Effects Of Curcumin And Mir-100-5p On Arsenic-induced HaCaT Malignant Phenotypic Cells And Its Mechanism

Research objectives: In this study,we prepared arsenic-induced HaCaT malignant phenotype cells(AS-TM cells)and treated AS-TM cells with different concentrations of curcumin to study the expression of vascular endothelial growth factor(VEGF)in AS-TM cells before and after curcumin treatment.Intensity to explore the mechanism by which curcumin inhibits AS-TM cell proliferation.Methods:1.HaCaT cells were cultured in complete medium containing arsenic at a concentration of 100 n M,and AS-TM cells were harvested 180 days later.2.The AS-TM cells were divided into 2 groups,and the cells were treated with 0ug/ml and 20 ug/ml curcumin respectively.After 24 hours,RT-PCR was used to detect the intensity of VEGF m RNA expression in AS-TM cells before and after curcumin treatment.Results:1.The expression of VEGF m RNA in the curcumin group was significantly lower than that in the control group by RT-PCR,and the expression level was 0.44 times that of the control group.Conlustions:1.This experiment successfully established a model of arsenic-induced HaCaT malignant phenotype cells(called AS-TM cells).2.Curcumin may inhibit the angiogenesis of AS-TM cells by down-regulating the expression of VEGF,thereby inhibiting the proliferation of AS-TM cells.Objective:This experiment intends to transfect miR-100-5p inhibitors and its mimics by Lipofectamine 2000 to study the effect of miR-100-5p on proliferation and apoptosis of HaCaT malignant phenotype cells induced by arsenic and its relationship with autophagy-related factors.Methods:1.The expression of miR-100-5p in HaCaT cells and arsenic-induced HaCaT malignant phenotype cells(AS-TM cells)was detected by RT-PCR.2.AS-TM cells were randomly divided into five groups,namely,miR-100-5p mimics group,mimics NC group,miR-100-5p inhibitors group,inhibitors NC group and blank group.Mi R-100-5p inhibitors,mimics and their respective negative controls were transfected into AS-TM cells by Lipofectamine 2000,respectively,and the blank group was not transfected.3.The expression of miR-100-5p in the five groups of cells after 48 hours of transfection was detected by RT-PCR.4.The proliferation capacity of AS-TM cells in each group after 48 hours of transfection was detected by CCK8 assay.5.Apoptosis of five groups of AS-TM cells was detected by flow cytometry after48 hours of transfection.6.RT-PCR was used to detect the m RNA expression of autophagy-related genes m TOR,Beclin1 and LC3 B in each group after 72 hours of transfection.Results:1.By RT-PCR,the expression of miR-100-5p in arsenic-induced HaCaT malignant phenotype cells was higher than that in HaCaT cells,and the expression level was about 1.4 times of that in HaCaT cells.2.The expression level of miR-100-5p in the mimics group was significantly higher than that in the blank group and the control group(mimics NC group).The expression level of miR-100-5p in miR-100-5p inhibitors group was lower than that of blank group and control group(inhibitors NC group).Mimics NC group and inhibitors NC group showed no significant difference compared with blank group.3.The results of CCK8 assay showed that the proliferation capacity of AS-TM cells in the miR-100-5p mimics group showed no significant change compared with the blank group and the control group(mimics NC group).The proliferation capacity of AS-TM cells in miR-100-5p inhibitors group was lower than that of blank group and control group(inhibitors NC group).The proliferation ability of AS-TM cells in mimics NC group,inhibitors NC group and blank group showed no significant difference.4.Flow cytometry was used to detect the apoptosis rate 48 h after transfection,the apoptosis rates of each group were: control group(4.89%+8.76%),miR-100-5p inhibitors(7.7%+17.13%),inhibitors NC group(4.12%+12.12%),miR-100-5p mimics group(5.05%+13.13%),and mimics NC group(4.26%+16.91%),indicating that down-regulation of miR-100-5p expression can promote apoptosis of AS-TM cells.5.mRNA expression levels of the phagocytic genes m TOR,Beclin1 and LC3 B in each group were detected by RT-PCR after 72 hours of transfection.The results showed that the expression level of m TOR m RNA in the miR-100-5p mimics group was lower than that in the mimics NC group and the blank control group,and the difference was statistically significant.Compared with the inhibitors NC group and the blank control group,the inhibitors group had no significant differences;Comparing the mimics NC group,inhibitors NC group and blank control group,there were no significant differences.The expressions of LC3 B and Beclin1 m RNA in the miR-100-5p mimics group and the inhibitors group were compared with the respective negative control group and the blank control group,while the negative control group showed no statistical significance compared with the blank control group.Conclusion:1.The expression of miR-100-5p in arsenic-induced HaCaT malignant phenotype cells was higher than that in HaCaT cells.2.Downregulation of miR-100-5p significantly inhibited the proliferation of as-tm cells and promoted their apoptosis;The up-regulation of miR-100-5p had no significant effect on the proliferation and apoptosis of as-tm cells.Mi R-100-5p may play a similar role as an oncogene in the malignant transformation of arsenic-induced HaCaT.3.Up-regulated or down-regulated miR-100-5p had no significant effect on autophagy related genes Beclin1 and LC3 B in AS-TM cells at the gene level.Up-regulation of miR-100-5p inhibited the expression of m TOR m RNA,and miR-100-5p may affect autophagy of AS-TM cells through m TOR,which is worthy of further study.4.MiR-100-5p may be a new target for the diagnosis and treatment of arsenic-induced skin cancer.