| Endometrial carcinoma(EC)is one of the three most prevalent gynecological malignancies in women around the world.According to whether estrogen is involved in the pathogenesis and biological characteristics,ECs could be classified into type I and type II,the former is estrogen-dependent with endometrioid adenocarcinoma mainly;the latter is estrogen-independent or called special pathological types.Although the two types of EC are different in histological grading,surgicalpathologic staging,infiltration degree,estrogen receptor status,etc.,plenty of researchs have confirmed that overexpression of specific genes or proteins,which promote cell proliferation and inhibit cell apoptosis,making endometrial cells escape growth control that may be a common key link in the pathogenesis of type I and type II EC.Therefore,the thorough research of such specific overexpressed protein or molecules and their supervision law and rules will not only help to further reveal the mechanism of the occurrence and progression of EC,but will also discover new targets and directions for its effective and accurate clinical treatment.The DJ-1 gene is firstly discovered by Japanese scholar in 1997 as a new mitogen-dependent oncogene,the protein encoded by DJ-1 is extremely conserved among various species,it’s also expressed abroad in more than 22 human organs and tissues.DJ-1 gene participates in various cellular life activities in the form of dimers,such as transcription regulation,oxidative stress response,fertilization,mitochondrial regulation,inflammatory fibrosis formation,glycosylation damage prevention,promotion of cell growth and proliferation,inhibition of cell apoptosis and "molecular chaperones",etc.According to our recent study,the expression level of DJ-1 protein in EC,which is remarkably higher than in normal endometrial tissue,is related to invasion depth,lymph node metastasis,and clinical stage of EC.Moreover,our previous studies have discovered that silencing DJ-1 expression by RNAi in EC cells could clearly strengthen cell apoptosis and resist cell proliferation.However,the mechanism of molecular regulation that leading DJ-1 up-regulation in EC cells remains to be clearly elucidated.At present,plenty of researchs have confirmed that DNA methylation-mediated miRNAs silencing is one of the considerable mechanisms for the up-regulation of tumor-related gene expression.Therefore,we speculated that the up-regulation of DJ-1 expression in EC was probably caused by certain tumor-suppressive miRNAs which silenced through aberrant DNA methylate on the CpG island of promoter region.To screen for epigenetically silenced miRNAs gene targeting DJ-1,we previously performed computational searches of miRNA target databases(micro RNA.org,Pic Tar,Target Scan Human 7.0,and Cometa)and choosed conserved and overlapping miRNAs targeting DJ-1.Then we screened human genome database for the existence of CpG islands surrounding these miRNAs by using the online prediction software(http://cpgislands.usc.edu/)and identified that miR-128-2 was the optimum candidate gene.Subsequently,it was found by qRT-PCR that the miR-128-2expression in EC tissues were statistically lower than that of normal endometrial tissues.During the same period of time,by reviewing literature,we also found that miR-128-2 was indeed reported to exist DNA methylation modification and involved in the regulation of multiple tumor cell proliferation as a tumor suppressor.In addition,it is noteworthy that some scholars recently found that miR-128-2expression was down-regulated and DJ-1 expression was up-regulated in HCC cells.Moreover,miR-128-2 overexpression significantly down-regulated DJ-1 protein expression and promoted the hepatocyte apoptosis induced by multi-kinase inhibitor sorafenib,suggesting that miR-128-2 may be a key regulator of DJ-1 expression.Obviously,the above evidence strongly suggests that the up-regulation of DJ-1expression in EC cells is likely to be closely related to the deletion of miR-128-2expression regulated through DNA methylation.However,whether DNA methylation could directly mediates miR-128-2 silencing in EC cells,and then directly regulates the expression of DJ-1,remains to be further clarified from multiple levels and perspectives.Therefore,firstly,this study tried to find the evidence that the up-regulation of DJ-1 expression is related to DNA methylation status of miR-128-2 gene promoter in EC tissues.Secondly,at the level of endometrial cancer cell lines,a series of molecular biological methods were adopted to further clarify whether high DNA methylation in the promoter region of miR-128-2 gene in EC cells mediates miR-128-2 expression silence which may directly lead to up-regulation