| BackgroundThe esophageal cancer(EC)is a very common cancer of digestive tract.The incidence and mortality rate of esophageal cancer in China ranks first in the world.Among them,most patients are male,and the onset age is more than 40 years old.At present,the main treatments of esophageal cancer still are surgery,radiotherapy,chemotherapy.In recent years,although some new targeted and immune drugs have appeared,the survival rate of patients with advanced esophageal cancer is still very low.Recurrence and distant metastasis are the main reasons of death.Therefore,how to control the invasion and metastasis of tumor cells,so as to improve the control of esophageal cancer is very important.In the past ten years,the metastasis mechanism of cancer genome has been known more deeply.However,there are still a large number of mechanisms about cancer invasion and metastasis that can not be explained by the known gene,which leads to attempts to clarify the mechanism of cancer metastasis from new perspectives such as epigenetics.Some recent studies have shown that RNA methylation and its function play a very important role in the occurrence or development of cancer,providing new ideas for the research of cancer metastasis mechanism.Currently,more than 100 modifications of RNA are understood.In eukaryotes,maintaining the stability of mRNA mainly depends on its internal modification.Among all modifications of mRNA,m6A is the most common and reversible one,accounting for 80%of RNA methylation modifications.Currently,it has been found that microRNA,circRNA,rRNA,tRNA and snoRNA all have m6A modification.M6A modification of RNA is involved in a variety of pathological processes,such as cancer cell growth,neurological diseases,and so on.Its possible role in malignant tumors in particular has attracted great attention of scientists.The m6A modification mainly occurred in adenine.The function is determined by"Writer","Eraser" and "Reader".The "Writer",the methyltransferase complex,is mainly composed of four components,and METTL3 is the key one of this complex,and it contains a total of 580 amino acid sequences,of which both the zinc finger domain(ZFD)and the methyltransferase domain are required for enzyme activity.ZFD domain is responsible for recognition of target,while the methyltransferase domain,known as the MT-A70,is primarily responsible for the transfer of methyl groups.A number of studies have shown that abnormal expression of METTL3 can affect tumor development by affecting the m6A modification level of RNA or by interacting with the mRNA translation initiation device.However,abnormal levels of METTL3 play a complex and variable role in many types of cancer,and by regulating different target genes,they may affect the development of different types of tumors,and are expected to become a new candidate target for tumor therapy.Current studies have found that it plays the role of oncogene in most tumors,but a little of literatures have reported that METTL3 plays the role of tumor suppressor gene in some tumors.For example,Lin SB reported that METTL3 prom ote the proliferation,invasion and metastasis of lung cancer cells,thus playing the oncogene role.But Li X et al.found that METTL3 can inhibit the proliferation and metastasis of renal tumor cells,so play the role of tumor suppressor genes.However,the role of METTL3 in esophageal cancer and its mechanism have not been studied.The first part:Effect of METTL3 on behavior of esophageal carcinoma cell linesAimsIn order to investigate the effect of METTL3 on the esophageal cancer cell growth,invasion and metastasis,and clear up its molecular mechanism,we tested the expression of METTL3 in HEEC?Eca-109?KY-SE150 cell lines and esophageal squamous cell carcinoma tissue.The influences of METTL3 on proliferation,invasion,migration,apoptosis of E C cells were detected,by interference or overexpression of METTL3 in EC cell lines.The results were further validated in the subcutaneous xenograft model of mice.Methods1.We use RT-qPCR to detect the expression of METTL3 in HEEC?Eca-109 and KY-SE150 cell lines.2.The expression of METTL3 was detected by immunohistochemistry.According to the staining score,the difference of METTL3 expression level between the two groups was analyzed.3.By transfecting esophageal cancer cell lines Eca-109 and KY-SE 150 with sh-METTL3 and shRNA,pcDNA3.1-METTL3 and control pcDNA3.1,stable low and high expression of METTL3 were established.Subsequently,we use Western blot to detect the METTL3 expression.4.The effects of cell proliferation in METTL3-KD and METTL3-OVgroups were detected by CCK-8 and colony formation assay.Flow cytometry was performed to detect the apoptosis of