| Background:The occurrence of tumors often involves the inactivation of multiple tumor suppressor genes.The treatment of single genes is not enough to inhibit tumor growth,which is one of the main reasons for the poor effect of tumor targeting gene therapy.Epigenetic mechanisms mean the gene expression level or function has undergone a genetic change with no change in the underlying DNA sequence.Epigenetic mechanisms include DNA methylation,histone modification,chromatin remodeling and RNA interference.Epigenetic mechanisms modificate the entire genome rather than a specific gene,its regulation can simultaneously restore multiple tumor suppressor gene expression,and reduce the incidence of gene mutation,improve genomic stability.It may be targeted for tumor therapy and provide a new target point.With the understanding of epigenetic changes,combined use of epigenetic drugs and gene drugs will be a new starting point for cancer therapy.By catalyzing and removal of methyl groups on the N-terminal lysine residues of histones,histone lysine demethylase genes(KDMs)participate in the regulation of chromatin function,and play an important role in the development and progression of tumor.The state of histone demethylation regulates the expression of the gene,which is an important epigenetic regulation.The histone demethylation mainly lies in the multiple lysine sites of histone H3,H4,which mediates gene silencing or activation(figure 1).Therefore,when the expression of KDMs has abnormalities,histone demethylation state also has abnormal changes,that are likely to cause abnormal gene activation or silence,and these changes are often closely related to the occurrence and development of malignant tumors.These changes exist in the entire process of cancer.Thus,cancer is,to some extent,an epigenetic disease.Epithelial ovarian cancer has the highest mortality rate in gynecological malignancies.In recent years,with the development of ovarian cancer research,ovarian cancer treatment has also been greatly developed.The standard treatment program includes a satisfactory cytoreductive surgery with the combined using of platinum and paclitaxel-based chemotherapy.Although the treatment for ovarian cancer has gained great progress,the overall survival rate of ovarian cancer was not significantly improved.The 5-year survival rate of advanced ovarian cancer was still low,and the 5-year survival rate of stage III and ? patients was only 29% and 13%.The reasons are mainly related to chemotherapy resistance,invasion and metastasis.Therefore,it has important clinical significance to explore the molecular mechanism of ovarian cancer,to find a new and reasonable and effective treatment for ovarian cancer.KDM5A,also known as Retinoblastoma Binding Protein 2(RBP2)or JARID1 A,can act specifically on 'Lys-4' of histone H3(H3K4me2 and H3K4me3)and demethylate it.KDM5 A was known as p Rb-binding protein,and it was also called RBP2,before it was found to be demethylase activity in 2007.It consists of Jmj N,Jmj C,ARID(ie,AT-rich interaction domain),C5HC2,non-T/E1 A,LXCXE tail(Leu-X-Cys-X-Glu motif)and 2 to 3 PHD domains.Jmj N,Jmj C is the specific demethylase activity region of KDM5A;ARID can bind to the tail of DNA CCGCCC to inhibit the transcription and translation of target genes,thus affecting human tumor development process.On the one hand,the PHD domain specifically recognizes H3K4me2/3 and binds it.On the other hand,C5HC2,LXCXE tail and non-T / E1 A structures can mediate with p Rb,p107,TBP and other proteins,multiple functional domains work together to cause target gene silencing.As an important member of KDMs,KDM5 A can play a role in the development of tumors through a variety of ways.In 2010,Zeng J and other studies found that: KDM5 A is overexpressed in gastric cancer,and have some relationship with gastric cancer cell proliferation and aging.By using RNA interference technology to silence KDM5 A,it can activate the cell cycle-dependent kinase inhibitor(cyclin-dependent kinase inhibitors,CDKIs),which induced gastric cancer cell aging and regulated gastric cancer cell proliferation.At the same time,KDM5 A can directly bind to the promoter of integrin ?1(ITGB?1)or up-regulate the expression of N-cadherin and Snail by activating the Akt signaling pathway and regulate malignant biological behavior such as invasion and metastasis of cancer cells.The KDM5 A also up-regulates the expression of cyclin D1,cyclin E1 and integrin?1,and