目的 探究丝氨酸蛋白酶抑制剂1(serine proteinase inhibitor family E member 1,SERPINE1)在结肠癌(colon adenocarcinoma,COAD)中的表达、意义及对COAD细胞增殖与凋亡的影响,了解其在COAD进展中发挥的生物学功能.方法 通过 Gene Expression Profilling Interactive Analysis 2.0(GEPIA 2.0)数据库分析 SERPINE1 在 COAD中的表达与分期及预后关系.收集作者医院5例COAD患者癌组织与对应的正常结肠组织标本,应用Western blot实验验证SERPINE1在COAD中的表达;接着构建SERPINE1过表达与干扰表达的慢病毒载体,进行细胞转染,通过实时荧光定量逆转录聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)和 West-ern blot对实验细胞筛选并验证过表达和干扰表达的效率;进而通过细胞计数试剂盒(cell counting kit 8,CCK-8)和Ho-echst 细胞核染色检测细胞增殖与凋亡;最后通过Western blot实验了解其对增殖标志物Ki-67(marker of proliferation Ki-67,MKI67)、Bcl-2 关联 X 蛋白(Bcl-2 associated X protein,Bax)与 B 细胞淋巴瘤 2 基因(B cell lymphoma 2,Bcl-2)比值的影响.结果 通过GEPIA 2.0数据库查找并分析COAD中SERPINE1表达与临床分期和预后关系发现,SER-PINE1在COAD中表达水平高于正常结肠组织,SERPINE1的表达水平与临床分期正相关,而与预后生存总生存期负相关(P均＜0.05).CCK-8实验结果表明,oe-RKO组和oe-LoVo组COAD细胞在48h时间点吸光度(optical density,OD)值大于oe-RKO-NC组和oe-LoVo-NC组,sh-RKO-822组和sh-LoVo-822组COAD细胞在48 h时间点OD值小于sh-RKO-NC和sh-LoVo-NC组(P均＜0.05).Hoechst凋亡细胞核染色结果表明,oe-RKO组和oe-LoVo组COAD细胞凋亡数量低于oe-RKO-NC组和oe-LoVo-NC组,sh-RKO-822组和sh-LoVo-822组COAD细胞凋亡数量明显高于sh-RKO-NC 组和 sh-LoVo-NC 组(P均＜0.05).Western blot 结果显示,oe-RKO 组和 oe-LoVo 组 COAD 细胞 MKI67 蛋白表达明显高于 oe-RKO-NC 组和 oe-LoVo-NC组,Bax/Bcl-2 明显低于 oe-RKO-NC 组和 oe-LoVo-NC 组;sh-RKO-822组和sh-LoVo-822组COAD细胞MKI67蛋白表达明显低于sh-RKO-NC组和sh-LoVo-NC组,Bax/Bcl-2明显高于sh-RKO-NC组和sh-LoVo-NC组(P均＜0.05).结论 SERPINE1在COAD中高表达、与临床分期正相关,而与患者预后负相关;SERPINE1可能通过影响COAD细胞MKI67表达和Bax/Bcl-2促进COAD RKO和LoVo细胞的增殖,抑制其凋亡.
Effects of SERPINE1 on Proliferation and Apoptosis of Colon Adenocarcinoma Cells RKO and LoVo
Objective To explore the expression and significance of serine proteinase inhibitor family E member 1(SERPINE1)in colon adenocarcinoma(COAD)and its effects on proliferation and apoptosis of COAD cells,and to ex-plore its biological function in the progression of COAD.Methods The relationship between SERPINE1 expression and clinical stage and prognosis of COAD was analyzed by Gene Expression Profilling Interactive Analysis 2.0(GEPIA 2.0)database.Cancer tissues and corresponding normal colonic tissues were collected from 5 patients with COAD,the expres-sion of SERPINE1 in COAD was verified by Western blot;then SERPINE1 overexpression and interference lentiviral vec-tors were constructed for cell transfection,real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to screen the experi-mental cells and verify the overexpression and interference expression efficiency;cell proliferation and apoptosis were detected by cell counting kit 8(CCK-8)and Hoechst nu-clear staining;finally,the effects of the inhibitor on the marker of proliferation Ki-67(MKI67),Bcl-2 associated X protein(Bax)and B cell lymphoma 2(Bcl-2)gene ratio were detected by Western blot.Results The GEPIA2 database was used to search and analyze the relationship between the expression of SERPINE1 in COAD and clinical stage as well as prognosis,it was found that the expression level of SERPINE1 in COAD was higher than that in normal colonic tis-sues,the expression level of SERPINE1 was positively correlated with clinical stage,but negatively correlated with prog-nosis and overall survival(all P＜0.05).The results of CCK-8 assay showed that the optical density(OD)value of COAD cells in oe-RKO group and oe-LoVo group at 48h was higher than that of oe-RKO-NC group and oe-LoVo-NC group,the OD value of COAD cells in sh-RKO-822 group and sh-LoVo-822 group at 48h was lower than that of sh-RKO-NC and sh-LoVo-NC groups(all P＜0.05).Hoechst staining showed that the number of apoptotic COAD cells in oe-RKO group and oe-LoVo group was lower than that in oe-RKO-NC group and oe-LoVo-NC group,the number of apoptotic COAD cells in sh-RKO-822 group and sh-LoVo-822 group was significantly higher than that in sh-RKO-NC group and sh-LoVo-NC group(all P＜0.05).Western blot results showed that the expression of COAD cells in MKI67 protein in oe-RKO group and oe-LoVo group was significantly higher than that in oe-RKO-NC group and oe-LoVo-NC group,and the Bax/Bcl-2 was significantly lower than that in oe-RKO-NC group and oe-LoVo-NC group;the expression of COAD cells in MKI67 protein in sh-RKO-822 group and sh-LoVo-822 group was significantly lower than that in sh-RKO-NC group and sh-LoVo-NC group,and the Bax/Bcl-2 was significantly higher than that in sh-RKO-NC group and sh-LoVo-NC group(all P＜0.05).Conclusion SERPINE1 is highly expressed in COAD and is positively correlated with clinical stage,but nega-tively correlated with prognosis;SERPINE1 may promote the proliferation and inhibit the apoptosis of COAD RKO and LoVo cells by affecting the expression of COAD cells in MKI67 and Bax/Bcl-2.
Colon adenocarcinomaSerine proteinase inhibitor family E member 1ProliferationApoptosisBio-logical function
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