of DJ-1expression.Subsequently,observe after DJ-1 expression is regulated by DNA methylation in the promoter region of miR-128-2 gene,whether it has a substantial effect on the biological behavior of EC cells(such as proliferation,apoptosis,migration and invasion,etc.).The completion of this study is conducive to further revealing the role of DJ-1 in the development of endometrial cancer and the molecular regulation mechanism of its expression,as well as discovering new targets and directions for clinical precision treatment of EC.Part Ⅰ Studies on the correlations among miR-128-2 methylation,miR-128-2expression level,DJ-1 expression level,and clinicopathologic parameters of EC patientsObjective:To find relevant evidences that miR-128-2 gene promoter DNA methylation may be involved in the regulation of DJ-1 expression in EC from the histological level by analyzing the level of miR-128-2 DNA methylation,the expressions of miR-128-2and DJ-1,and their correlation in EC tissues and adjacent normal tissues.Methods:The methylation level of miR-128-2 and the expression of miR-128-2 and DJ-1mRNA were detected by quantitative methylation-specific PCR(qMSP)and qRT-PCR,respectively,in 63 pairs of clinical EC tissues and adjacent non-cancerous tissues from Maternal and Child Health Affiliated Hospital of Nanchang University.At the same time,the expression of DJ-1 protein was determined by Western Blot in the same panel tissues.Moreover,the correlations among miR-128-2 methylation,miR-128-2 and DJ-1 expression,and clinicopathologic parameters of EC patients were analyzed.Results:Compared with normal endometrial tissue adjacent to cancer,the DNA methylation level of miR-128-2 promoter region in EC tissue was significantly increased(P<0.01),while the expression of miR-128-2 was decreased and DJ-1mRNA or protein expression were regulated to increase(P<0.01);through correlation analysis,we found that the status of DNA methylation in the promoter region of miR-128-2 gene in EC tissues were interrelated with the miR-128-2expression negatively(Pearson correlation coefficient r =-0.9693,P<0.01)and positively interrelated with DJ-1 mRNA expression(Pearson correlation coefficient r= 0.8762,P<0.01).In addition,by reviewing clinicopathological data,it was found that the methylation level of miR-128-2,miR-128-2 or DJ-1 mRNA expression levels were not related to the pathological type and histological differentiation of patients with EC,but all of them were related to the patients’ tumor invasiveness,lymph node metastasis and surgical-pathological staging(P<0.05).Conclusion:At the histological level,our evidence suggested that DNA methylation of miR-128-2 gene promoter may be involved in the up-regulation of DJ-1 expression in EC by silencing the expression of miR-128-2.Part Ⅱ Effect of miR-128-2 silencing mediated by DNA methylation on the expression of DJ-1 in endometrial cancer cellsObjective:To further clarify that miR-128-2 expression silencing caused by DNA methylation of miR-128-2 gene promoter was an important regulatory mechanism for the up-regulation of DJ-1 expression in EC cells.Methods:1.The methylation level of miR-128-2 and the expressions of miR-128-2 and DJ-1 mRNA were detected by qMSP and qRT-PCR,respectively,in normal endometrial cell line(ESC)and 4 EC cell lines(Ishikawa,RL95-2,and KLE respectively stands for the type I EC cell lines with various differentiation;and SPEC-2 stands for the type II EC cell line).At the same time,western blot was used to detect the protein expression level of DJ-1 in all above cell lines.Furthermore,the correlation among miR-128-2 methylation,miR-128-2 expression,and DJ-1 mRNA expression in EC cell lines was statistically analyzed by Pearson’s correlation analysis.2.To clarify whether miR-128-2 could regulate the expression of endogenous DJ-1,trancfection of miR-128-2 mimics or miR-128-2 inhibitors toward Ishikawa and SPEC-2 EC cell lines was made in sequence separately,and set corresponding negative controls.Then,change of miR-128-2 and DJ-1 mRNA expression were determined by qRT-PCR,and level shift of DJ-1 protein expression was detected by WB.3.To test and verify whether DJ-1 was a direct target gene of miR-128-2,the recombinant luciferase reporter vectors(pGL3-DJ-1-3’UTR-wt containing wild-type DJ-1-3 ’UTR and pGL3-DJ-1-3’ UTR-mut containing mutant DJ-1-3 ’UTR)were constructed.The vectors(DJ-1-3’UTR-wt and DJ-1-3’UTR-mut)were independently co-trasnfected into Ishikawa and SPEC-2 cells with miR-128-2 mimic or its negative control RNA(miR-NC),appling dual luciferase reporter gene assay kit to examine the activity change of luciferase in the transfected cells.4.To clarify whether miR-128-2 gene promoter DNA methylation silences