circ_0003926对食管鳞状细胞癌的调控作用及临床意义

Regulation and clinical significance of circ_0003926 for esophageal squamous cell carcinoma

目的探讨circ_0003926在食管鳞状细胞癌(ESCC)中的作用机制及临床意义。方法收集2022-05-01-2023-01-30在华北理工大学附属医院就诊的60例ESCC患者及同时期体检的年龄相仿、性别相同的60名健康者血浆标本。荧光原位杂交实验检测circ_0003926和miR-139-3p在ESCC组织芯片表达水平,并分析其临床相关性。采用Sanger测序和背靠背实验以及核糖核酸外切酶(Rnase R)实验验证circ_0003926环状结构和稳定性。采用实时荧光定量聚合酶链式反应(qRT-PCR)检测circ_0003926和miR-139-3p在ESCC细胞系和血浆中表达水平。应用细胞计数试剂盒8(CCK8)法、克隆形成实验、Transwell侵袭迁移实验和划痕实验及流式细胞术分别检测circ_0003926和miR-139-3p对ESCC细胞增殖、克隆、侵袭、迁移和凋亡的影响,双荧光素酶报告基因实验和qRT-PCR验证circ_0003926与下游miRNAs结合。裸鼠皮下移植瘤实验检测circ_0003926对移植瘤生长的影响。结果Sanger测序、背靠背和Rnase R实验验证了circ_0003926的环状结构。荧光原位杂交结果显示,circ_0003926在癌及癌旁组织中表达水平分别为18.00±5.10和11.80±4.22(t=8.792,P<0.001);miR-139-3p的表达水平分别为18.26±2.81和26.23±2.97(t=17.713,P<0.001)。circ_0003926高表达与临床分期(χ^(2)=5.312,P=0.021)有关联,与生存率也有关联(χ^(2)=9.501,P=0.002);miR-139-3p表达水平与淋巴结转移(χ^(2)=4.114,P=0.043)和病理分级(χ^(2)=4.267,P=0.039)有关联。qRT-PCR结果显示,在ESCC患者血浆和健康者血浆中,circ_0003926的表达水平分别为3.54±2.15和1.03±0.05(t=6.993,P<0.001);miR-139-3p的表达水平分别为0.36±0.17和1.03±0.03(t=27.001,P<0.001)。与正常细胞系HEEC相比,circ_0003926在ESCC细胞系中的表达水平升高(F=281.577,P<0.001),而miR-139-3p表达水平下降(F=87.034,P<0.001)。敲低circ_0003926后,KYSE-150和KYSE-410细胞的增殖、迁移和侵袭能力下降,凋亡水平升高(均P<0.05)。抑制miR-139-3p的表达可以逆转抑制circ_0003926表达后对细胞增殖、凋亡、迁移和侵袭能力的作用(均P<0.05)。裸鼠皮下移植瘤实验结果显示,si-circ_0003926组的肿瘤体积和体质量小于si-NC组,10 d~28 d差异均有统计学意义,均P<0.001,及si-circ_0003926+antagomiR-NC组的肿瘤体积和体质量小于si-circ_0003926+antagomiR-139-3p组,10 d~28 d差异均有统计学意义,P<0.001。结论circ_0003926在ESCC组织、血浆和细胞中均高表达,通过miR-139-3p调控ESCC的进展,有可能成为ESCC诊断、治疗和预后的一种新的生物标志物。

Objective To investigate the role and mechanism of circ_0003926 in esophageal squamous cell carcinoma(ESCC).Methods Plasma samples from 60 ESCC patients and 60 age-and gender-matiched healthy controls were collected at Affiliated Hospital of North China University of Science and Technology from May 1,2022,to January 30,2023.The expression levels of circ_0003926 and miR-139-3p were detected by fluorescence in situ hybridization in a ESCC tissue microarray and their clinical relevance was analyzed.The loop structure and stability of circ_0003926 were verified by Sanger sequencing,back-to-back primer validation and ribonuclease R(Rnase R)tolerance assay.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detected the expression levels of circ-0003926 and miR-139-3p in ESCC cell lines and plasma.Counting Kit-8(CCK-8),colony formation,transwell invasion and migration assays,wound healing assays,and flow cytometry assay were used to measure the effects of circ_0003926 and miR-139-3p on cell proliferation,migration,invasion and apoptosis,respectively.The interaction between circ_0003926 and miR-139-3p was further identified by dual luciferase reporter gene assay and qRT-PCR.Nude mouse subcutaneous xenograft tumor model was used to investigate the impact of circ_0003926 on tumor growth.Results The circular structure of circ_0003926 was identified by Sanger sequencing,back-to-back primer and Rnase R assay.The results of fluorescence in situ hybridization showed that the mean fluorescence intensity of circ_0003926 expressed in ESCC tissues and adjacent tissues were 18.00±5.10 and11.80±4.22(t=8,792,P<0.001),respectively,and the mean fluorescence intensity of miR-139-3p were 18.26±2.81and 26.23±2.97(t=17.713,P<0.001),respectively.The expression level of circ_0003926 was positively correlated with the clinical stage(χ^(2)=5.312,P=0.021),but negatively related to overall survival time,the differences were statistically significant,P=0.002;while the expression level of miR-139-3p was positively correlated with lymph node metastasis(χ^(2)=4.114,P=0.043)and pathological grade(χ^(2)=4.267,P=0.039).qRT-PCR results demonstrated that the expression of circ_0003926 in the ESCC and normal plasma samples was 3.54±2.15 and 1.03±0.05(t=6.993,P<0.001),respectively,and the expression level of miR-139-3p was 0.36±0.17,and 1.03±0.03(t=27.001,P<0.001),respectively.Compared with HEEC,circ_0003926 was highly expressed in ESCC cell lines(F=281.577,P<0.001),and the expression of miR-139-3p was low(F=87.034,P<0.001).After knock-down of circ_0003926,the proliferation,migration and invasion abilities of KYSE-150 and KYSE-410 cells were significantly decreased,and the level of apoptosis was significantly increased(all P<0.05).The miR-139-3p silencing rescued the proliferation,apoptosis,migration and invasion abilities in circ_0006168-silenced ESCC cells(P<0.05).The results of subcutaneous transplanted assay in nude mice demonstrated that the tumor volume and tumor weight of si-circ_0003926 group were lower than those in si-NC group,and the differences were statistically significant from day 10 to day 28(all P<0.001);and the tumor volume and body weight in the si-circ_0003926+antagomiR-NC group were lower than those in the si-circ_0003926+antagomiR-139-3p group,and the differences were statistically significant from day 10 to day 28(P<0.001).Conclusion circ_0003926 was highly expressed in ESCC tissue,plasma,and cells,and and regulates the progression of ESCC through miR-139-3p,which could become a new biomarker of diagnosis,treatment,and prognosis for ESCC.