【摘要】 目的 探究基因间长链非编码RNA626（LINC00626）通过Janus激酶1（JAK1）/信号转导和转录激活因子3（STAT3）/KH型剪切蛋白（KHSRP）信号通路对食管鳞状细胞癌（ESCC）转移的恶性进程的影响及潜在的分子机制。方法 选取河南大学淮河医院2020-01-01-2022-12-31微创腔镜手术切除的144例ESCC患者中ESCC组织及癌旁正常组织（距离肿瘤>5 cm）标本作为研究对象。采用实时荧光定量聚合酶链反应（qRT-PCR）法检测ESCC组织、癌旁正常组织、人食管黏膜上皮细胞Het-1A和4株食管癌细胞（EC-9706、OE-33、KYSE-450和SK-GT-4）中LINC00626和KHSRP的表达。慢病毒转染分为LINC00626敲低慢病毒（sh-LINC00626）组及其空转病毒（sh-NC）组、sh-LINC00626+KHSRP组、LINC00626过表达慢病毒（LINC00626）组及其空转病毒（vector）组，qRT-PCR法检测其转染效率。采用细胞计数盒8（CCK8）增殖实验、Transwell小室实验检测LINC00626对ESCC细胞生长的影响，qRT-PCR法检测LINC00626对JAK/STAT家族mRNA水平表达的影响，蛋白质印迹法检测KHSRP对JAK/STAT家族蛋白表达的影响。LINC00626、KHSRP在ESCC组织和癌旁正常组织中的表达差异，LINC00626对细胞迁移能力影响的Transwell实验、分子机制实验中的qRT-PCR实验和蛋白质印迹法结果分析采用两独立样本t检验；LINC00626、KHSRP在人食管黏膜上皮细胞和食管癌细胞系中的表达差异，转染sh-NC、sh-LINC00626+vector、sh-LINC00626+KHSRP后LINC00626表达量的差异，回复实验中的Transwell实验结果分析采用单因素方差分析；CCK8实验结果分析采用双因素方差分析。结果 qRT-PCR结果显示：ESCC组织中LINC00626表达量高于癌旁正常组织，差异有统计学意义，t=12.68,P<0.001;4株食管癌细胞中LINC00626的表达量高于人食管黏膜上皮细胞Het-1A,F=38.19,P=0.017;ESCC组织中KHSRP表达量高于癌旁正常组织，差异有统计学意义，t=19.23,P<0.001;4株食管癌细胞中KHSRP的表达量高于人食管黏膜上皮细胞Het-1A,差异有统计学意义，t=103.20,P<0.001。CCK8增殖实验结果显示：LINC00626组细胞生长率高于vector组，且24、48、72 h差异有统计学意义，F_{不同组别×时间}=1 909.00,P<0.05;sh-LINC00626组细胞生长率低于sh-NC组，且24、48、72 h差异均有统计学意义，F_{不同组别×时间}=94.50,P<0.001;回复实验中，sh-LINC00626+KHSRP组细胞生长率高于sh-LINC00626+vector组，且24、48、72 h细胞生长率差异有统计学意义，EC-9706细胞中F_{不同组别×时间}=2 653.00,SK-GT-4细胞中F_{不同组别×时间}=543.60,均P<0.05。Transwell小室实验显示：LINC00626组穿过小室的细胞数量高于vector组，差异有统计学意义，t=460.30,P<0.001;sh-LINC00626组穿过小室的细胞数量少于sh-NC组，差异有统计学意义，t=44.00,P<0.001;回复实验中，sh-LINC00626+KHSRP组穿过小室的细胞数量高于sh-LINC00626+vector组，差异有统计学意义，EC-9706细胞中F=252.80,SK-GT-4细胞中F=690.00,均P<0.001。分子机制相关实验结果显示，LINC00626、KHSRP过表达和敲降均可影响JAK1、STAT3、p-JAK1、p-STAT3的表达，差异有统计学意义，均P<0.05。结论 LINC00626、KHSRP在ESCC组织和细胞中均表达上调，且LINC00626可以通过JAK1/STAT3调控KHSRP影响ESCC转移的恶性进程。更多还原

【Abstract】 Objective To explore the impact and potential molecular mechanisms of LINC00626 on the malignant progression of esophageal squamous cell carcinoma（ESCC） through the Janus kinase 1（JAK1）/signal transduction and transcription activator 3（STAT3）/KH-type splicing regulator protein（KHSRP） signal axis. Methods ESCC tissue and adjacent normal tissue（>5 cm away from the tumor） samples from 144 ESCC patients who underwent minimally invasive endoscopic surgery at Huaihe Hospital, Henan University from January 1, 2020 to December 31, 2022 were selected as the research subjects. Quantitative reverse transcription polymerase chain reaction（qRT-PCR） was used to detect the expression of LINC00626 and KHSRP in ESCC tissue, adjacent esophageal normal tissue, human esophageal mucosal epithelial cells Het-1A and 4 strains of esophageal cancer cells（EC-9706, OE-33, KYSE-450, and SK-GT-4）. The lentivirus transfection was divided into LINC00626 knockdown lentivirus（sh-LINC00626） group and its empty vector virus（sh-NC） group and（sh-LINC00626+KHSRP） group, LINC00626 overexpression lentivirus（LINC00626） group and its empty（vector） group, and the transfection efficiency was detected by qRT-PCR. The effect of LINC00626 on the growth of ESCC cells was detected using cell counting kit 8（CCK8） proliferation assay and Transwell cell assay. Effect of LINC00626 on JAK/STAT family mRNA expression was detected by qRT-PCR