the miR-128-2 expression and thereby up-regulates the DJ-1 expression in EC cells,Ishikawa and SPEC-2 cells were treated with methylation inhibitor 5-Aza-d C(5 μM)alone for 48 h;or Ishikawa and SPEC-2 cells were pretreated with 5-Aza-d C 5 μM for 48 h,then transfected with miR-128-2 inhibitor for 48 h right after.Subsequently,the degree of miR-128-2 methylation was detected by qMSP,the expression status of miR-128-2 and DJ-1 mRNA were determined by qRT-PCR,and the protein expression levels of DJ-1 was detected by WB.Results:1.Comparing with ESC cells,the degree of miR-128-2 promoter DNA methylation were significantly increased in in four different human EC cell lines(Ishikawa,RL95-2,KLE,and SPEC-2)(P<0.01);Meanwhile,the miR-128-2 expression was decresed(P<0.01)while the DJ-1 mRNA and protein expressions were up-regulated(P<0.01).Through correlation analysis,it was found that the methylation level of miR-128-2 was significantly inversely correlated with miR-128-2 expression(Pearson correlation coefficient r =-0.9124,P<0.01),and correlated with the DJ-1 mRNA expression positively(Pearson correlation coefficient r = 0.8304,P<0.01).In addition,the expression level of miR-128-2 was inversely correlated with that of DJ-1 mRNA(Pearson correlation coefficient r =-0.8963,P<0.01).2.With miR-128-2 inhibitor transfecting in Ishikawa and SPEC-2 cells,DJ-1 mRNA and protein expression levels were significantly higher than those in the control group(P<0.01).While miR-128-2 mimic transfected cells,the expression levels of DJ-1 mRNA and protein were significantly down-regulated compared with the control group(P<0.01).3.After co-transfection of miR-128-2 mimic and pGL3-DJ-1-3’ UTR-wt,the luciferase activity was significantly restrained as contrasted to control group(co-transfection of miR-NC and pGL3-DJ-1-3’ UTR-wt)(P<0.01).However,the luciferase activity after co-transfection of pGL3-DJ-1-3’ UTR-mut and miR-128-2 mimic was comparable to that in control group(transfection with miR-NC and pGL3-DJ-1-3’ UTR-mut)(P>0.05).The results suggested that miR-128-2 might bind to the DJ-1 3’ UTR directly.4.After pretreatment Ishikawa and SPEC-2 cells for 48 h with 5 μM 5-Aza-d C,the level of miR-128-2 promoter DNA methylation was significantly decreased(P<0.01);Meanwhile,the expression of miR-128-2 was increasd(P<0.01),at the same time DJ-1 mRNA/protein expressions were decreased(P<0.01).Interestingly,we found that the miR-128-2 up-regulation or the DJ-1 mRNA/protein down-regulation following 5-Aza-d C pretreatment could be reversed by miR-128-2 inhibitor(P<0.01),but the reduction of miR-128-2 methylation by 5-Aza-d C pretreatment was not affected by miR-128-2 inhibitor.Conclusions:miR-128-2 can directly target and negatively regulate DJ-1 expression in EC cells.DNA methylation-mediated silencing of miR-128-2 expression is a crucial regulatory mechanism for up-regulation of DJ-1 expression in EC cells.Part Ⅲ Effects of DJ-1 expression regulated by miR-128-2 methylation on the biological behaviors of endometrial cancer cellsObjective:To observe whether DNA methylation silencing miR-128-2 expression thereby up-regulating DJ-1 expression affects cell proliferation,apoptosis,migration and invasion of EC cells.Methods:With the help of methylation inhibitor 5-Aza-d C,miR-128-2 inhibitor and DJ-1 overexpression lentiviral vector LV-DJ-1,after 5-Aza-d C pretreatment or co-treatment with 5-Aza-d C and miR-128-2 inhibitor or LV-DJ-1,further detection of Ishikawa and SPEC-2 endometrial cancer cells changes were made to clarify how the DNA methylation of miR-128-2 gene promoter to silencing its expression and then up-regulating the expression of DJ-1,whether it have a substantial impact on the biological behavior of endometrial cancer cells.(1)The changes of cell proliferation were assayed by CCK-8 experiment;(2)The changes of cell apoptosis were determined by flow cytometry;(3)The changes of cell migration and invasion ability were determinated by the Transwell chamber experiment.Results:After 5-Aza-DC pretreatment of Ishikawa and Spec-2 cells for 48 h,the cell proliferation was significantly decreased(P<0.01),and the rate of cell apoptosis was statictically increased(P<0.01),and the abilities of cell invasion and migration were inhibited(P<0.01).The important thing was that the aforesaid effects of 5-Aza-DC pretreatment could respectively suppressed by miR-128-2 inhibitor and LV-DJ-1 transfection(P<0.01).Conclusion:After miR-128-2 DNA methylation silences the expression of miR-128-2 and thereby up-regulates the expression of DJ-1 in EC cells,cell apoptosis was inhibited and cell proliferation,cell migration or invasion were promoted. |
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