Expression Of LSD1 In Nasopharynx Cancer And The Mechanism Of Induction Of Resistance To Radiotherapy

【Objective】 To research the level of Histone demethylase 1(LSD1,also known as KDM1A)in Nasopharynx Cancer(NPC)and its effect on the value-added,migration and invasion of NPC cells,and to discuss the role of LSD1 and DNA-Damage Repair,DDR)in radiotherapy-tolerant NPC cells,and the potential mechanisms for inducing resistance to radiotherapy.【Methods】(1)Analysis of LSD1 expression levels in squamous cell carcinoma of the head and neck tissues and normal tissues by the GEPIA2 database.The expression intensity and staining range of LSD1 in Nasopharynx Cancer tissues and adjacent tissues were analyzed by immunohistochemical staining.LOGpc survival analysis database was used to verify the relationship between LSD1 expression intensity and poor prognosis of patients with squamous cell carcinoma of the head and neck.Protein immunoblotting experiments were performed to select appropriate Nasopharynx Cancer cell lines for subsequent experiments according to the expression of LSD1.(2)The Nasopharynx Cancer cell lines CNE1 and CNE2 were treated with Various concentrations of LSD1 inhibitor(GSK-LSD1).CCK-8 assay,Wound healing assay,and Transwell assay were used to detect the effect of LSD1 on cellular proliferation,migration and invasion ability of CNE1 and CNE2.The appropriate IC50 drug concentration of GSK-LSD1 was selected by CCK-8 assay for subsequent experiments.(3)The m RNA expression levels of DDR indicators in Nasopharynx Cancer cells treated with 8Gy ionizing radiation were detected by q-PCR and compared with the control group;Correlation of LSD1 with DNA-damage response(DDR)indicators was analyzed by the GEPIA2 database;Western blot and agarose gel electrophoresis were used to verify the knockdown of LSD1 in Nasopharynx Cancer cells after transfection and used for subsequent experiments.Expression levels of DDR indicator m RNA were detected by q-PCR in the control group,radiotherapy group,GSK-LSD1-treated group and radiotherapy combined with GSK-LSD1-treated group,and verified simultaneously using sh-LSD1-stabilized Nasopharynx Cancer cell.The value-added of Nasopharynx Cancer cells in the control group,radiotherapy group,GSK-LSD1-treated group and radiotherapy combined with GSK-LSD1-treated group were detected by CCK-8 methodand verified simultaneously using sh-LSD1-stabilized Nasopharynx Cancer cells;γ-H2 AX is considered as a Marker of DNA damage.Protein immunoblotting assay was performed to detect the expression of γ-H2 AX in Nasopharynx Cancer cells in the control group,radiotherapy group,GSK-LSD1-treated group and radiotherapy combined with GSK-LSD1-treated group.【Result】(1)High expression of LSD1 in Nasopharynx Cancer was also associated with poor patient prognosis.The GEPIA2 database showed that LSD1 expression was higher in head and neck squamous cell carcinoma tissues than in normal tissues;Immunohistochemical staining experiments showed that LSD1 was highly expressed in Nasopharynx Cancer tissues;LOGpc database showed that high expression of LSD1 in head and neck squamous cell carcinoma was also associated with poor patient prognosis;Protein immunoblotting experiments the results showed high levels of LSD1 protein in CNE1 and CNE2,which were used for follow-up experiments.(2)Inhibition of LSD1 inhibited the maligant progression of Nasopharynx Cancer cells.1)Inhibition of LSD1 inhibited the proliferation of Nasopharynx Cancer cells.CCK-8 assays showed that GSK-LSD1 inhibited the proliferation of CNE1 in a timeand dose-dependent manner,whereas for CNE2 the appropriate treatment time and dose needed to be selected to better exploit the inhibitory effect of GSK-LSD1.2)Inhibition of LSD1 inhibited the migration of Nasopharynx Cancer cells.Cell scratching assay and Transwell(without Matrigel matrix gel)assay showed a decrease in the migration rate of CNE1 and CNE2 as well as the number of cells migrating out of the chambers compared to the control group,indicating that inhibition of LSD1 inhibited the migration of Nasopharynx Cancer cells.3)Inhibition of LSD1 inhibited the invasion of Nasopharynx Cancer cells.Transwell assay(pavement of Matrigel stromal gel)showed that CNE1 and CNE2 lysed a significantly lower number of cells that penetrated from the stromal gel chambers compared to the control group,indicating that inhibition of LSD1 inhibited the invasion of Nasopharynx Cancer cells.(3)LSD1 enhances DNA repair and induces radiation therapy resistance in Nasopharynx Cancer cells after radiotherapy.1)LSD1 correlated with DNA damage response.The q-PCR results showed that the m RNA expression levels of DDR indicators were significantly higher in Nasopharynx Cancer cells after radiotherapy compared with the control group;the GEPIA2 database showed that LSD1 correlated with DDR indicators.2)LSD1 is involved in DNA repair after radiotherapy.The q-PCR results showed that the indexes of DDR were significantly decreased after LSD1 inhibition or knockdown compared with the radiotherapy group.3)Inhibition of LSD1 increased the sensitivity of cancer cells to radiotherapy.CCK-8 assay shows that inhibition of LSD1 followed by radiotherapy can better inhibit the cellular activity of Nasopharynx Cancer cells compared to radiotherapy alone.4)Inhibition of LSD1 enhanced DNA damage induced by post-radiation therapy in Nasopharynx Cancer cells.Protein immunoblotting experiments showed that inhibition of LSD1 significantly suppressed γ-H2 AX expression in Nasopharynx Cancer cells.【Conclusions】LSD1 is highly expressed in Nasopharynx Cancer and is associated with poor prognosis of patients.At the same time,inhibition of LSD1 can inhibit the proliferation,migration and invasion of Nasopharynx Cancer cells.In addition,LSD1 is involved in DNA repair and resistance to radiotherapy in Nasopharynx Cancer cells after radiotherapy.Therefore,targeting LSD1 in Nasopharynx Cancer is an effective strategy for sensitization by radiotherapy.