Mechanism Of Aloe-emodin Inhibiting The Progression Of Melanoma By Regulating SNHG16/miR-146b-5p/β-catenin

Objective: To explore the inhibitory effects of aloe-emodin on melanoma and analyze its molecular mechanism of inhibiting melanoma through SNHG16/miR-146b-5p/β-catenin.Methods: Firstly,the effect and molecular mechanism of aloe-emodin on melanoma through β-catenin were analyzed.(1)0 μg/m L,5 μg/m L,10 μg/m L,15 μg/m L,20 μg/m L,25 μg/m L aloe-emodin were to the cell culture medium of A375 and SK-MEL-28.The cell viability was calculated by CCK-8,so as to find the optimal experimental concentration(2)20 μg/m L aloe-emodin were to the cell culture medium of A375 and SK-MEL-28.Cell viability,clone formation ability,cell cycle,apoptosis,migration,invasion,and epithelialmesenchymal transition capabilities were tested.Western blot was used to evaluate the levels of Wnt/β-catenin pathway in the cells.(3)A rescue experiment was conducted to clarify the mechanism of β-catenin in the inhibition of melanin by aloe-emodin.The cell model of β-catenin overexpression was constructed by transfection.The effects of aloeemodin on cell viability,migration,invasion and apoptosis by inhibiting β-catenin were verified.(4)In order to clarify the inhibitory effect of aloe-emodin on melanoma throughβ-catenin in vivo,A375 and SK-MEL-28 cells transfected with OE-NC and OE-β-catenin were injected subcutaneously to construct tumor-bearing nude mice models.Gavage intervention of aloe-emodin(50 mg/kg).After 28 d,the tumor growth,proliferation,apoptosis,and epithelial-mesenchymal transition marker proteins were detected.Then,based on the principle of competitive endogenous RNA,the molecular mechanism of regulating the expression of β-catenin protein in melanoma cells was analyzed.(5)The upstream Lnc RNAs and miRNAs that regulate β-catenin were searched based on bioinformatics analysis.The target relationship between Lnc RNA SNHG16,miR-146b-5p and β-catenin was verified by dual luciferase report.(6)Then cell experiments were applied to analyze that miR-146b-5p could inhibit melanoma by targeting β-catenin,A375 and SK-MEL-28 cells were divided into 4 groups: NC group,mimic group,mimic+β-catenin group and β-catenin group.The biological behavior of cells in each group was tested.(7)In order to further analyze that SNHG16 regulates β-catenin by targeting miR-146b-5p,thereby affecting the biological behavior of melanoma cells,A375 and SK-MEL-28 cells were divided into 4 groups: NC group,mimic group,mimic+SNHG16 group and SNHG16 group.The biological behavior of each group of cells was tested.Finally,based on the above-mentioned regulation mechanism of SNHG16/miR-146b-5p/β-catenin,the molecular mechanism of aloe-emodin inhibiting the expression of β-catenin was analyzed.(8)Firstly,the effects of 20 μg/m L aloe-emodin on the m RNA and protein expressions of SNHG16,miR-146b-5p,β-catenin in A375 and SK-MEL-28 cells were detected.(9)In order to clarify the mechanism of aloe-emodin inhibiting β-catenin in melanoma cells through SNHG16,rescue experiments were performed,A375 and SK-MEL-28 cells were divided into control group,aloe-emodin group,and aloe-emodin+OE-SNHG16 group.The biological behavior of each group of cells was tested,and the expressions of miR-146b-5p,β-catenin were detected.Results:(1)When the concentration of aloe-emodin is within 20 μg/m L,aloe-emodin inhibited cell viability in a dose-dependent manner.(2)The cell viability,clone formation ability,the proportion of cells in G0/G1 and S phase,migration and invasion abilities of the20 μg/m L group were significantly lower than those of the 0 μg/m L group,and the proportion of cells in the G2/M phase and the apoptotic rate were significantly higher than those in the 0 μg/m L group(P<0.05).(3)As for cell viability,migration and invasion: 20μg/m L+OE-β-catenin > 20 μg/m L+OE-NC;as for apoptosis rate: 20 μg/m L+OE-β-catenin< 20 μg/m L+OE-NC(P<0.05).Overexpression of β-catenin significantly blocked the inhibitory effect of aloe-emodin on melanoma cells.(4)In vivo,the tumor quality of the aloe-emodin+OE-NC group was significantly lower than that of the NC+OE-NC group(P<0.05).The tumor quality of the aloe-emodin+OE-β-catenin group was significantly higher than that of the aloe-emodin+OE-NC group(P<0.05).The expression levels of cyclin D1,c-myc,Bcl-2,ICAM1,Vimetin m RNA and protein in the aloe-emodin+OE-β-catenin group were significantly higher than those in the aloe-emodin+OE-NC group,while cleaved-caspase-3,Bax and E-cadherin m RNA and protein expression was significantly lower than those in the aloe-emodin+OE-NC group(P<0.05).Overexpression of β-catenin significantly blocked the inhibitory effect of aloe-emodin on melanoma in vivo.(5)In melanoma cells,SNHG16 was positively correlated with β-catenin,while miR-146b-5p was negatively correlated with SNHG16 and β-catenin,respectively.The verification results showed that SNHG16 targeted miR-146b-5p,and miR-146b-5p targeted β-catenin.SNGH16 could promote the expression of β-catenin protein by targeting miR-146b-5p.(6)-(7)The cell viability,clone formation,migration and invasion: mimic+β-catenin > mimic,SNHG16 > NC,mimic+SNHG16 < SNHG16(P<0.05).Overexpression of β-catenin blocked the inhibitory effect of miR-146b-5p on melanoma.Overexpression of miR-146b-5p blocked the promotion of SNHG16 on melanoma and β-catenin expression.(8)SNHG16,β-catenin m RNA and protein: 20 μg/m L < 0 μg/m L,aloe-emodin < control,aloeemodin+OE-SNHG16 > aloe-emodin;miR-146b-5p: 20 μg/m L > 0 μg/m L(P<0.05).(9)miR-146b-5p,apoptosis: aloe-emodin+OE-SNHG16 < aloe-emodin.β-catenin m RNA and protein,cell viability,migration and invasion: aloe-emodin+OE-SNHG16 > aloe-emodin(P<0.05).Conclusion: The mechanism of aloe-emodin inhibiting the progression of melanoma is related to the inhibition of β-catenin pathway.SNHG16 promotes the expression of β-catenin by targeting miR-146b-5p to improve the proliferation and metastasis of melanoma cells.Aloe-emodin may play an anti-melanoma effect by regulating SNHG16/miR-146b-5p/β-catenin.