Mechanism Of Cantharidin Inhibits A375 Cell Proliferation By Regulating The MiR-21/PTEN Pathway

BackgroundMelanoma is a kind of skin malignant tumor,which is characterized by rapid progression,easy metastasis and high mortality.The risk factors of melanoma include age,sex,race,skin color,even geographical area.In recent years,the prevalence of melanoma has been increasing rapidly all over the world.The mechanism of melanoma is a complex biological process involving multiple factors,which abnormal expression of multiple genes and genetic factors are involved in the process of tumor genesis and development in a certain probability.However,its exact etiology and pathogenesis have not been clarified,increasing the difficulty of treating melanoma,especially the lack of targeted treatment for metastatic melanoma,resulting in unsatisfactory clinical efficacy.It is critical for dermatology to thoroughly elucidate the pathogenesis of melanoma and formulate the best treatment plan according to the key target.Current drugs used to treat metastatic melanoma are mainly high-dose or pegylated IFN-a and IL-2.These drugs have antitumor effects on only about 20%of patients,but have little impact on long-term survival and are prone to significant adverse reactions.A large number of studies have suggested that mutations of BRAF,NRAS,NF1 and PTEN genes in patients with melanoma lead to activation of MAPK and PI3K/AKT signaling pathways,resulting in programmed necrosis and the occurrence of melanoma.The identification of these genes is important for early intervention and treatment of advanced malignant melanoma.Therefore,molecular targeted drugs to these gene mutations have obtained important research focus in the treatment of melanoma.Recently,targeted therapies include BRAF inhibitors(Verofinib),MAPK/ERK signal pathway inhibitors(Trimetinib),c-Kit and PI3K/AKT/mTOR inhibitors.However,these drugs often cause digestive system toxicity or autoimmune diseases.Therefore,the immunotherapy of melanoma has gradually attracted the attention of researchers.At present,the research of immunotherapy focuses on PD-1 monoclonal antibody.Although PD-1 antibody improves the survival rate of patients,there are also some shortcomings,such as low efficiency and expensive price.PD-1 antibody is only effective for about 20%of patients in clinical treatment.MicroRNAs(miRNAs)could inhibit the translation of genes transcription or inhibit the translation of protein-coding genes by binding to the 3,-untranslated region(3,-untranslated regions,3,-UTR)of target gene mRNA.miRNAs account for about 1%genome and regulate about one third of the genes in the human genome.It has been proved that the variation of miRNAs caused by genetic mutation,epigenetic changes or chromosomal aberrations,play an important role in cell regeneration and differentiation.Recent studies have confirmed that miRNAs are involved in the development of many kinds of tumors.miR-21,the earlier found miRNA,is involved in cell growth,development and apoptosis.It is highly expressed in many malignant tumors,including lung cancer,digestive system cancers,and cervical cancer.It has been found that miR-21 could exert its biological function by regulating anti-oncogenes,Bcl-2.In addition,miR-21 could participate in tumor genesis and development via regulating transforming growth factor-?,protomyosin-1.Cantharidin(CTD)is an effective component extracted from Cantharis,a traditional Chinese medicine,which is a selective protein phosphatase 2A(PP2A)inhibitor.PP2A is an important phosphatase,which could regulate cell proliferation and transcription.CTD and its derivatives have been proved to have anti-tumor activity in various solid tumors,such as chronic myelogenous leukemia.CTD and its derivatives have been applied in clinical use involves compound CTD capsules,disodium CTD injection and norcantharidin.Some researchers have suggested that CTD has a certain toxic effect on colorectal cancer and other malignant tumors by activating and regulating the immune response.The anti-tumor mechanisms of CTD include blocking cell cycle progression,inducing cell apoptosis,reducing tumor angiogenesis,and inhibiting invasion and metastasis of tumor cells.It has been reported that norcantharidin could increase the expression of miR-16,miR-34a,miR-34b,miR-34c,miR-17 and miR-125 in K562 cells,and decrease the expression of miR-106 and miR-150.CTD could regulate migration,invasion and apoptosis of melanoma cells A375-S2 by blocking cell cycle and inducing apoptosis.Whether CTD can cause changes in miR-21 expression in melanoma cells has not been reported.We investigated the effect of CTD on proliferation of melanoma cells by regulating mir-21/PTEN pathway and explored its potential anti-tumor mechanism.Aim1.The expression of miR-21 and PTEN in melanoma tissue and normal control tissue was studied.After down-regulation of miR-21 expression,the changes of PTEN in A375 cells were observed,and the potential mechanism of miR-21/PTEN in proliferation,invasion and metastasis of A375 cells was explored.2.Whether CTD can inhibit proliferation of A375 cells by targeting miR-21/PTEN pathway and its potential antitumor mechanism were explored.Part I:miR-21 affects A375 cell proliferation by targeting PTENMethods1.In 20 melanoma tissues and normal skin tissues,The expression of miR-21 and PTEN was tested to show that the expression levels were different.In this experiment,RT-PCR was mainly used for detection.Western blot(WB)was used to detect PTEN protein expression in 20 melanoma tissues and normal skin tissues.2.The effect of miR-21 