Expression and Clinical Significance of PLCβ4 Gene in Hepatocellular Carcinoma Analyzed Based on TCGA Database and Experimental Validation
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摘要:目的
基于癌症基因组图谱（TCGA）数据库分析肝细胞癌中PL-Cβ4基因mRNA的表达及其临床意义。
方法基于TCGA数据库中的424例临床样本资料（包括肝细胞癌组织374例，非肿瘤肝组织50例），采用Kaplan-Meier法、Cox回归分析、免疫浸润分析，评估PLCβ4基因与HCC患者临床特征及生存预后的关系。应用PLCβ4基因与24种免疫细胞的相关性分析，观察PLCβ4基因与免疫细胞浸润的关系及与肝癌高频突变基因TP53 mRNA表达水平的相关性。另外收集新疆医科大学第一附属医院病理科HCC患者的高、中、低分化肿瘤组织以及正常肝组织石蜡切片各20例，HE染色法进行组织病理学观察，免疫组织化学法对各临床样本中PLCβ4、Ki-67蛋白表达水平进行验证。
结果PLCβ4基因在HCC中的表达水平显著高于正常组织（P<0.01），且PLCβ4高表达患者的总生存期均明显长于低表达者（P<0.05），提示PLCβ4显著影响HCC患者预后。相关性分析结果显示PLCβ4基因的表达水平与免疫细胞浸润高度相关；且PLCβ4与TP53基因的表达水平高度相关。HCC组织样本中的PLCβ4基因表达显著高于正常组织（P<0.001），且与分化程度呈负相关关系。
结论PLCβ4可作为肝细胞癌的一个独立预后因素，有望成为HCC治疗的新型分子靶点。
Abstract:ObjectiveTo analyze the PLCβ4 gene mRNA expression and its clinical significance in hepatocellular carcinoma (HCC) based on TCGA database.
MethodsBased on the data on 424 clinical samples (including 374 cases of HCC tissues and 50 cases of nontumor liver tissues) in the TCGA database, Kaplan–Meier method, Cox regression analysis, and immune infiltration analysis were performed to evaluate the relationship between PLCβ4 gene and the clinical characteristics and survival prognosis of HCC patients. Correlation analysis between PLCβ4 gene and 24 types of immune cells was applied to investigate the relationship between PLCβ4 gene and immune cell infiltration and mRNA expression level of TP53 gene, a high-frequency mutation gene in HCC. In addition, paraffin sections of highly, moderately, and poorly differentiated tumor tissues and normal liver tissues from HCC patients were collected. The histopathological observation was carried out via HE staining method, and the expression levels of PLCβ4 and Ki-67 proteins in each clinical sample were verified through the immunohistochemical method.
ResultsThe expression level of PLCβ4 gene in HCC was significantly higher than that in normal tissues (P<0.01), and all patients in the PLCβ4 high-expression group had a significantly longer overall survival than those in the low-expression group (P<0.05), which suggested that PLCβ4 substantially affected the prognosis of HCC patients. Correlation analysis showed that the expression level of PLCβ4 gene was highly correlated with immune cell infiltration and the expression level of TP53 gene. As verified by clinical sample experiments, HE staining experiments and immunohistochemical results revealed that PLCβ4 gene expression in HCC tissue samples was significantly higher than that in normal tissues (P<0.001), and it was negatively correlated with the degree of differentiation.
ConclusionPLCβ4 may serve as an independent prognostic factor in HCC and is expected to be a novel molecular target for HCC treatment.
