LncRNA DGCR5 Silencing Enhances The Radio-Sensitivity Of Human Esophageal Squamous Cell Carcinoma Via Negatively Regulating The Warburg Effect

Background and purposeEsophageal cancer is one of the common malignant tumors of the digestive tract,and its incidence and mortality rate are among the highest in the world,and esophageal squamous cell carcinoma(ESCC)is one of the main histological types.At present,radiation therapy is one of the main treatments for esophageal cancer,which can significantly improve the local control rate of ESCC,reduce the recurrence rate of ESCC patients and prolong the survival time of patients.However,radiation therapy resistance is a major problem in radiation therapy for ESCC,and radiation therapy resistance often directly leads to the failure of ESCC treatment.An increasing number of studies have confirmed that radiotherapy and chemotherapy mediate the progression of multiple malignancies through the regulation of the lncRNA/miRNA/m RNA molecular axis.In addition,lncRNA DGCR5,miR-195 and Warburg effect have been reported to play an important role in the radiosensitivity of tumor cells,but their role in radiation therapy for ESCC is unclear.Hexokinase 2(HK2)is a key protease in glycolysis and is closely related to the Warburg effect in tumor cells.However,the lncRNA DGCR5,miR-195 and HK2 molecular axis have not been validated in ESCC,and the relationship between this molecular axis and the efficacy of radiotherapy in ESCC needs to be further explored.Objective1.To compare the expression levels of lncRNA DGCR5,miR-195 and HK2 in ESCC tissues and normal tissues.2.To verify the regulatory relationship between lncRNA DGCR5,miR-195 and HK2 signaling pathways and analyze their correlation with the clinicopathological characteristics of ESCC patients.3.The lncRNA DGCR5/miR-195/HK2 molecular axis was further elucidated to regulate the Warburg effect of ESCC cells,which in turn affects the sensitivity of ESCC cells to radiotherapy.Methods1.The TCGA(The Cancer Genome Atlas)database data were used to analyze the expression levels of lncRNA DGCR5,miR-195 and HK2 in cancerous and paracancerous tissues.Identification of potential miRNA targets for lncRNA DGCR5 and miR-195 through the ENCORI database.2.The cancerous and para-cancerous tissue samples and corresponding clinical data of 78 esophageal cancer patients were collected for retrospective analysis.The expression levels of lncRNA DGCR5,miR-195 and HK2 in cancer and para-cancerous tissue samples were measured using q RT-PCR and correlated with the corresponding clinical data.3.In the cell experiment,TE-1 and KYSE150 ESCC cell lines were selected,and the expression levels of lncRNA DGCR5,miR-195 and HK2 in the cell lines were detected by q RT-PCR.Dual luciferase reporter gene assay to verify the targeting relationship between the three.4.The cells were treated with different radiation doses,and the best radiation dose was finally selected.According to the experimental design,sh DGCR5,pc DNA3.1-DGCR5,miR-195 mimic,miR-195 inhibitor and pc DNA3.1-HK2 were transfected into TE-1 and KYSE150 cells.5.The growth,proliferation and apoptosis of each group of cells were detected using CCK-8,MTT assay,plate clone formation assay,and Annexin V-FITC/PI doublestained flow assay.Using Western blot to detect the expression level of apoptosisrelated proteins in each group.And then,the glucose uptake kit and the lactate secretion kit were used to detect glucose uptake and lactate secretion in each group of ESCC cells.Results1.In ESCC tissues,lncRNA DGCR5 and HK2 were highly expressed and miR-195 was lowly expressed and significantly correlated with tumor size,vascular invasion,lymph node or distant metastasis and TNM stage in patients with ESCC.2.LncRNA DGCR5 promotes the proliferation of ESCC cells and inhibits apoptosis.3.Knock down lncRNA DGCR5 can inhibit the Warburg effect of ESCC cells,thus enhancing the radiosensitivity of ESCC cells.4.LncRNA DGCR5 can target binding to miR-195/HK2 molecular axis and regulate Warburg effect in ESCC cells.5.Knockdown of miR-195 or overexpression of HK2 both reversed the enhanced effect of knockdown of lncRNA DGCR5 on the sensitivity of ESCC cells to radiotherapy.ConclusionThis study showed that lncRNA DGCR5 interacted with miR-195 and HK2,and knocking down lncRNA DGCR5 could inhibit the Warburg effect of ESCC cells by targeting the miR-195/HK2 molecular axis,which in turn enhanced the sensitivity of ESCC cells to radiotherapy.