inhibitor on A3 75 cells proliferation was evaluated by cell count kit-8(CCK-8).Cell scratch assay was used to detect the effects of miR-21 inhibitor on A375 cells migration.3.After transfection of A3 75 cells with mir-21 inhibitor,Western blot was used to detect the expression changes of PTEN protein,AKT,mTOR and other signaling pathway proteins.The targeting relationship between PTEN and mir-21 in A375 cells was determined by double luciferase reporter gene assay.Results1.RT-PCR suggested that the expression of miR-21 was significantly increased,whereas PTEN was significantly decreased in cutaneous melanoma compared with normal skin tissue.Meanwhile,Western blot showed that the expression of PTEN in malignant melanoma was decreased in protein level.2.Down-regulation of miR-21 expression inhibited migration and proliferation and increased PTEN expression in A375 cells.Treatment A375 cells with miR-21 inhibitor for 48h could reduce migration and proliferation detected by cell scratch experiment and CCK-8 assay.RT-PCR and WB results showed that miR-21 inhibitors could significantly increased PTEN mRNA and protein expression in A3 75 cells in vitro.3.Luciferase reporter gene test was carried out and verified that miR-21 targeted the 3'UTR region of PTEN gene and inhibited PTEN gene expression,indicated PTEN was the downstream target gene of miR-21.4.Down-regulation of miR-21 expression inhibited the expression of p-AKT and p-mTOR in A375 cells.Treatment of A375 cells with miR-21 inhibitors could reduce the expression of p-Akt and p-mTOR detected by Western blot,but no significant change could be found in the expression of total-AKT and total-mTOR.ConclusionmiR-21 expression increased and PTEN expression decreased in malignant melanoma.miR-21 significantly promoted A375 cell proliferation and migration mainly by targeting inhibition of PTEN expression and regulating AKT and mTOR signaling pathway.Part II:Cantharidin inhibited A375 cell proliferation by regulating the MiR-21/PTEN pathway.Methods1.A375 cells cultured in vitro were treated with different doses of CTD at 48h,72h,and 96h by CCK-8 test detected the cell proliferation.2.Colony formation experiments was carried out to detect the number of cells after treatment with CTD.3.The growth of tumor after CTD treatment was detected by subcutaneous xenotransplantation.4.Apoptosis of A375 cells by treated CTD was detected by flowcytometry and the expression of Bcl2,Bax and caspase 3 in A375 cells were detected by Western blot.5.After treatment with CTD,RT-PCR was used to detect the expression of miR-21 and PTEN in A375 cells,while Western blot was used to detect the expression of PTEN and pAKT.6.A375 cells were cultured and transfected with miR-21 agomir,miR-21 antigomir and PTEN siRNA,then treated with CTD.The proliferation of A375 cells was observed by CCK-8.Results1.CTD inhibited the proliferation of A375 cells and the tumorigenicity of xenograft mouse model.A375 cells were treated with different doses of CTD(0.2,1 and 5 ?M)for 48h,72h,and 96h and detected cell proliferation by CCK-8 assay.The results showed that CTD could significantly inhibit the proliferation of A375 cells.With the increase of the dose,the inhibitory effect was dose-dependent,and the inhibitory effect of 5 ?M was the strongest among three doses.Colony formation experiments demonstrated that CTD can reduce the number of A375 cell populations.A nude mouse subcutaneous xenograft tumor model was established and the changes in tumor volume after CTD treatment were measured.The results showed that the volume and growth rate of xenograft tumors in the CTD treatment group were significantly slower than those in the control group,which showed that CTD can effectively inhibit the growth of melanoma in mice.2.CTD induced apoptosis of A375 cells.Annexin v-fitc/PI staining was used to observe the apoptosis number of A375 cells after CTD treatment.The flow cytometry results showed that the apoptosis number of cells in the CTD treatment group increased compared to the control group.Furthermore,observation of the changes in expression of apoptosis-related proteins showed that the expression of anti-apoptotic protein bcl-2 was decreased after CTD treatment,whereas the expression of corresponding pro-apoptotic proteins Bax and active caspase-3 were significantly increased.The results of these two experiments indicated that CTD could induce and promote the apoptosis of A375 cells.3.CTD inhibited miR-21 and promoted PTEN expression in A375 cells.After CTD treatment of A3 75 cells in vitro,downregulation of miR-21,upregulation of PTEN and inhibition of AKT phosphorylation could be detected by RT-PCR and Western blot.4.CTD inhibited A375 cell proliferation through targeting miR-21/PTEN.To further verify the relationship between down-regulation of miR-21 and CTD-induced inhibition of A3 75 cell proliferation,miR-21 agomir,miR-21 antagomir and PTEN siRNA were used,respectively.The effect of CTD on A375 proliferation and tumorigenesis was observed after different treatment.CCK-8 assay showed that miR-21 agomir inhibited the inhibitory effect of CTD on A3 75 cell proliferation;on the contrary,miR-21 antagomir enhanced the effect of CTD.And combination with overexpression or inhibition of PTEN reversed the above results,respectively,indicating that CTD inhibited proliferation of A375 cells by targeting miR-21/PTEN ConclusionCTD inhibited proliferation of A375 cells by reducing the activity of miR-21,increasing PTEN and decreasing the activity of AKT signaling pathway.Besides,CTD can inhibit the growth of melanoma in xenograft mice.