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Competing interests: The authors declare that they have no competing interests.利益冲突声明：所有作者均声明不存在利益冲突。作者贡献：文丽梅：实验实施、数据分析、论文撰写郭娅丽：实验实施、数据分析侯强、郑董璇、代武、高翔：数据收集及整理杨建华、胡君萍：指导研究方案、论文修改
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图 1 基于TCGA数据库分析PLCβ4基因在HCC组织中的表达
Figure 1 Analysis of PLCβ4 gene expression in HCC tissues based on TCGA database
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图 2 基于TCGA数据库分析PLCβ4基因的表达对HCC患者预后的影响
Figure 2 Effect of PLCβ4 gene expression on the prognosis of HCC patients analyzed based on TCGA database
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图 3 基于TCGA数据库分析PLCβ4基因对HCC诊断能力的ROC曲线
Figure 3 ROC curve of diagnostic ability of PLCβ4 gene for HCC analyzed based on TCGA database
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图 4 基于TCGA数据库分析PLCβ4的表达与HCC患者临床病理参数的相关性分析
Figure 4 Correlation of PLCβ4 expression with clinicopathologic parameters of HCC patients based on TCGA database analysis
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图 5 基于TCGA数据库分析PLCβ4与免疫微环境的关系
Figure 5 Analysis of relationship between PLCβ4 and immune microenvironment based on TCGA database
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图 6 基于TCGA数据库分析PLCβ4与TP53之间的相关性分析
Figure 6 Correlation analysis between PLCβ4 and TP53 based on TCGA database
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图 7 临床样本组织实验验证结果 (免疫组织化学法，×200)
Figure 7 Experimental validation of clinical tissue samples (IHC, ×200)
表 1 HCC患者临床特征与PLCβ4基因表达的单因素和多因素Cox回归分析
Table 1 Univariate and multivariate Cox regression analyses of clinical characteristics and PLCβ4 gene expression in HCC patients
Clinical characteristics n Univariate analysis Multivariate analysis HR(95% CI) P HR(95% CI) P T stage T1-T2 277 Reference T4-T3 93 2.598 (1.826-3.697) <0.001 1.606 (0.212-12.182) 0.647 N stage N0 254 Reference N1 4 2.029 (0.497-8.281) 0.324 M stage M0 268 Reference M1 4 4.077 (1.281-12.973) 0.017 1.447 (0.339-6.166) 0.618 Pathologic stage Ⅰ-Ⅱ 259 Reference Ⅲ-Ⅳ 90 2.504 (1.727-3.631) <0.001 1.291 (0.171-9.743) 0.804 Tumor status Tumor free 202 Reference With tumor 152 2.317 (1.590-3.376) <0.001 2.706 (1.616-4.531) <0.001 Gender Female 121 Reference Male 252 0.793 (0.557-1.130) 0.200 Race Asian 159 Reference Black or African American 17 1.585 (0.675-3.725) 0.290 White 185 1.323 (0.909-1.928) 0.144 Age (years) ≤60 177 Reference >60 196 1.205 (0.850-1.708) 0.295 Weight(kg) ≤70 184 Reference >70 161 0.941 (0.657-1.346) 0.738 Height(cm) <170 201 Reference ≥170 139 1.232 (0.849-1.788) 0.272 BMI(kg/m2) ≤25 177 Reference >25 159 0.798 (0.550-1.158) 0.235 Residual tumor R0 326 Reference R1 17 1.448 (0.705-2.972) 0.313 1.218 (0.439-3.378) 0.704 R2 1 11.749 (1.595-86.516) 0.016 Histologic grade G1-G2 233 Reference G3-G4 135 1.091 (0.761-1.564) 0.636 AFP (ng/ml) ≤400 215 Reference >400 64 1.075 (0.658-1.759) 0.772 Prothrombin time(sec) ≤4 207 Reference >4 89 1.335 (0.881-2.023) 0.174 Adjacent hepatic tissue inflammation None 118 Reference Mild 101 1.204 (0.723-2.007) 0.476 Severe 17 1.144 (0.447-2.930) 0.779 Albumin(g/dl) <35 69 Reference ≥35 230 0.897 (0.549-1.464) 0.662 Child-Pugh grade A 218 Reference B 21 1.595 (0.757-3.361) 0.219 C 1 2.138 (0.294-15.544) 0.453 Fibrosis Ishak score 0-2 106 Reference 3-6 108 0.740 (0.445-1.232) 0.247 Expression of PLCβ4 Low 187 Reference High 186 0.677 (0.478-0.959) 0.028 0.551 (0.328-0.925) 0.024 Note: Data related to patients with unclear clinical characteristics and missing survival times have been excluded from the table above. 
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