lncRNA GAS5通过miR-26a靶向调控JAK1/STAT3信号通路对子宫内膜样癌细胞凋亡及侵袭的影响

The effect of lncRNA GAS5 on apoptosis and invasion of endometrioid carcinoma cells through miR-26a targeting regulation of JAK1/STAT3 signaling pathway

目的 探讨lncRNA GAS5通过miR-26a靶向调控JAK1/STAT3信号通路对子宫内膜样癌(EC)细胞凋亡及侵袭的影响。方法 选取2018年4月—2021年5月经我院首次确诊并进行广泛性子宫切除及腹膜后淋巴结清扫术治疗的新鲜无菌EC患者癌灶及相对癌旁组织(距离肿瘤5 cm外)共45对。使用定量聚合酶链反应(qRT-PCR)检测EC患者癌灶及相对癌旁组织、人子宫内膜癌细胞系ISK(高分化)、HEC-1-B(中分化)、人EC细胞系KLE(低分化)、子宫内膜腺上皮细胞(正常细胞)中lncRNA GAS5的表达。依据转染不同分为NC组(阴性对照质粒)、lncRNA GAS5组(转染lncRNA GAS5过表达质粒)、anti-miR-26a组(转染miR-26a干扰质粒)、lncRNA GAS5+miR-26a组(转染lncRNA GAS5过表达质粒+miR-26a过表达质粒)。使用EdU实验、AnneinV-FITC/PI双染流式细胞术检测各组细胞的凋亡、细胞活力和增殖能力;使用荧光素酶报告基因分析荧光素酶活性;通过qRT-PCR和Western blot分析各组细胞中lncRNA GAS5、miR-26a、蛋白酪氨酸激酶(JAK1)、信号转导和转录活化因子(STAT3)、程序性死亡配体-1(programmed death ligand-1,PD-L1)、基质金属蛋白酶(MMP9)、E-钙黏蛋白(E-cadherin)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达情况;利用动物试验进行皮下移植,测定肿瘤重量与体积。结果 lncRNAGAS5在EC组织中显著低表达(P=0.001);与子宫内膜腺上皮细胞相比,EC细胞系中的表达显著降低(P<0.05);双荧光素酶报告基因分析表明,miR-26a是lncRNA GAS5潜在的靶向miRNAs。与NC组相比,lncRNA GAS5组和anti-miR-26a组EdU阳性细胞率显著降低,凋亡细胞数量显著增加,小鼠的肿瘤体积和质量显著减少,JAK1、STAT3、PD-L1、MMP9表达显著上调,E-cadherin、Caspase-3表达显著下调(P<0.05)。与lncRNA GAS5组相比,lncRNA GAS5+miR-26a的EdU阳性细胞率显著升高,凋亡细胞显著减少,小鼠肿瘤体积和质量显著增加,JAK1、STAT3、PD-L1、MMP9表达显著下调,E-cadherin、Caspase-3表达显著上调(P<0.05)。结论 lncRNAGAS5在EC组织中低表达,低表达的lncRNAGAS5可能通过上调miR-26a,抑制JAK1/STAT3信号通路的激活,影响下游E-cadherin、PD-L1、Caspase-3、MMP9的表达,从而调节细胞增殖和凋亡。

Objective To study the effect of lncRNA GAS5 on apoptosis and invasion of endometrioid carcinoma cells regulated by miR-26a targeting JAK1/STAT3 signaling pathway.Methods From April 2018 to May 2021,45 pairs of fresh aseptic EC patients with cancer foci and relative paracancerous tissues(5 cm away from the tumor) were selected and treated with extensive hysterectomy and retroperitoneal lymph node dissection for the first time in our hospital.qRT-PCR was used to detect the expression of lncRNA GAS5 in cancer foci and paracancerous tissues of EC patients,human endometrial carcinoma cell lines ISK(well differentiated),HEC-1-B(moderately differentiated),human endometrioid carcinoma cell line KLE(poorly differentiated) and endometrial glandular epithelial cells(normal cells).According to the difference of transfection,the cells were divided into NC group(negative control plasmid),lncRNA GAS5 group(transfection of lncRNA GAS5overexpression plasmid),anti-miR-26a group(transfection of miR-26a interference plasmid) and lncRNA GAS5+miR-26a group(transfection of lncRNA GAS5 overexpression plasmid+miR-26a overexpression plasmid).Apoptosis,cell viability and proliferation were detected by EdU test and AnneinV-FITC/PI double staining flow cytometry.Luciferase activity was analyzed by luciferase report.The expression of lncRNA GAS5,miR-26a,JAK1,STAT3,PD-L1,MMP9,E-cadherin and Caspase-3 in cells of each group was analyzed by qRT-PCR and Western blotting;subcutaneous transplantation was carried out in animal experiment to determine the weight and volume of tumor.Results The expression of lncRNA GAS5 in EC tissue was significantly lower than that in endometrial glandular epithelial cells(P=0.001),and the expression in EC cell line was significantly lower than that in endometrial glandular epithelial cells(P<0.05).Double luciferase report analysis showed that miR-26a was a potential targeting microRNAs for lncRNA GAS5.Compared with NC group,the rate of EdU positive cells in lncRNA GAS5 group and anti-miR-26a group decreased significantly(P<0.05).The number of apoptotic cells increased significantly(P<0.05).The tumor volume and weight of mice decreased significantly(P<0.05).The expression of JAK1,STAT3,PD-L1 and MMP9 increased significantly,and the expression of E-cadherin and Caspase-3 decreased significantly.Compared with lncRNA GAS5 group,the rate of EdU positive cells in lncRNA GAS5+miR-26a down regulated significantly(P<0.05).The number of apoptotic cells decreased significantly(P<0.05).The volume and weight of tumor increased significantly(P<0.05).The expression of JAK1,STAT3,PD-L1 and MMP9 down regulated significantly,while the expression of E-cadherin and Caspase-3 up regulated significantly(P<0.05).Conclusion The low expression of lncRNA GAS5 in EC tissues and the low expression of lncRNA GAS5 may regulate cell proliferation and apoptosis by up-regulating miR-26a,inhibiting the activation of JAK1/STAT3 signal pathway and affecting the expression of downstream E-cadherin,PD-L1,Caspase-3 and MMP9.