LHPP在人胃腺癌组织中的表达及意义

Expression of LHPP and its clinic pathological significance in gastric adenocarcinoma

目的通过检测LHPP(phospholysine phosphohistidine inorganic pyrophosphate phosphatase)在人胃腺癌组织和胃腺癌细胞系,以及幽门螺杆菌(Helicobacter pylori,H.pylori)感染正常胃上皮细胞系后的表达水平,评估LHPP表达及LHPP与胃腺癌的关系。方法 (1)使用TCGA(The Cancer Genome Atlas)数据库和GEO(Gene Expression Omnibus)数据库中的胃腺癌基因表达数据,分析LHPP在胃腺癌组织和正常胃黏膜组织中的表达差异。(2)选取2017年3月至2018年5月行胃部分切除术或全胃切除术的41例胃腺癌患者,取胃癌组织及匹配的正常胃黏膜上皮组织,应用免疫组化检测LHPP在胃腺癌组织和正常胃黏膜组织中的表达差异。(3)取常规传代培养的第三代正常胃黏膜上皮细胞系GES-1和胃腺癌细胞系AGS,qRT-PCR检测两组LHPP基因表达差异。(4)应用H.pylori P12标准菌株以感染复数(multiplicity of infection,MOI)50感染GES-1细胞系6、12、18和24 h后,Western blot检测各个时间点LHPP和H.pylori毒力蛋白CagA表达量。结果 TCGA数据库中的LHPP表达数据表明LHPP在胃腺癌中表达下调(t=7.064,P<0.0001),来自GEO数据库中GSE65801表达数据的芯片测序结果得到相同的结论(P=0.001)。免疫组化证实LHPP在低分化胃腺癌中低表达,且表达与分化程度相关(χ~2=5.559,P=0.018),而与性别(χ~2=2.304,P=0.129)、年龄(χ~2=0.001,P=0.986)、浸润深度(χ~2=1.007,P=0.316)、淋巴结转移(χ~2=0.693,P=0.405)及肿瘤分期(χ~2=0.003,P=0.953)不相关。LHPP在胃腺癌细胞系AGS中表达低于正常胃上皮细胞系GES-1(t=10.38,P=0.005)。H.pylori感染GES-1细胞后LHPP表达随着感染时间延长总体有升高趋势。结论 LHPP在胃腺癌中低表达并与腺癌分化程度相关,有望成为胃腺癌诊断治疗的新靶点。

Objective To evaluate the correlation between LHPP(phospholysine phosphohistidine inorganic pyrophosphate phosphatase) and gastric tumorigenesis. Methods(1) Human Cancer Genome Atlas(TCGA) and Gene Expression Omnibus(GEO) were used to analyze the expression of LHPP in gastric adenocarcinoma and paired normal tissue.(2) Gastric adenocarcinoma specimens were obtained from 41 patients who had undergone curative total gastrectomy or subtotal gastrectomy from March 2017 to May 2018;the available cancer and paired normal tissue specimens were collected, and immunohistochemistry was used to identify different expressions of LHPP protein.(3) Normal gastric epithelial cell line GES-1 and gastric adenocarcinoma cell line AGS were cultured, and total mRNA was extracted from the passage 3 cells with Trizol method. The LHPP gene expression in different cell lines were detected using real-time quantity PCR.(4) After GES-1 cells were infected with H. pylori P12 with multiplicity of infection at 50 for 6 hours, 12 hours, 18 hours and 24 hours, Western blot was used to detect the expression levels of LHPP protein and CagA, a characteristic toxic protein of H. pylori. Results The expression data from TCGA database showed LHPP was downregulated in gastric cancer tissue(t=7.064, P<0.0001). Further, the same results were obtained from the sequencing results of the GSE65801 expression data in the GEO database(P=0.001). According to immunohistochemistry, low expression of LHPP shows significant correlations with poor differentiation(χ~2=5.559, P=0.018), but not with the gender(χ~2=2.304, P=0.129), age(χ~2=0.001, P=0.986), depth of invasion(χ~2=1.007, P=0.316), lymph node metastasis(χ~2=0.693, P=0.405), and the stage(χ~2=0.003, P=0.953). The expression of LHPP in gastric cancer cell line AGS was lower than in normal gastric epithelial cell line GES-1, and increased in H. pylori infected GES-1 cells. Conclusion The expression of LHPP is decreased in gastric adenocarcinoma and is associated with the degree of adenocarcinoma differentiation, which is expected to become a new target for the diagnosis and treatment of gastric cancer.