| Mycosis fungoides(MF)is the most common T-cell lymphoma in cutaneous malignant lymphoma,which can involve the whole body in the advanced stage and has a poor prognosis,so the research on the pathogenesis and treatment of MF is still a hot spot.Tumorigenesis is not only related to gene mutation,but also to post-transcriptional modification of DNA,including histone methylation and acetylation,which also shows a synergistic effect with each other.Till now a variety of histone deacetylase inhibitors(HDACi)have been used clinically in the treatment of T-cell lymphoma,unfortunately without obvious effectiveness.It has been found that histone methyltransferase EZH2(enhancer of zeste homolog 2)is involved in the regulation of physiological or pathological processes such as cell cycle,cell senescence,cell differentiation and cancer.It is highly expressed in many tumors,including T-cell lymphoma.So that we used immunohistochemical method and found that the expression of EZH2 was high in 20 cases of MF tissue specimens,especially in tumor stage.In this study,we further expanded the sample to detect the expression of EZH2 in tissues,peripheral blood mononuclear cells(PBMCs)of patients with different stages and cutaneous T-cell lymphoma(CTCL)cell lines.Besides we observed the changes of biological behavior of T-cell lymphoma cell line treated by combined use of HDAC and EZH2 inhibitors,in order to explore the molecular mechanism of anti-tumor effect of combined therapy.Part 1:The expression and significance of EZH2 in MF tissues,PBMCs and cell linesIn order to explore the relationship between the expression of EZH2 and the pathogenesis and development of MF,and to analyze the relationship with clinicopathological features of MF,we used immunohistochemical methods to detect the expression of EZH2 in 59 cases of MF(including the 20 cases)and control group(lichen planus and normal skin).The results showed that the expression of EZH2 was significantly increased in MF,and was positively correlated with clinical stage and Ki67 expression,especially higher with lymphatic metastasis and large cell transformation.At the same time,real-time qPCR and western blot were used to detect the expression of EZH2 mRNA and protein,respectively.The results showed that both mRNA and protein of EZH2 were overexpressed in MF tissues(3 cases)and PBMCs(9 cases)compared with paracancerous tissues and PBMCs of normal control(3 cases of psoriasis vulgaris and erythrodermic psoriasis).These results confirmed the abnormal high expression of EZH2 in MF tissues,PBMCs and cell lines,and suggested a relationship between EZH2 expression and the progression of MF disease.Part 2:The influence of combination of HDACi and EZH2i on biological behavior in MF cell linesWestern blot was used to detect expression of EZH2,cell proliferation ability was detected by CCK8 assay,and cell apoptosis and cycle were detected by Flow cytometer.The biological behaviors of MF cells in each group were evaluated and compared,respectively.These results manifested that combination of HDACi and EZH2i obviously down-regulated the expression of EZH2,which inhibited proliferation of MJ and HUT78 cells and significantly promoted cell apoptosis and induced S-phase arrest.The above studies suggested that combination of HDACi and EZH2i could further inhibit tumorigenesis.Part 3:The effects of combination of HDACi and EZH2i on the expression of PcG and H3K27me3Since the previous experimental results have confirmed that the expression of EZH2,as the core element of PRC2 complex,was related to the progress and prognosis of MF,the expression of other members of PRC complex including EED,SUZ12 and so on remained to be verified.Therefore,we detected the expression level of PcG family related proteins through western blot,only to find that the expression of EED and SUZ12 were significantly inhibited in MJ and HUT78 cells under the treatment of combination of HDACi and EZH2i,which indicated that EZH2 and its chaperone subunits EED and SUZ12 played a key role in mechanism of lymphoid tumors.In addition,we detected the expression of H3K27me3,a protein related to methyltransferase activity.It turned out that its expression was barely expressed,so we speculated that EZH2 was independent of its methyltransferase activity in the carcinogenesis of MF.Part 4:Combination of HDACi and EZH2i changes the activities of JAK-STAT pathwayJAK-STAT pathway,as a signal transduction pathway,involved in tumorigenesis and development,of which the continuous activation can lead to atypical growth of tumor cells.Till now,although some studies have found that JAK-STAT pathway may be related to the pathogenesis of MF,the specific mechanism remains to be further studied.Besides it is necessary to clarify whether the effect of combination therapy on cell biological behavior depends on the inhibition of the activity of JAK-STAT pathway.The results showed that the combined therapy could inhibit the phosphorylation of STAT3 and STAT5,indicating that the combination further inhibited the activation of JAK-STAT pathway.ConclusionsThe expression of EZH2 was up-regulated in MF tissues and PBMC,and it was closely related to the clinical stage and disease progression.At the same time,EZH2 was also significantly expressed in CTCL cell lines.The combination of HDACi and EZH2i could cause changes in biological behaviors such as proliferation,apoptosis and cycle of tumor cells,thus participated in the occurrence and development of tumor.In addition,although EZH2 and its chaperone subunits EED and SUZ12 jointly played a key role in pathogenesis of lymphoid tumors,the carcinogenic role of EZH2 in MF was independent of its methyltransferase activity.This study confirmed that the abnormal activation of STAT3 and STAT5 is closely related to the occurrence of MF,and through the combination of HDACi and EZH2i,the activation of it could be further inhibited.Therefore,we speculated that the inhibitory effects of HDACi and EZH2i on proliferation and apoptosis of CTCL cells might be related to the inhibition of JAK-STAT pathway activity,which probably provided a new target for the treatment of MF. |
PDF Full Text Request