promotes the proliferation,migration and invasion and metastasis of lung cancer cells.At the same time,KDM5 A also participate in the chemotherapy resistance.Jinling Hou and other studies have found that overexpression of KDM5 A in breast cancer is related to the chemo-resistance.The use of sh RNAs to inhibit the expression of KDM5 A in breast cancer cells leads to an increase in H3K4 methylation and activation of CDKIs,which enhances the antitumor effect of EGFR(epidermal growth factor receptor)inhibitors,suggesting that KDM5 A may be a potential target for breast cancer treatment.However,the role and molecular mechanism of KDM5 A in the development and progression of ovarian cancer has not been reported.Micro RNA(mi RNA)is an endogenous non-coding small RNA,about 18-25 nt in length.It is highly conserved in evolution.MiRNAs complement with the target gene,resulting in the target gene cleavage and translation inhibition and thus regulating the expression of target genes.MiRNAs do not regulate the target gene one-to-one match.One mi RNA can directly regulate about 200 target m RNA,while multiple mi RNAs can also control the same target gene.The human genome contains thousands of mi RNAs that can control nearly 33% of the protein-coding genes.MiRNAs play a very important role in human gene expression.Recent studies have shown that mi RNAs play an important role in the development of tumors,and about 50% of mi RNAs are associated with a variety of human tumors.This is not an accidental incident.The current research results of mi RNA have proved that mi RNA can play the role of oncogenes or tumor suppressor gene in the tumor development and metastasis of the various stages.These days,researches about the post-transcription role of mi RNA are one of the hot spots.In recent years,the role of mi RNAs in the development of ovarian cancer has also received more and more attention.By using microarrays or massively parallel signature sequencing,it has been found that there are a series of abnormal up-regulated or down-regulated mi RNAs in ovarian cancer.However,downregulation of mi RNAs is more common,especially in advanced and high-grade lesions.Although mi RNA differential expression profiles in ovarian cancer have not been consistently studied,there are still some mi RNAs that have the same abnormal expression pattern in multiple studies.For example,Let-7 is a definite tumor suppressor mi RNA that is down-regulated in almost all epithelial ovarian cancer.The target genes of let-7 include embryonic genes HMAG2,Mlin-41 and IMP-1 and the famous oncogene ras.Similar to the embryonic development process,there is often re-expression of the embryonic mesenchymal gene in the early stages of cancer.So the low expression of let-7 can result in the dedifferentiation of cancer cell,which may be the early event of epithelial ovarian cancer.In contrast,EMT is one of the important mechanisms of epithelial-mesenchymal transition(EMT)in the process of epithelial ovarian cancer metastasis.MiR-200 is a key regulator of EMT,and the decrease of mi R-200 expression is associated with the development of epithelial ovarian cancer metastasis.This process was caused by mi R-200 enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2.These findings suggest that down-regulation of mi R-200 plays some roles in the progression and metastases of epithelial ovarian cancer.In addition,some abnormal expression of mi RNAs that regulate cell apoptosis(such as mi R-34s)or proliferation(eg,mi R-143)promotes the development and progression of epithelial ovarian cancer by causing tumor cell proliferation and apoptosis.It can be seen that the abnormal expression of mi RNAs in epithelial ovarian cancer is related to the changes of DNA copy number,epigenetic factors,transcription factor regulation and changes of mi RNA processing proteins.These results suggest that mutations and abnormal expression of mi RNA are associated with the development and progression of various human tumors.MiRNAs can inhibit the expression of a variety of important tumor-related genes and play a role as an oncogene or tumor suppressor gene.And recent studies have found that mi RNAs can target the KDMs family and participate in the regulation of tumorigenesis,invasion and metastasis.Recently,Professor Wang XF from Duke University found that hypoxia-induced mi R-215 can reprogram glioma initiating cells(GICs)to accommodate to hypoxic microenvironment by inhibiting the