| Background:Esophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumors in China.ESCC has the characteristics of insidious onset,high malignancy,and easy recurrence and metastasis.At present,the treatment of ESCC is mainly divided into surgical resection and medical treatment,but its curative effect is still unsatisfactory.This is mainly due to the complex pathogenesis of ESCC and the drug resistance.Therefore,it is essential to elucidate the pathogenesis of ESCC and find new safe and effective anti-ESCC drugs.Human telomerase reverse transcriptase(hTERT)is a catalytic subunit of telomerase that plays a key role in the occurrence and progression of tumors.It has been reported that hTERT is a major determinant regulating telomerase activity and is highly expressed in most tumors.However,the role of hTERT in ESCC and its effect on ESCC have rarely been reported.Therefore,clarifying hTERT expression and its influence on ESCC may provide some theoretical basis for the treatment of ESCC.Dihydroartemisinin(DHA)is a derivative of artemisinin.Besides its antimalarial effect,DHA also has anti-tumor activity.Studies have shown that DHA can promote esophageal cancer cell apoptosis and inhibit esophageal cancer cell migration by inducing autophagy;and inhibit esophageal cancer cell proliferation by enhancing sensitivity to cisplatin and blocking the cell cycle.However,the role of hTERT in DHA inhibition of ESCC and its molecular mechanism have not been reported yet.Therefore,the aim of this study was to investigate the expression of hTERT in ESCC and its effect on the biological behavior of ESCC cells,to explore the potential molecular mechanism of DHA in the treatment of ESCC using hTERT as a target,and to provide a new theoretical basis for DHA as an anti-esophageal cancer drug.Objective:1.to explore the expression of hTERT in esophageal squamous cell carcinoma and its correlation with prognosis,as well as the effect of hTERT on the biological behavior of tumor cells;2.to study the effect of dihydroartemisinin(DHA)on the proliferation and metastasis of esophageal squamous cell carcinoma and its regulation of hTERT expression;3.To explore the potential molecular mechanism of hTERT as a target in dihydroartemisinin(DHA)treatment of esophageal squamous cell carcinoma.Methods:1.The expression of hTERT in esophageal cancer and its correlation with patient prognosis were analyzed using the ENCORI tool based on the TCGA database.2.The tissue samples of 32 patients with ESCC diagnosed by histopathology were collected from the Department of thoracic surgery of the Affiliated Hospital of North Sichuan Medical College from November 2018 to may 2019.The mRNA expression level of hTERT in 32 pairs of ESCC cancer tissues and paired adjacent tissues was quantitatively detected by qRT-PCR,and the correlation between the mRNA expression level of hTERT and the clinicopathological characteristics of patients was analyzed.3.pcDNA3.1-3HA-TERT plasmid was transiently transfected in Eca109 and TE1 cells,and the effect of hTERT overexpression was verified by Western blot;CCK8 assay and plate colony formation assay were used to detect the changes of viability and proliferation ability of Eca 109 and TE1 cells overexpressing hTERT;cell scratch assay and transwell assay were used to detect the changes of cell migration ability.4.After Eca109,KYSE150 and TE1 cells were treated with DHA(100μM),the changes of cell viability and proliferation ability were detected by CCK8 assay and plate colony formation assay;the changes of cell migration ability were detected by scratch assay and transwell assay.5.qRT-PCR and Western blot were used to detect the effects of DHA(100 μM)on the mRNA and protein levels of hTERT in Eca 109,KYSE150 and TE1 cells.6.Overexpression of hTERT in Eca109 cells followed by DHA(25 μM)treatment,CCK8 and plate colony formation assay were used to detect the changes of cell viability and proliferation ability;scratch assay and transwell assay were used to detect the migration ability of Eca109 cells.7.Actinomycin D assay was used to evaluate the stability of hTERT mRNA in Eca109,KYSE150 and TE1 cells treated with DHA(100 μM);qRT-PCR and Western blot were used to detect the effects of DHA(100 μM)on the mRNA and protein expression of the intracellular transcription factor SP1 gene in Eca109,KYSE150 and TE1 cells.8.overexpression of SP1 in Eca109,KYSE150 and TE1 was used to detect its effect on hTERT protein expression;CCK8,plate colony formation,scratch assay and transwell assay were used to detect the changes of proliferation and migration ability of Eca109 and TE1 cells after overexpression of SP1;the same method was used to detect the changes of proliferation and migration of Eca109 cells overexpressing SP1 after treatment with DHA(25 μM).9.the effects of DHA on the content of intracellular reactive oxygen species(ROS)in Eca109,KYSE150 and TE1 cells were detected by fluorescence microscopy and flow cytometry;and the changes of ROS and the changes of hTERT and SP1 protein expression were detected by the same method after pretreatment with antioxidant NAC.10.Using Eca109 cell ESCC xenograft model,100 mg/kg DHA was intraperitoneally injected(once every two days),and the transverse diameter and diameter of the transplanted tumor tissues were measured using a vernier caliper;After 19 days,the transplanted tumors were sacrificed,dissected,and the weight of the transplanted tumors was weighed by an electronic balance;the effects of DHA on the mRNA and protein levels of hTERT and SP1 genes in the transplanted tumor tissues were detected by Western blot and qRT-PCR;the effects of DHA on the expression of hTERT,SP1 and Ki67 proteins in the transplanted tumor tissues were detected by IHC;and the toxicity of DHA on important organs such as heart,liver,lung and kidney was evaluated by HE.Results:1.Bioinformatics analysis showed that the expression of hTERT in esophageal cancer was significantly higher than that in adjacent tissues(P<0.05),and the shorter the overall survival time of esophageal cancer patients with hTERT expression;the expression level of hTERT was significantly higher than that in 32 pairs of clinically collected ESCC patients.Further analysis showed that there were no correlations between hTERT mRNA expression and clinicopathological features such as patient age,gender,tumor location,differentiation grade,tumor diameter,lymph node metastasis and TNM stage.2.after overexpression of hTERT in Eca109 and TE1 cells,the proliferation and migration ability were significantly increased.3.DHA significantly inhibited the proliferation and migration of Eca109,KYSE150 and TE1 cells,and down-regulated the mRNA and protein expression levels of hTERT gene in a time-and dose-dependent manner.4.overexpression of hTERT partially reversed the inhibition of DHA on the proliferation and migration of Eca109 cells.5.DHA significantly down-regulated the mRNA and protein expression of SP1 gene in Eca109,KYSE150 and TE1 cells,but had no effect on hTERT mRNA stability.6.After overexpression of SP1 in Eca109 and TE1 cells,the expression level of hTERT protein was significantly increased,its proliferation and migration ability were significantly enhanced,and the inhibition of DHA on the proliferation and migration ability of Eca109 cells could be partially reversed.7.DHA significantly induced ROS generation in Eca109,KYSE150 and TE1 cells and could be inhibited by antioxidant NAC;and inhibition of ROS production could partially reverse the down-regulation of hTERT and SP1 proteins by DHA.8.DHA could significantly inhibit the growth of tumor tissues in ESCC xenograft model,down-regulate the mRNA and protein expression levels of hTERT and SP1 genes in tumor tissues,and also significantly down-regulate the expression of Ki67,a cell proliferation marker.In addition,DHA had no significant effect on the histomorphology of important organs such as heart,liver,lung and kidney.Conclusion:1.High expression of hTERT in ESCC promotes the proliferation and migration of tumor cells,and is positively correlated with the poor prognosis of ESCC patients.2.DHA down-regulates hTERT expression in ESCC cells through the ROS-SP1 signaling axis,and hTERT is a potential target for DHA treatment of ESCC. |
