to determine the potential pathogenic mechanisms of HAPLN3 in ccRCC via enrichment analysis. The expressions of HAPLN3 in ccRCC tissues and adjacent tissues were detected by immunohistochemistry, and the mRNA and protein expressions of HAPLN3 in different ccRCC cell lines (OS-RC-2 and ACHN) were detected by qRT-PCR and Western blotting. The cell lines were transfected with specific small interfering RNA to silence the expression of HAPLN3, and the effectiveness of HAPLN3 gene silencing in different ccRCC cell lines was detected by qRT-PCR. In addition, CCK-8 assay was performed to determine the effects of HAPLN3 gene silencing on the proliferation of different ccRCC cell lines.Results The bioinformatic analysis showed that the mRNA expression of HAPLN3 in ccRCC was significantly higher than that in normal kidney tissues (P < 0.05). In ccRCC with different WHO/ISUP grades, it was found that the expression of HAPLN3 in G4 group was significantly higher than that in G3 group (P < 0.05). Kaplan-Meier survival curve exhibited that high expression of HAPLN3 significantly shortened overall survival (OS) (P < 0.05), and that the OS of the G4 group was significantly shorter than that of the G3 group (P < 0.05). Univariate regression analysis showed that high HAPLN3 expression H^R = 1.548 (95% CI: 1.325, 1.801) ], advanced age H^R = 1.029 (95% CI: 1.016, 1.042) ], high pT stage H^R = 1.923 (95% CI: 1.632, 2.265) ], high pN stage H^R = 3.425 (95% CI: 1.818, 6.456) ], high pTNM stage H^R = 1.867 (95% CI: 1.638, 12.12) ], and high tumor grade H^R = 2.29 (95% CI: 1.870, 2.806) ] were risk factors for poor prognosis of ccRCC patients (P < 0.05). The multivariable regression analysis demonstrated that high HAPLN3 expression H^R = 1.430 (95% CI: 1.144, 1.789) ], advanced age H^R = 1.006 (95% CI: 1.006, 1.043) ], high pTNM stage H^R = 1.661 (95% CI: 1.205, 2.289) ], and high tumor grade H^R = 1.486 (95% CI: 1.070, 2.065) ] were independent poor prognostic factors (P < 0.05). The mRNA expression of HAPLN3 was positively correlated to the infiltration of B cells, CD4⁺ T cells, CD8⁺ T cells, neutrophils, macrophages, and dendritic cells (r_s =0.284, 0.532, 0.584, 0.617, 0.323 and 0.620, all P =0.000). The mRNA expressions of CTLA4, LAG3, PDCD1, and TIGIT in the G4 group were higher than those in the G3 group (P < 0.05). The TIDE score of the G4 group was also higher than that of the G3 group (P < 0.05). Gene Set Enrichment Analysis (GESA) showed that HAPLN3 was associated with multiple immune and tumor pathways in ccRCC. Immunohistochemistry showed that HAPLN3 was highly expressed in ccRCC tissues compared with adjacent tissues (P < 0.05). The mRNA and protein expressions of HAPLN3 in ccRCC cell lines OS-RC-2 and ACHN were higher than those in HK-2 cells (P < 0.05). As demonstrated by the CCK-8 assay, the OD values of OS-RC-2 and ACHN cells from si-NC group and si-HAPLN3 group among distinct time points were compared via the repeated measures ANOVA, which revealed that the OD values of OS-RC-2 and ACHN cells were different among the time points (F = 481.158 and 292.321, both P = 0.000) and between the two groups (F =27.471 and 255.219, both P = 0.002), and that the change trends of OD values of OS-RC-2 and ACHN cells were different between the two groups (F = 20.799 and 11.301, P = 0.017 and 0.040).Conclusions HAPLN3 is upregulated in ccRCC and correlated with tumor proliferation. It may be an independent prognostic marker for ccRCC patients and a potential target for immunotherapy.