epigenetic regulatory factor KDM1 B and regulating the activity of multiple signaling pathways.This process plays an important role in the occurrence,development and metastasis of gliomas.Therefore,it is important to study the expression and interaction of mi RNA and KDMs family in ovarian cancer,and it is helpful for the diagnosis,treatment and prognosis of ovarian cancer.The first part: the expression and biological function of KD5A in ovarian cancer carcinoma Objective:Detect the expression level of KDM5 A in ovarian cancer tissues;analyze the biological function in the occurrence and development of ovarian cancer Method:1.Based on bioinformatics,microarray analysis software(BRB-Arraytools)and GEO and TCGA public databases were used to analyze the expression of KDM5 A in ovarian cancer tissues and normal ovarian tissues.2.The expression of KDM5 A m RNA in 51 specimens normal ovary tissues and 51 specimens epithelial ovarian cancer(EOC)tissues was detected by RealtimePCR.3.Immunohistochemistry and tissue microarray technology were used to determine the protein expression of KDM5 A in ovarian cancer tissues,as well as analyzed the relationship between the expression level of KDM5 A and clinical features and prognosis of ovarian cancers patients.4.By using RNA interference to silence the gene of KDM5 A in OVCAR-8 and SKOV-3 cells,and proliferation,apoptosis,migration and invasion were investigated to learn the functions of KDM5 A in ovarian cancer cell lines.5.All data was present by mean+/-standard deviation.The comparisons between two groups were analyzed by Student's t-test or the Mann-Whitney's inspection.Disease-free survival and overall survival were analyzed by Kaplan Meier-survival curves,alpha <0.05 as the inspection level.Results:1.The expression level of KDM5 A in GEO database: Compared with normal ovarian tissue(12 cases),the expression of KDM5 A in ovarian cancer tissues(57 cases)was significantly increased(P <0.0006).Kaplan-Meier analysis showed that survival time of ovarian cancer patients with KDM5 A low expression was significantly better than KDM5 A high expression patients in TCGA database.It suggested that overexpression of KDM5 A in ovarian cancer patients is closely related with clinical prognosis.2.The m RNA expression of KDM5 A in ovarian cancer tissues was significantly higher than that in normal ovarian tissues(P <0.05).3.According to the results of ovarian cancer tissue microarray,Kaplan-Meier analysis showed that the expression level of KDM5 A was closely related to the prognosis of ovarian cancer patients.The results of univariate analysis showed that KDM5 A expression was associated with FIGO stage(P=0.012),lymph metastasis(P = 0.005).4.After KDM5 A knockdown,the proliferation ability,cell migration,invasion ability and tumorigenic ability were significantly inhibited.The apoptosis rate of ovarian cancer cell line was significantly increased after KDM5 A knockdown.In conclusion1.The expression level of KDM5 A in ovarian cancer tissues was significantly higher than those in normal ovarian tissues.2.The high expression level of KDM5 A has the correlation with the malignant phenotype of ovarian cancer patients and the shorter overall survival period.KDM5 A may play an important role in the development of ovarian carcinoma.3.KDM5 A gene silencing can significantly inhibit ovarian cancer cell proliferation,migration,and invasive ability,significantly induced ovarian cancer cell apoptosis.The second part: Screening and identifying the mi RNA gene targeting KDM5 A and investigating the biological behavior of the mi RNA gene in ovarian cancer cell lines Objective:Using bioinformatics techniques to screen and identify the mi RNA gene targeting KDM5 A.Investigating the biological behavior of the mi RNA gene in ovarian cancer Method:1.By using the online prediction tool(Targetscan,mi Randa,mi Rwalk),mi RNAs that could target KDM5 A was predicted.MiR-421 of 51 ovarian cancer and 51 normal ovary tissues samples were analyzed.2.The relationship between the expression of mi R-421 in ovarian cancer and the follow-up data of ovarian cancer was analyzed using the data of mi RNA chip expression in TCGA data.3.After overexpression of mi R-421,the m RNA and protein expression of KDM5 A was detected in ovarian cancer cells and the proliferation ability of ovarian cancer cells was observed.4.Luciferase reporter was used to verify the negative regulatory role of mi R-421 targeting for the KDM5 