序列相似性家族107成员A抑制前列腺癌进展的实验研究

Family with sequence similarity 107 member A suppresses prostate cancer progression:an experi-mental study

目的 探讨序列相似性家族107成员A(FAM107A)调控缺氧诱导因子3A(H1F3A)对前列腺癌(PCa)迁移、侵袭、凋亡的影响.方法 通过生物信息学技术筛选目的基因并进行分析.体外培养人PCa细胞PC-3,采用反转录病毒稳定转染,采用蛋白质印迹法(Western blot)技术验证HIF3A及DNA甲基转移酶1(DNMT1)的表达量;流式细胞仪检测细胞周期及凋亡;焦磷酸测序检测启动子甲基化程度;细胞功能试验验证对PCa迁移和侵袭的影响.两组间差异表达采用独立样本t检验方法比较.结果 生信分析提示FAM107A基因在PCa组织中表达低于正常前列腺组织(P<0.01),Gleason评分(GS)越高,FAM107A表达量越低(P<0.01).划痕实验、Transwell侵袭实验提示过表达 FAM107A会抑制 PCa 细胞的迁移(C4-2:0.337±8.930 比 29.334±2.651,t=-7.260,P<0.05;PC-3:18.672±10.494 比 52.043±3.512,t=-6.024,P<0.05)、侵袭(DU145:117.703±12.845 比 242.303±21.401,t=10.273,P<0.05;PC-3:51.674±9.502 比 139.303±13.394,t=16.025,P<0.05),促进凋亡(17.180±0.700 比 0.527±0.121,t=40.600,P<0.05).细胞周期实验表明oe-FAM107A-ENZ组细胞G1期比例高于对照组(80.410±0.786比35.583±0.714,t=73.130,P<0.05),S 期比例低于对照组(11.233±0.374 比 38.747±1.809,t=25.790,P<0.05),细胞分裂被阻滞在S期.蛋白免疫印迹实验证明过表达FAM107A组的DNMT1表达量低于对照组(0.277±0.032 比 0.900±0.026,t=25.93,P<0.05),HIF3A 表达量高于对照组(0.657±0.040 比0.523±0.043,t=4.041,P<0.05).sh-FAM107A-5-aza 组的 HIF3A 的表达量明显恢复(0.550±0.020比0.427±0.067,t=3.073,P<0.05).蛋白免疫印迹实验证明过表达FAM107A时E-钙黏蛋白(E-cadherin)表达量高于对照组(0.773±0.095 比 0.307±0.046,t=7.683,P<0.05),N-钙黏蛋白(N-cadherin)表达量低于对照组(0.380±0.060 比 0.733±0.045,t=8.154,P<0.05),波形蛋白(Vimentin)表达量低于对照组(0.347±0.107 比 0.700±0.040,t=5.361,P<0.05).结论 FAM107A高表达可抑制HIF3A启动子甲基化促进其表达,进而抑制PCa的迁移、侵袭,是PCa进展的生物标志物.

Objective To investigate the effects of family with sequence similarity 107 member A(FAM107A)on the migration,invasion and apoptosis of prostate cancer(PCa)by regulating hypoxia in-ducible factor 3 subunit alpha(HIF3A).Methods The target genes were screened and analyzed using bioinformatics tools.Human prostate cancer cells PC-3 were cultured in vitro,then stably transfected with retrovirus and divided into separate groups.The expression levels of HIF3A and DNA methyltransferase 1(DNMT1)were confirmed through Western blotting.Flow cytometry was employed to detect cell cycle and apoptosis.In addition,pyrosequencing was employed to evaluate promoter methylation levels.Cell func-tional assays were performed to investigate the effects on migration and invasion of PCa cells.Differential expression between two groups was compared using independent samples t-test.Results Bioinformatics a-nalysis revealed that the expression of FAM107A gene was lower in PCa tissues than in normal prostate tis-sues(P<0.01),and its expression level decreased with higher Gleason score(GS)(P<0.01).Scratch test and Transwell invasion assay demonstrated that overexpression of FAM107A inhibited the migration(C4-2:0.337±8.930 vs.29.334±2.651,t=-7.260,P<0.05;PC-3:18.672±10.494 vs.52.043±3.512,t=-6.024,P<0.05)and invasion(DU145:117.703±12.845 vs.242.303±21.401,t=10.273,P<0.05;PC-3:51.674±9.502 vs.139.303±13.394,t=16.025,P<0.05)of PCa cells,while promoting apoptosis(17.180±0.700 vs.0.527±0.121,t=40.600,P<0.05).Cell cycle experiment results indicate that the proportion of cells in the G1 phase in the oe-FAM107A-ENZ group was higher than in the control group(80.410±0.786 vs.35.583±0.714,t=73.130,P<0.05),while the proportion of cells in the S phase was lower than in the control group(11.233±0.374 vs.38.747±1.809,t=25.790,P<0.05),and cell division was arrested at the S phase.Western blotting analysis demonstrated that the expression level of DNMT1 in the overexpressed FAM107A group was lower than in the control group(0.277±0.032 vs.0.900±0.026,t=25.93,P<0.05),and the expression level of HIF3A was higher than in the control group(0.657±0.040 vs.0.523±0.043,t=4.041,P<0.05).The expression level of HIF3A was significantly restored in the sh-FAM107A-5-aza group(0.550±0.020 vs.0.42±0.067,t=3.073,P<0.05).Western blotting analysis demonstrated that the expres-sion level of E-cadherin was higher than in the control group when FAM107A was overexpressed(0.773±0.095 vs.0.307±0.046,t=7.683,P<0.05),while the expression level of N-cadherin was lower than in the control group(0.380±0.060 vs.0.733±0.045,t=8.154,P<0.05),and the expression level of Vimentin was also lower than in the control group(0.347±0.107 vs.0.700±0.040,t=5.361,P<0.05).Conclusion High expression of FAM107A can inhibit the methylation of the HIF3A promoter,promoting its expression,thereby inhibiting the migration and invasion of PCa.This may serve as a poten-tial biomarker for the progression of PCa.