Clinicopathological Study Of EZH2 Protein And MRNA Expression In Invasive Pituitary Adenomas

Introduction:EZH2 is catalytic subunit of the multi-protein HMT complex known as PRC2,which catalyzes the trimethylation of H3K27.Upregulation and dysfunction of EZH2 caused by mutation has been identified in various human neoplasms.EZH2 was also found to play critical roles in the tumorigenesis,proliferation,invasion and stemness in human tumors.However,the expression of EZH2 in human pituitary adenomas remain unknown.In the present study,we explored the expression level of EZH2 mRNA and protein in pituitary adenomas.Next,the discrepancy of EZH2 expression between invasive and non-invasive pituitary adenomas was compared.We also further studied the correlation of EZH2 and MT1-MMP proteins with various clinicopathological parameters,such as age,gender,tumor size and hormone secretion.Materials and Methods:We retrospectively selected 62 patients with histologically confirmed gliomas who underwent surgical resection at Shandong Provincial Hospital between July 2006 and September 2015.The cohort include 30 male patients and 32 females,the average age was 42.0±10.2 years.Complete clinicopathological information was retrieved from the medical records.Written consent to use stored specimens was obtained from all living patients.Immunohistochemistry was employed to examine the expression of EZH2,Ki-67,and MT1-MMP proteins following standard procedures.Immunohistochemical evaluation was executed according to the criteria as follows:Each slide was evaluated using light microscopy by 2 independent observers blinded to the clinicopathological background of the patients.Sections on which there were differing evaluations were reevaluated simultaneously by observers using a double-headed microscope.The EZH2 staining was categorized on the basis of the percentage of positive cells on the slide(0,no expression;+,1%-10%;++,10%-50%;+++,>50%).Expression of Ki-67 and mtl-MMP was likewise recorded by using the percentage of immunopositive cells.The MIB-1 labeling index was calculated as the percentage of immunopositive glioma cells.Isolation of total RNA,cDNA preparation and PCR reaction were performed according to manuals from manufacturers.Relative expression level of EZH2 mRNA was examined by PCR with ?-actin serves as internal control.Two-sided student t test was used to analyze the difference of EZH2 expression between invasive pituitary adenomas and non-invasive pituitary adenomas statistically.Relative EZH2 protein expression was also explored using western blot(wet transfer)with ?-actin protein serves as internal control.Two-sided student t test was used to analyze the difference of EZH2 protein expression between invasive pituitary adenomas and non-invasive pituitary adenomas statistically.Image processing were performed using Photoshop CC and Graphpad Prism version 6.0.All statistical analyses were performed using SPSS version 20.0.Results:1.Expression of EZH2 and MT1-MMP proteins in pituitary adenoma formalin-fixed paraffin-embedded slides and their correlation with clinicopathological parametersImmunopositive EZH2 proteins could be found in nuclei of pituitary adenoma tumor cells with partial expression in cytoplasm.The immunopositive rate of EZH2 protein in the total cohort was 88.7%(55/62),with 100%(30/30)in invasive pituitary adenomas and 78.1%(25/32)in non-invasive pituitary adenomas.Judging by tumor size,EZH2-immunopositive cases accounted for 93.6%(44/47)in macroadenomas(>1 cm)and 73.3%in microadenoma(<1cm).Spearman rank correlation test revealed that EZH2 protein expression was stronger in invasive pituitary adenomas than in non-invasive pituitary adenomas(P = 0.008).Similarly,EZH2 protein expression was stronger in macroadenomas than in microadenomas(P = 0.018).However,no correlation was found between EZH2 protein expression with age(P = 0.439),gender(P = 0.694),and hormone secretions(P = 0.416).ANOVA unveiled that Ki-67 expression varies with EZH2 expression intensity(P<0.001).More specifically,Dunnet T3 post hoc test revealed that Ki-67 expression was weaker in EZH2(-)group than in EZH2(+)group(P = 0.023),EZH2(++)group(P = 0.002),and EZH2(+++)group(P = 0.004).Moreover,Ki-67 expression was stronger in EZH2(+++)group than in EZH2(+)group(P = 0.024).On the contrary,no discrepancy was found between EZH2(+++)group than in EZH2(++)group(P = 0.238),and EZH2(++)group than in EZH2(+)group(P = 0.293).Immunopositive MT1-MMP proteins could be found in cytoplasm and membrane of pituitary adenoma tumor cells.The immunopositive rate of MT1-MMP protein in the total cohort was 86.7%(50/62),with 86.7%(26/30)in invasive pituitary adenomas and 75%(24/32)in non-invasive pituitary adenomas.Strong correlation was found between EZH2 and MT1-MMP proteins(P = 0.002).Spearman rank correlation test revealed that MT1-MMP protein expression was stronger in invasive pituitary adenomas than in non-invasive pituitary adenomas(P = 0.009).Nevertheless,no correlation was found between MT1-MMP protein expression with age(P = 0.652),gender(P = 0.186),tumor size(P = 0.631),and hormone secretions(P = 0.859).Similarly,discrepancies of Ki-67 expression within MT1-MMP expression groups were not revealed(P all>0.05).2.EZH2 mRNA and protein expressions in pituitary adenomas examined by PCR and western blotEZH2 mRNA levels were found to be upregulated in invasive pituitary adenomas than in non-invasive adenomas(6.07-fold,P<0.001).Similarly,EZH2 protein levels were also higher in invasive pituitary adenomas than in non-invasive pituitary adenomas(3.61-fold,P<0.001).Conclusion1.EZH2 and MT1-MMP expression was up-regulated in pituitary adenomas with a high frequency.2.EZH2 and MT1-MMP expression correlated with infiltrating behavior in human pituitary adenomas,in other words,their expression was stronger in invasive pituitary adenomas than in non-invasive pituitary adenomas.Furthermore,EZH2 expression was associate with Ki-67 expression,and EZH2 expression was stronger in macroadenoma(? 1 cm)than in microadenoma(<1 cm).3.EZH2 and MT1-MMP might play critical role in the mechanism of invasive behavior of invasive pituitary adenomas.EZH2 might involve in the proliferation of pituitary adenomas.EZH2 is a potential target for genetic therapy of pituitary adenomas.