| Objective:Gastric cancer is the third most lethal cancer in the world and it’s also one of the highest incidence of cancer in our country.The incidence of this disease is increasing year by year and has the tendency of younger people.The molecular mechanisms which influence the occurrence and development of gastric cancer are very complex.Therefore,studying the key signaling molecules of gastric cancer and find the downstream target genes and understand the molecular mechanism of its action can help us to understand gastric cancer deeply and provide a theory for the diagnosis and treatment of gastric cancer.Histone demethylase includes LSD1 and JMJC family.Among them,histone demethylase of Jmj C family needs iron ion and a-ketoglutarate to participate in the reaction,which can make Monomethylation,Dimethylation or Trimethylation of lysine demethylated.Demethylation is an important modification of epigenetic and may be involved in the regulation of proliferation,aging,tumorigenesis and other important biological processes.Human JMJD1 A is a member of the histone demethylase family that contains a typical Jmj C domain and contains a C6-type zinc finger structure that is characteristic of the transcription factor,and that JMJD1 A is capable of acting via hydroxylation Monomethylation and dimethylation of Histone H3K9(H3K9me1 / 2)demethylation,thereby lifting the histone inhibitory effect,regulating gene expression.JMJD1A may play different roles in different organizations.Studies have shown that the expression of JMJD1 A in prostate cancer,liver cancer,colorectal cancer and renal cell carcinoma is higher than that in adjacent tissues,and the difference is significant.Hypoxic environment up-regulated JMJD1 A regulate a variety of hypoxia-related gene expression,in order to promote the growth potential of tumor cells in hypoxia.In human germ cell tumors,nasopharyngeal carcinoma,esophageal cancer and breast cancer tissues,JMJD1 A is low expressed,and its low expression is associated with poor prognosis.However,the role of JMJD1 A in gastric cancer cells is not clear.To reveal the role of JMJD1 A in the development and progression of gastric cancer,we constructed stable cell lines by lentivirus RNA interference,including flow cytometry,MTT,CCK-8,Cell invasion and metastasis assay,soft agar colony formation,nude mice tumorigenesis,A series of experiments in vitro proved that JMJD1 A affect gastric cancer cell cycle,inhibit the growth of gastric cancer cells and inhibit the migration and invasion of gastric cancer cells.The downstream gene of JMJD1 A was searched by RNA-seq technology.The RUNX3 gene is a new potential downstream gene that is regulated by JMJD1 A expression by RNA-seq technology.Runx3 is a member of Runt-related transcription factor 3,a member of the Runt domain family of transcription factors,also known as polybronchial enhancer binding protein 2(PEBP2)/ corebinding factor(CBF).Studies have shown that RUNX3 plays a role as a tumor suppressor in gastric cancer.Western Blot and RT-PCR experiments verified that JMJD1 A up-regulated RUNX3 expression in gastric cancer.Overexpression of RUNX3 in gastric cancer cells stably silenced JMJD1 A markedly reduced the subcutaneous tumors in nude mice.Overexpression of JMJD1 A can induce the changes of downstream genes of RUNX3,inhibit the growth of gastric cancer cells.The RUNX3 gene is located in the human chromosome 1p36 region and its transcription is regulated by two different promoter regions(RP1,RP2).The results showed that JMJD1 A could bind to the potential binding sites of RUNX3 promoter and CREB and ETS-1,respectively.Luciferase reporter assay showed that JMJD1 A and ETS-1 significantly increased the activity of RUNX3 promoter by accumulating.Chromatin co-immunoprecipitation demonstrated that JMJD1 A binds to RUNX3 promoter via ETS-1.Immunohistochemical detection of gastric cancer tissue chip showed that patients with low expression of JMJD1 A gastric cancer risk a greater risk of adverse prognosis.Low expression of JMJD1 A was correlated with regional lymph node metastasis(N),clinical stage and pathological stage in gastric cancer,and positively correlated with RUNX3 expression in gastric cancer cells.This study,for the first time,found a new tumor suppressor JMJD1 A in gastric cancer,which revealed that JMJD1 A inhibits the growth of gastric cancer cells by regulating the RUNX3 transcriptional mechanism by affecting the activity of RUNX3 promoter.Provides a new basis for the molecular mechanism for the diagnosis and treatment of gastric cancer provides a new way of thinking.Methods:To study the role of JMJD1 A in the development and progression of gastric cancer cells,stable gastric cancer cell lines JMJD1 A and JMJD1 A were overexpressed in gastric cancer SGC-7901 cells and MGC-803 cells by lentivirus RNA interference.The effect of JMJD1 A on the cell cycle of gastric cancer cells was detected by flow cytometry.MTT,colony formation and soft