Effect Of Gypenoside LI And MiR-128-3p On The Proliferation Of Melanoma Cells

Melanoma is a skin tumor caused by hyperproliferation of melanocytes.It has a high degree of malignancy,easy metastasis,poor prognosis and high mortality rate.At present,the main clinical treatment methods are surgery,chemotherapy,immunotherapy and targeted therapy,but the treatment effect is still not satisfactory.Therefore,its incidence is also one of the malignant tumors which have increased rapidly in recent years.Currently available drugs for the treatment of melanoma are Vemurafenib,Dalafenib,Trametinib,Temozolomide,Interleukin-2 and so on.Gynostemma is a perennial herbaceous liana,the main active substance is gypenoside.At present,the known effects are anti-inflammatory,liver,anti-fungal,anti-virus and anti-tumor effect.In addition,it also has the effect of lowering blood lipids,preventing cardiovascular and cerebrovascular diseases and immune regulation.Gypenoside LI is a gypenoside saponin monomer whose pharmacological effects have not been reported.Although studies have shown that gypenosides have anti-tumor function,no studies have reported that gypenoside LI induces apoptosis of melanoma cells.Therefore,it is particularly important to explore the role of gypenoside LI in melanoma.miRNA is a type of endogenous non-coding RNA found in eukaryotes,the length is 18-24bp.Its mechanism of action is binding to the 3'-UTR of the target gene,reducing the stability of the target gene mRNA,inhibiting the expression of the target protein s ynthesis,It plays an important role in cell proliferation,differentiation,development and apoptosis.There is a large amount of datas show that miRN As are abnormally expressed in many tumors,and these finding can be applied to clinical diagnosis and treatment.It has been reported that miR-128-3p is abnormally expressed in cancers such as lung cancer,liver cancer,and lymphatic leukemia,but it has not been reported whether this phenomenon exists in melanoma.Therefore,this topic will explore the effect of miR-128-3p on melanoma proliferation and explore its regulatory mechanisms.Objective:To investigate whether gypenoside LI inhibits the proliferation of A375 and SK-MEL-28 cells via the Wnt/?-catenin signaling pathway,and to explore the role of gypenoside LI in the proliferation of melanoma;To investigate the expression of miR-128-3p in melanoma cells;To study the effect of miR-128-3p on the proliferation of melanoma cells;To investigate the molecular mechanism of miR-128-3p influencing the proliferation of melanoma cells.Method 1:cck-8 was used to detect the toxic effects of gypenoside LI and C isplatin on HaCat,A375 and SK-MEL-28 cells;Gypenoside LI acts on morphological observations of melanoma for 24 hours at an IC₅₀ drug concentration;The apoptosis of melanoma cells induced by gypenoside LI was detected by AV/PI apoptosis assay;The effect of gypenoside LI on the cycle of A375 and SK-MEL-28 was detected by flow cytometry;Cell colony assay was used to detect the proliferation ability of gypenoside LI on melanoma;Western blot was used to detect the expression of apoptosis related proteins?PARP,Caspase 9,BID,FLIP and Bcl-2?,cyclin-dependent protein?CDK 2 and p-CDK2?,Wnt/?-catenin signaling pathway related protein??-catenin,DVL-2 and C yclin D1?and the expression of cytokine IL-24.Method 2:qPCR was used to detect the expression of miR-128-3p in human normal immortalized epidermal cells HaCat and melanoma A375 cells;The effect of miR-128-3p on the proliferation of A375 cells was detected by cck-8;The influence of miR-128-3p on HaCat and A375 cells in colonies and migration?transwell assay,cell scratch assay?;TargetScan software predicts miR-128-3p target genes;After miR-128-3p was transfected,Western blot was used to detect the expression of target protein in HaCat and A375 cells and the expression of related proteins such as cell proliferation.Results 1:The IC₅₀ of gypenoside LI on A375 cells was 75?g/ml by cck-8,and the IC₅₀ of SK-MEL-28 cells was 29.71?g/ml;Observation of cell morphology after treatment by gypenoside LI in 24 hours,cell apoptosis was time-dependent;Flow cytometry detected the early apoptosis of A375 cells was 45.3%;Flow cytometry showed that gypenoside LI arrested A375 and SK-MEL-28 cells in S phase;Meanwhile,colony-forming experiments showed that gypenoside LI could inhibit the proliferation of melanoma cells;Western blot results showed that the expression levels of apoptotic protein PARP,Caspase 9 a nd BID in the treated group were significantly higher than those in the control group,and the expression levels of FLIP and Bcl-2 were decreased compared with the control;Wnt/?-catenin signal pathway-related proteins?-catenin,DVl-2 and C yclin D1 were also decreased in the treatment group compared with the control;While IL-24 was increased in treatment group.Results 2:The results of qPCR showed that miR-128-3p was overexpressed in HaCat cells and lower in A375 melanoma cells.cck-8 data showed that overexpression of miR-128-3p inhibited the proliferation of A375 cells.After miR-128-3p inhibitor was transfected,the colony number of HaCat cells increased compared with NC group,and the cell migration?Transwell,scratch test?increased compared with NC group.After miR-128-3p was overexpressed,the number of colonies of A375 cells decreased compared with NC group,and the cell migration rate?Transwell,scratch test?became smaller;However,the expression of MMP2 and MMP9,which are related to cell migration,also changed.In HaCat cells,which downregulated of miR-128-3p,the expression of MMP2 and MMP9 were increased.In A375 cells,which overexpressed of miR-128-3p,the expression of MMP2 and MMP9 were decreased.TargetScan software predicts that the target genes of miR-128-3p are DVL2 and CDC6;After down-regulated the expression of miR-128-3p,Western blot results showed that the expression of DVl-2 and Cdc6 in HaCat cells were increased;While the expression of DV1-2 and Cdc6 protein in A375 cells after overexpression of miR-128-3p was decreased.The expression of?-catenin protein,which is related to Wnt/?-catenin signaling pathway,increased in HaCat cells?miR-128-3p inhibitor?and decreased in A375 cells?miR-128-3p mimic?.Expression of CDK2 increased in HaCat cells transfected with miR-128-3p inhibitor,but decreased in A375 cells after transfected with miR-128-3p mimic.Conclusion:1.Gypenoside LI can inhibit proliferation of melanoma cells through Wnt/?-catenin signaling pathway,which can arrest cells in the S phase and induce apoptosis.2.miR-128-3p inhibits melanoma cell proliferation by regulating the expression of DVL2 and CDC6 genes.