第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因在前列腺癌组织中的表达及荷载该基因的溶瘤腺病毒的构建

Expression of phosphatase and tensin homologue deleted on chromosome ten gene in prostate cancer tissues and construction of oncolytic adenovirus loaded with phosphatase and tensin homologue deleted on chromosome ten gene

目的构建荷载第10号染色体上缺失与张力蛋白同源的磷酸酯酶(PTEN)基因的溶瘤腺病毒验证其在前列腺癌中表达水平及靶向抗肿瘤效果。方法挖掘公开数据库和利用Galaxy在线工具处理本中心去势抵抗性前列腺癌(CRPC)标本基因测序数据, 分析PTEN基因在前列腺癌组织中的表达差异。收集2019年3月至2022年8月徐州医科大学附属医院79例前列腺癌患者的病理样本和临床信息, 免疫组织化学法检测前列腺癌组织及癌旁组织中PTEN蛋白的表达。同源重组法构建溶瘤腺病毒ZD55-PTEN, 细胞计数试剂盒(CCK-8)法、Hoechst-33258和划痕实验检测其靶向抗肿瘤效果。分别采用卡方检验和t检验对定性资料和定量资料进行统计分析, 不满足正态分布的数据采用Mann-WhitneyU检验;Spearman分析PTEN表达水平与临床病理特征的关系。结果前列腺癌组织中PTEN表达水平明显低于癌旁组织, 差异有统计学意义(501例比52例, Z=-19.25, P<0.05);PTEN的mRNA表达水平与淋巴结转移(n=425, r_(s)=-0.98, P<0.05)和Gleason评分(n=497, r_(s)=-1.25, P<0.05)呈负相关。CRPC阶段PTEN缺失明显高于激素敏感性前列腺癌(HSPC)阶段(79例比125例, Z=-0.89, P<0.05)。前列腺癌组织的PTEN蛋白缺失率高于癌旁组织(45.6%比16.5%, t=10.98, P<0.01), 术前血清PSA水平(n=79, r_(s)=-0.47, P<0.01)和术后Gleason评分(n=79, r_(s)=-0.31, P<0.05)与PTEN的表达呈负相关。PTEN缺失的前列腺癌患者, 确诊时PSA水平(36例比43例, χ^(2)=12.22, P<0.05)和根治术后Gleason评分(36例比43例, χ^(2)=6.92, P<0.05)显著高于PTEN蛋白完整患者。成功构建溶瘤腺病毒ZD55-PTEN, ZD55-PTEN组细胞存活率低于对照组[ZD55-EGFP组(49.67±4.19)%、ZD55-PTEN组(29.33±3.84)%, t=8.68, P<0.05];细胞凋亡率高于对照组[PBS组(12.86±5.23)%、ZD55-EGFP组(48.18±4.22)%、ZD55-PTEN组(68.93±5.88)%, t=5.90, P<0.05];划痕愈合率低于对照组[PBS组(79.13±3.98)%、ZD55-EGFP组(61.02±4.73)%、ZD55-PTEN组(47.92±5.97)%, t=6.34, P<0.05]。结论 PTEN基因低表达于前列腺癌组织中, 且具有抑制DU145细胞的增殖、迁移并诱导凋亡的功能。

Objective To construct oncolytic adenovirus carrying gene of phosphatase and tensin homolog deleted on chromosome ten(PTEN)and verified its expression level in prostate cancer and targe-ted anti-tumor effects.Methods Mined public databases and used the Galaxy online tool to process gene sequencing data of castration-resistant prostate cancer(CRPC)specimens from our center,and analyzed the differences in PTEN gene expression in prostate cancer tissues.Collected pathological samples and clin-ical information of 79 prostate cancer patients from March 2019 to August 2022 at the Affiliated Hospital ofXuzhou Medical University,and detected the expression of PTEN protein in prostate cancer tissues and adjacent tissues by using immunohistochemistry.Constructed the oncolytic adenovirus ZD55-PTEN by using homologous recombination,and used the cell counting kit-8(CCK-8)method,Hoechst-33258,and scratch assay to detect its targeted anti-tumor effects.Chi-square test and t-test were used for statistical analysis of qualitative and quantitative data,respectively:Mann-Whitney U test was used for data that does not meet normal distribution.Spearman's analysis was used to assess the relationship between PTEN expression levels and clinicopathological features.Results The expression level of PTEN in prostate cancer tissues was significantly lower than that in adjacent non-cancerous tissues,with a statistically significant difference(501 cases vs.52 cases,Z=-19.25,P<0.05).The mRNA expression level of PTEN was negatively correlated with lymph node metastasis(n=425,r,=-0.98,P<0.05)and Gleason score(n=497,r,=-1.25,P<0.05).The loss of PTEN in the CRPC stage was significantly higher than in the hormone-sensitive prostate cancer(HSPC)stage(79 cases vs.125 cases,Z=-0.89,P<0.05).The rate of PTEN protein loss in prostate cancer tissues was higher than that in adjacent non-cancerous tissues(45.6%vs.16.5%,t=10.98,P<0.01),and preoperative serum PSA levels(n=79,r,=-0.47,P<0.01)and postoperative Gleason score(n=79,r,=-0.31,P<0.05)were negatively correlated with PTEN expression.Prostate cancer patients with PTEN loss had significantly higher PSA levels at the time of diagnosis(36 vs.43 cases,t=12.22,P<0.05)and postoperative Cleason scores(36 cases vs.43 cases,t=6.92,P<0.05)compared to patients with intact PTEN protein.The oncolytic adenovirus ZD55-PTEN was successfully constructed,and the cell viability in the ZD55-PTEN group was lower than that in the control group[ZD55-EGFP group(49.67±4.19)%,ZD55-PTEN group(29.33±3.84)%,t=8.68,P<0.05];the apoptosis rate was higher than that in the control group[PBS group:(12.86±5.23)%,ZD55-EGFP group(48.18±4.22)%,ZD55-PTEN group(68.93±5.88)%,t=5.90,P<0.05];and the scratch healing rate was lower than that in the control group[PBS group(79.13±3.98)%,ZD55-ECFP group(61.02±4.73)%,ZD55-PTEN group(47.92±5.97)%,t=6.34,P<0.05].Conclusion The PTEN gene is underexpressed in prostate cancer tissues and exhibits the function of inhibiting the proliferation and migration of DU145 cells and inducing apoptosis.