Mechanism of LINC00626 in regulating metastasis of esophageal squamous cell carcinoma metastasis through the JAK1/STAT3/KHSRP axis
Objective To explore the impact and potential molecular mechanisms of LINC00626 on the malignant progres-sion of esophageal squamous cell carcinoma(ESCC)through the Janus kinase 1(JAK1)/signal transduction and transcrip-tion 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 endo-scopic 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 expres-sion of LINC00626 and KHSRP in ESCC tissue,adjacent esophageal normal tissue,human esophageal mucosal epithelial cells Het-IA and 4 strains of esophageal cancer cells(EC-9706,OE-33,KYSE-450,and SK-GT-4).The lentivirus trans-fection 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 Trans well 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 e-sophageal cell lines and esophageal cancer cell lines,as well as the differences in LINC00626 expression levels after trans-fection with sh NC,sh LINC00626+vector,and sh LINC00626+KHSRP,the Transwell experiment in the rescue ex-periment 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 esoph-ageal tumor cells(EC-9706,OE-33,KYSE-450,SK-GT-4)was higher than that in esophageal normal cells(Het-1 A),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-1 A),F=103.20,P＜0.001.The results of CCK8 pro-liferation 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,Fdifferentgroup,×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,Fdifferent 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 sig-nificant difference in cell growth rates at 24,48,and 72 h,in EC-9706 cells Fdifferentgroupsxtimes=2 653.00,in SK-GT-4 cells Fdifferent 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 num-ber 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-JAKl,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.
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