A.5.All data was present by mean+/-standard deviation.The comparisons between two groups were analyzed by Student's t-test or the Mann-Whitney's inspection.Disease-free survival and overall survival were analyzed by Kaplan Meier-survival curves,alpha <0.05 as the inspection level.Result:1.The expression level of mi R-421 in ovarian cancer was significantly decreased(P<0.0001),and KDM5 A might be the target gene of mi R-421.The activity of the fluorescent reporter gene of mi R-421 binding KDM5 A binding site was further confirmed by luciferase reporter assay.2.Kaplan-Meier analysis showed that the expression level of mi R-421 was significantly correlated with the clinical outcome of ovarian cancer patients(P= 0.016),and the survival time of patients with low expression of mi R-421 was significantly shorter than that of patients with high expression of mi R-421,suggesting that mi R-421 expression and clinical prognosis in ovarian cancer patients is closely related.3.Overexpression of mi R-421 had a significant effect on the expression of KDM5 A.After the overexpression of mi R-421,the proliferation ability of ovarian cancer cell lines decreased significantly.This was similar to that of KDM5 A knockout.Thus it was confirmed that mi R-421 is involved in the invasion and metastasis of ovarian cancer by transcriptional regulation of KDM5 A Conclusions: 1.MiR-421 is low expressed in ovarian cancer,which suggests it may be a tumor suppressor gene.The expression level of mi R-421 is closely related to the clinical prognosis of ovarian cancer patients.2.MiR-421 can specifically bind to KDM5 A,and mi R-421/KDM5 A pathway may play an important role in the development and progression of ovarian cancer.Part 3: Effects of mi R-421 on the biological behavior of ovarian cancer cells in vivo Objective:To study the effect of mi R-421 targeting KDM5 A on the biological behavior of ovarian cancer cells in vivo Methods: 1.The effect of mi R-421 on the biological behavior of ovarian cancer was observed by transfection of the mi R-421 stable overexpression group(Lenti-mi R-421)and the empty virus control group(Lenti-mock).2.To investigate the effect of mi R-144 in vivo,the tumor growth curve were observed and recorded.3.The tumor volume and weight of nude mice were observed and recorded.4.By using RT-PCR and Western blot,the expression level of mi R-421,KDM5A and Ki67 was detected.5.All data was present by mean+/-standard deviation.The comparisons between two groups were analyzed by Student's t-test or the Mann-Whitney's inspection.Disease-free survival and overall survival were analyzed by Kaplan Meier-survival curves,alpha <0.05 as the inspection level.Result: 1.After transfection of Lenti-mi R-421,the expression level of mi R-421 in the tumor tissue of nude mice was significantly increased(P<0.05).In vivo,the proliferation rate of ovarian cancer was significantly slower than that of the control group(P <0.05).The tumor volume was also decreased and the survival time was significantly increased.2.It was detected by immunohistochemistry that the expression of KDM5 A and Ki67 was decreased after overexpression mi R-421.The expression of KDM5A was negatively correlated with the expression of mi R-421.Conclusion: 1.In vivo experiments confirmed that compared with empty virus,overexpression of mi R-421 decreased the ovarian cancer proliferation rate significantly.2.KDM5 A is a downstream direct target gene of mi R-421,and mi R-421 inhibits ovarian cancer proliferation by targeting KDM5 A.Conclusions: 1.The expression of KDM5 A was significantly increased in ovarian cancer,and was closely related with FIGO stage,metastasis and clinical prognosis of patients. KDM5A played a role of cancer-promoting gene in the occurrence and development of ovarian cancer.2.The expression of mi R-421 in ovarian cancer was significantly reduced.The survival time of the patient with low expression of mi R-421 was significantly shorter than that with high expression of mi R-421,suggesting that mi R-421 plays an anti-tumor effect in the development and progression of ovarian cancer.3.MiR-421 regulates the progression of ovarian cancer by specifically binding to the 3'UTR region of KDM5 A and negatively regulating the protein expression of KDM5A.4.MiR-421/ KDM5 A pathway played an important role in the development of ovarian cancer,thus it may become a new target for treatment of ovarian cancer. |
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