Casticin Inhibits Stemness Of Hepatocellular Carcinoma Cells Via Modulating The Interaction Between DNMT1 And MiR-148a-3p

The existence of cells with cancer stem cell properties(hereafter referred to as stemness)in hepatocellular carcinoma(HCC)is the underlying cause of the difficulty for cure of this disease.Epigenetic regulation involves the maintenance of the stemness features of HCC cells.Casticin(CAS)is a polymethyl flavonoid from Vitex agnus,which has multiple pharmacological activities,including anticancer effects.Our team's previous study has demonstrated that CAS has pharmacological effects that inhibit self-renewal of liver cancer stem cells.Using the GEO database,bioinformatics analysis of the differential expression of DNA methyltransferase 1(DNMT1)and miR-148a-3p in HCC and their impact on the prognosis of HCC patients suggested that DNMT1 was highly expressed and miR-148a-3p was underexpressed in HCC tissues,which were negatively correlated.In addition,survival curve analysis of HCC patients showed that the lower DNMT1 expression or the higher miR-148a-3p expression were associated with an increase of the overall survival in HCC patients.Accordingly,we hypothesized that in HCC,DNMT1 and miR-148a-3p negatively regulate each other to promote the stemness features,while CAS can interrupt this negative regulatory feedback loop to inhibit the stemness features of HCC cells,thereby exerting the therapeutic efficacy in patient with HCC.To validate the above scientific hypothesis,we firstly evaluated the stemness features in two different HCC cell lines(MHCC97H and SKHep-1)and normal embryonic hepatocytes(LO2)as a control,using a series of experiments reflected to the stemness features of HCC cells.The evaluations included: expressions of the stemness-related markers(CD44,Ep CAM,Bmi1,Nanog,and Oct4)were used by flow cytometry,western blot and RT-q PCR as well as the self-renewal and anchorage-independent growth capacities were determined by spheroid formation and colony formation assay in HCC cells.Next,we used DNA methyltransferase activity assay kit,RT-q PCR and western blot to detect DNMT1 activity and expressions of DNMT1 and miR-148a-3p in HCC cells.We sequentially manipulated the expression levels of DNMT1 or miR-148a-3p using a lentiviral delivery system and a small RNA transfection,then analyzed the effect of DNMT1 or miR-148a-3p on the stemness features of HCC cells using the aforementioned evaluations.Then,we used methylation-specific PCR(MSP)and luciferase reporter assay to identify the modality of interaction between DNMT1 and miR-148a-3p.To confirm that the interaction between DNMT1 and miR-148a-3p is involved in the stemness feature changes of HCC cells,we also analyzed the stemness features in HCC cells overexpressing DNMT1 treated with miR-148a-3p mimic or transfected with miR-148a-3p mimic treated by desitabine(DAC),a DNMT1 inhibitor.Subsequently,we treated HCC cells with CAS at selected subcytotoxic concentrations to determine the effect of CAS on HCC cell stemness features.We also analyzed the changes of DNMT1 activity and expressions of DNMT1 and miR-148a-3p in HCC cells after CAS treatment.Furthermore,we treated HCC cells overexpressing DNMT1 or transfected with miR-148a-3p mimic followed by treatment with CAS to analyze the molecular mechanisms by which CAS inhibits the stemness features of HCC cells.Finally,we also performed the xenograft model in nude mice to assess the tumorigenicities in vivo for treatment with CAS and agomir-148a-3p alone or in combination.A novel pharmacological mechanisms underlying inhibition of CAS on the stemness features of HCC cells can be illustrated via the aforementioned experiments in vitro and in vivo.The results showed that there were a small fraction of HCC cells that exhibited higher expression of stemness-related markers(CD44,Ep CAM,Bmi1,Nanog,and Oct4),stronger spheroid formation and colony formation capacity,namely,more pronounced stemness features and had DNMT1 activation(increased activity and expression)and underexpression of miR-148a-3p.Overexpression DNMT1 elevated the stemness features of HCC cells,while knocking down DNMT1 had the opposite effect.Transfection of miR-148a-3p inhibitor enhanced the stemness of HCC cells,but transfection of miR-148a-3p mimic significantly attenuated the stemness of HCC cells.MSP and RT-q PCR confirmed that overexpression of DNMT1 enhanced promoter methylation and silencing of miR-148a-3p in HCC cells.Target Scan algorithm and luciferase assay confirmed that DNMT1 was a direct functional target of miR-148a-3p.Mi R-148a-3p mimic inhibited DNMT1 activity and expression in HCC cells.In addition,we also found that miR-148a-3p mimic could reverse the enhanced effect of DNMT1 overexpression on the stemness features of HCC cells,and DAC enhanced the inhibitory effect of miR-148a-3p mimic on HCC cell stemness features.In experiments investigating the anti-HCC pharmacological effects and mechanisms of CAS,we found that CAS inhibited the viability of HCC cells(MHCC97H and SK-Hep-1),but did not affect LO2 cell survival.Subcytotoxic concentrations of CAS significantly inhibited the stemness features of HCC cells and attenuated DNMT1 expression and activity and upregulated miR-148a-3p expression.Overexpression of DNMT1 could reverse the inhibitory effect of CAS on HCC cell stemness.Transfection of miR-148a-3p mimic in combination with CAS cooperatively attenuated HCC cell stemness features.The tumorigenicity experiments showed that treatment with CAS or agomir-148a-3p alone could inhibit the growth of xenograft tumors from the mice bearing MHCC97 H cell tumors,and the inhibitory effect of CAS combined with agomir-148a-3p was further enhanced.Immunohistochemical staining of the specimens from xenograft tissues showed that DNMT1 and CD44 protein expression were lower in the co-treated group than in the group treated with CAS and agomir-148a-3p alone.We also observed that DNMT1 activity as well as DNMT1 and CD44 m RNA levels were decreased in different treatment groups,but the decrease was the most robust in the co-treated group,and that as DNMT1 decreased,there was a corresponding increase in miR-148a-3p expression.Additionally,no significant changes of DNMT3 a and DNMT3 b activity were detected.Summing up the above results,it is reasonable to draw the following inferences.1.The reciprocal negative regulation between DNMT1 and miR-148a-3p promotes and maintains HCC cell stemness features as one of the important pathological mechanisms of HCC initiation and progression.2.The inhibition of HCC cell stemness features by interrupting the reciprocal negative regulation between DNMT1 and miR-148a-3p is one of the pharmacological mechanisms for anti-HCC effect of CAS.