methods.Western blot was used to detect the effect of KHSRP on the expression of JAK/STAT family proteins. The expression differences of LINC00626 and KHSRP in normal esophageal tissue and esophageal cancer tissue, the Transwell experiment on the impact of LINC00626 on cell migration ability, the qRT-PCR experiment and protein blotting method in the molecular mechanism experiment were conducted for two independent sample t-tests. The expression differences of LINC00626 and KHSRP in normal esophageal cell lines and esophageal cancer cell lines, as well as the differences in LINC00626 expression levels after transfection with sh NC, sh LINC00626+vector, and sh LINC00626+KHSRP, the Transwell experiment in the rescue experiment were conducted one-way ANOVA. The CCK8 experiment results were analyzed using two-way ANOVA.Results The qRT-PCR results showed that the expression level of LINC00626 in ESCC tissue was higher than that in normal esophageal tissue adjacent to the cancer, with t=12.68, P<0.001; The expression level of LINC00626 in esophageal tumor cells（EC-9706, OE-33, KYSE-450, SK-GT-4） was higher than that in esophageal normal cells（Het-1A）, F=38.19, P=0.017; The expression level of KHSRP in ESCC tissue was higher than that in adjacent esophageal normal tissue, t=19.23, P<0.001; The expression level of KHSRP in esophageal tumor cells（EC-9706, OE-33, KYSE-450, SK-GT-4） was higher than that in esophageal normal cells（Het-1A）, F=103.20, P<0.001. The results of CCK8 proliferation experiment showed that the cell growth rate of LINC00626 group was higher than that of vector group, and there was a statistically significant difference at 24, 48, and 72 h, F_{different groups×times}=1 909.00, P<0.05; The cell growth rate of the sh-LINC00626 group was lower than that of the sh-NC group, and the differences were statistically significant at 24, 48, and 72 h, F_{different groups×times}=94.50, P<0.001; In the rescue experiment, the cell growth rate of the sh LINC00626+KHSRP group was higher than that of the sh-LINC00626+vector group, and there was a statistically significant difference in cell growth rates at 24, 48, and 72 h, in EC-9706 cells F_{different groups×times}=2 653.00, in SK-GT-4 cells F_{different groups×times}=543.60, all P<0.05. The Transwell chamber experiment showed that the number of cells passing through the chamber in the LINC00626 group was higher than that in the vector group t=460.30, P<0.001; The number of cells passing through the compartments in the sh-LINC00626 group was less than that in the sh-NC group, t=44.00, P<0.001; In the rescue experiment, the number of cells passing through the compartment in the sh LINC00626+KHSRP group was higher than that in the sh LINC00626+vector group, in EC-9706 cells F=252.80, in SK-GT-4 cells F=690.00, P<0.001. The results of molecular mechanism related experimental protein immunoblotting showed that both overexpression and knockdown of LINC00626, KHSRP can affect the expression of JAK1, STAT3, p-JAK1, p-STAT3, all P<0.05. Conclusion LINC00626 and KHSRP are upregulated in ESCC tissues and cells, and LINC00626 can regulate the malignant progression of ESCC metastasis through JAK1/STAT3 regulation of KHSRP.更多还原