agar colony formation assay were used to detect the role of JMJD1 A in the growth and proliferation of gastric cancer cells.The effects of JMJD1 A on the migration and invasion ability of gastric cancer cells were detected by Cell migration and invasion(without spreading and spreading).Nude mouse tumorigenicity test to verify the effect of JMJD1 A on proliferation of gastric cancer cells in vivo.In order to find downstream genes regulated by JMJD1 A in gastric cancer cells.RNA-seq method was used to detect the gene expression changes of gastric cancer cells(SGC-7901-sh JMJD1A)stably silenced by JMJD1 A compared with control cells.The regulatory effect of JMJD1 A on downstream gene expression was verified by RT-PCR and Western Blot.IP experiments confirmed the binding between JMJD1 A and potential regulatory factors in RUNX3 promoter region.Double luciferase reporter gene experiment and chromatin immunoprecipitation experiment have confirmed the regulatory effect of JMJD1 A on RUNX3 promoter.The inhibitory effect of RUNX3 mediated JMJD1 A on proliferation and migration of gastric cancer cells was verified by subcutaneous tumor formation in nude mice.3.In order to analyze the expression and influence of JMJD1 A and RUNX3 in gastric cancer,we used immunohistochemistry to analyze the expression of JMJD1 A and RUNX3 in gastric cancer tissue.Statistical analysis of the relationship between the expression of JMJD1 A and RUNX3 in gastric cancer and clinicopathological features of gastric cancer.The relationship between the expression of JMJD1 A and RUNX3 in gastric cancer and the prognosis of patients with gastric cancer were statistically analyzed.The correlation between JMJD1 A and RUNX3 expression in gastric cancer tissues was analyzed by software.Results: 1.JMJD1 A plays an inhibitory role in the occurrence and development of gastric cancer cells.Flow cytometry results show that JMJD1 A can affect the cycle of gastric cancer cells.MTT,CCK-8,cell colony and soft agar colony formation experiments prove that JMJD1 A can inhibit the proliferation of gastric cancer cells.Cell migration and invasion experiment results show that JMJD1 A can inhibit the migration and invasion of gastric cancer cells.Tumor-forming experiments in nude mice show that JMJD1 A can over-inhibit the growth of gastric cancer cells in nude mice.2.JMJD1 A regulates the transcription of RUNX3 in gastric cancer.The expression of JMJD1 A detected by RNA-seq in gastric cancer is related to Wnt and TGF-β signal pathways.RT-PCR confirmed that the expression of JMJD1 A caused changes in m RNA level expression of downstream genes including RUNX3.Western Blot experiments confirmed that JMJD1 A upregulates RUNX3 expression in gastric cancer.Western Blot verified that JMJD1 A induced changes in downstream gene expression of RUNX3 in gastric cancer were consistent with up-regulation of RUNX3.Protein immuno-coprecipitation experiment proves that JMJD1 A binds to transcription factors on RUNX3 promoter The double luciferase reporter gene experiment proves that JMJD1 A and transcription factors ETS-1 and CREB both act to regulate transcription in RUNX3 promoter region.Chromatin immunoprecipitation technique showed that JMJD1 A and ETS-1 formed a complex,which promoted the transcription of RUNX3 by reducing the levels of histones H3K9me1 and H3K9me2 near RUNX3 promoter P1.Experimental results of subcutaneous tumor formation in nude mice show that JMJD1 A mediates its inhibitory effect on proliferation and migration of gastric cancer cells through RUNX3.3.Expression and influence of JMJD1 A and RUNX3 in gastric cancer tissues.Immunohistochemical analysis results showed that the expression of JMJD1 A in gastric cancer tissues was lower than that in adjacent tissues.The low expression of JMJD1 A was associated with adverse prognosis.The expression of JMJD1 A was associated with regional lymph node metastasis(N),clinical stage and pathological stage of patients.RUNX3 has been reported to be down-regulated in gastric cancer tissue and is a known tumor suppressor gene.We believe that the expression of JMJD1 A and RUNX3 may be correlated in gastric cancer.Conclusion: 1.JMJD1 A inhibits the proliferation,migration and invasion of gastric cancer cells.2.JMJD1 A upregulates the m RNA,protein levels and the activity of RUNX3 promoter P1 in gastric cancer cells.JMJD1 A promotes the expression of Bim,which is one of the target genes of RUNX3.3.JMJD1 A promotes RUNX3 transcription through co-activation of transcription factor ETS-1.4.JMJD1 A inhibits the growth of gastric cancer cells in vivo,partially dependent on RUNX3.5.JMJD1 A expression is decreased in gastric cancer tissues and the low expression of JMJD1 A is correlated with an aggressive phenotype and a poor prognosis in patients with gastric cancer.6.The expression of JMJD1 A is positively correlated with the expression of RUNX3 in gastric cancer samples. |
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