Inhibition Of Autophagy By MFN2 And Enhancement Of Cisplatin Sensitivity In Hypopharyngeal Cancer Cells

Objective As a common ENT malignant tumor,hypopharyngeal cancer has no obvious symptoms and hidden location in the early stage of its onset,so most of it has entered the middle and late stage when diagnosed,and the therapeutic effect is not ideal as expected.Therefore,surgical treatment combined with chemotherapy and radiotherapy has become the main treatment methods for hypopharyngeal cancer.Drug chemotherapy plays an important and irreplaceable role in the comprehensive treatment of hypopharyngeal cancer.However,drug resistance generated in the course of chemotherapy greatly reduces the effect of chemotherapy and limits the application of chemotherapy drugs.This situation may be related to drug resistance caused by enhanced self-protective autophagy of tumor cells.mitofusin2(MFN2)is a core protein that mediates mitochondrial fusion on the outer mitochondrial membrane(OMM)and plays an important role in autophagy.The purpose of this study was to explore the relationship between MFN2 mediated autophagy and cisplatin(DDP)sensitivity of hypopharyngeal cancer,providing new clues and ideas for further research on the treatment of hypopharyngeal cancer and improving the drug resistance of hypopharyngeal cancer.Method 1.Purchase human pharyngeal squamous cell cell line FaDu for culture and amplification.Three groups of cells,the control group,MFN2 overexpression type and MFN2 knockdown type,were constructed by lentivirus transfection.After preliminary observation of transfection efficiency with fluorescence microscope,purinomycin was used to screen,and three groups of stable strains were obtained and amplified cells.In order to further verify whether the obtained strains were stable,the expression level of MFN2 was detected by Western Blot.2.To explore the effect of MFN2 on autophagy of hypopharyngeal cancer cells,proteins were extracted from wild type,MFN2 overexpression type and MFN2 knockdown type cells.The expression levels of autophagy related proteins Beclin1,P62,LC3Ⅱ/LC3Ⅰ in the three groups were detected by Western Blot assay.3.Explore the time-effect relationship of cisplatin in hypopharyngeal cancer cells.The control group,MFN2 overexpression type and MFN2 knockdown type cells were treated with the same concentration of cisplatin.Result 1.Cell culture and culture into stable transgenic strains after transfection The fluorescence microscope observed that the green fluorescent cells were more than85% and the cells were in good condition.The Western blot test showed that the expression of MFN2 protein in the cells of the overexpression MFN2 group was significantly higher than that of the control group,and the expression of MFN2 protein in the cells of the knockdown MFN2 group was significantly lower than that of the control group,indicating that the transfection was successful;After screening with 1ug/ml purinomycin,stable transgenic plants with stable passage were obtained.2.Autophagy inhibition of MFN2 on FaDu cellsWestern blot was used to detect the expression of autophagy-related proteins Beclin1,P62,LC3Ⅱ/LC3Ⅰ in MFN2 overexpression group,MFN2 knockdown expression group and blank control group.The expression of Beclin1,LC3Ⅱ/LC3Ⅰ in MFN2 overexpression group was higher than that in MFN2 knockdown expression group,and the difference between control group and MFN2 knockdown expression group was more obvious.The expression of P62 was lower than that of the MFN2 knockdown expression group and the difference between the control group and the MFN2 knockdown expression group was more significant(the difference was statistically significant,P<0.05).The results suggest that MFN2 can inhibit autophagy of hypopharyngeal cancer cells at the cellular level under normal culture conditions.3.Effect of autophagy inhibition of MFN2 on cisplatin sensitivity of FaDu cells The overexpression MFN2 cell group,knock-down MFN2 expression cell group and blank control group were treated with cisplatin of the same concentration and different concentration gradients at the same time.The proliferation ability of cells in each group was detected by CCK-8,and the sensitivity of cells in different groups to cisplatin was detected by flow cytometry.The results showed that compared with the control group,under the stimulation of cisplatin of the same concentration,the activity of cells in the overexpression MFN2 group decreased significantly,and the number of cells apoptosis was more,It was proved that MFN2-mediated autophagy inhibition could increase the sensitivity of FaDu cells to cisplatin.Conclusion 1.MFN2 can inhibit autophagy of FaDu cells.2.The inhibition of autophagy mediated by MFN2 is an important mechanism to increase the sensitivity of hypopharyngeal cancer cells to cisplatin.