cells.The invasion and migration of cell lines were detected also.5.Eca-109-shMETTL3 and ECA-109-NC cells were injected subcutaneously in female BALB/c Nude mice at 6-8 weeks to observe the METTL3 effect on the tumor-forming ability of Eca-109 cells.Results1.METTL3 is highly expressed in human EC cells than HEEC cells.We firstly detected the expression of METTL3 in Eca-109 and KY-SE150 and HEEC by qRT-PCR assay.It showed that METTL3 was expressed in both normal esophageal cell line(HEEC)and esophageal cancer cell lines(Eca-109,KY-SE150).Moreover,METTL3 was highly expressed in Eca-109 and KY-SE 150 cells,compared into HEEC cells(P<0.05).2.METTL3 was highly expressed in EC tumor cells.Immunohistochemical results told that METTL3 was mainly expressed in the nuclei of tumor cells,with visible brown-yellow and brown particles.METTL3 was highly expressed in EC tissues tumor cells and low in normal tissues surrounding the tumor.(P<0.05).3.METTL3 promotes the viability of human EC cells.In order to investigate the effect of METTL3 in EC,loss-of-function and gain-of-function assays were performed.ShRNA was used to down-regulate METTL3 expression(METTL3-KD)in human Eca-109 and KY-SE150 cells.pcDNA 3.1-METTL3 cDNA recombinant plasmid(METTL3-OV)was used to over-express the METTL3.Western blot results showed that in EC cell lines,the expressions of METTL3 in the METTL3-KD groups were lower than that in NC groups.Among the two targets,METTL3-KD-2 target sequence was selected;Compared with the NC group,the expression of METTL3 was higher in the METTL3-OV group,which proved that the success of transfection.CCK8 assay suggested that the proliferation of EC cells were significantly inhibited in METTL3-KD group,while the proliferation of EC cells were significantly promoted in METTL3-OV group(P<0.05).Furthermore,the same conclusion was found in colony formation assay.4.METTL3 inhibits apoptosis of human EC cells.The proportion of apoptotic cells was detected.It was found that the apoptosis of EC cells was more in the METTL3-KD group than NC group,and the difference was statistically significant.On the contrary,the proportion of apoptosis of EC cells was significantly decreased in the METTL3-OV group compared with NC group,and the difference was significant.5.METTL3 promotes the invasion and migration of EC cells.It is known that,invasion and migration of tumor cells is complex,which play an important part in metastasis and recurrence of cancer.We continued to investigate the role of METTL3 on cell invasion and migration.The results of transwell assays told that the abilityof invasion and migration of EC cells were obviously inhibited in the METTL3-KD group,while the invasion and migration ability were obviously promoted in the METTL3-OV group.The results of wound healing assay showed that,when down-regulating the expression of METTL3 in EC cells,the healing wound width was dwarfed,while over-expression of METTL3 promoted the healing of wound.6.METTL3 promotes tumorigenesis of EC cells in vivo.By constructing the stable cell line Eca-109-shMETTL3 and the control group Eca-109-NC,the results showed that the tumor volume in the group of implanted Eca-109-shMETTL3 cells was significantly smaller than that in the group of implanted Eca-109-NC cells.The results of tumor weight and tumor volume were consistent,and the difference was statistically significant.Conclusion1.METTL3 was expressed in both normal esophageal cells and EC cells,and the expression was higher in EC cells.2.The expression of METTL3 in EC tissue was higher than that in paracercinoma normal esophageal mucosa tissue.3.METTL3 can promote the proliferation,invasion and migration of EC cells,and play a variety of malignant behaviors such as anti-apoptosis.4.Animal experiments confirmed that METTL3 could promote tumor formation of esophageal cancer cell Eca-109 in vivo.The second part:The mechanisms of METTL3 on apoptosis,migration and proliferation of EC cellsAimsTo explore the molecular mechanisms of the malignant biological effect of METTL3 on esophageal cancer cells,the expression of related pathway proteins was detected.In addition,the dual inhibitor of PI3K and phosphorylated mTOR(p-mTOR)was added to the overexpression of METTL3 esophageal cancer cells,so as to observe their effects of METTL3.By interfering with the upstream molecule EGFR of AKT signaling pathway,the effects of EGFR on the proliferation,migration and other malignant behaviors of overexpressing METTL3 EC cell lines were observed.Methods1.The expression changes of related apoptotic proteins and several proteins in the EC cell lines after interference and overexpression of METTL3 and tumor tissues were detected by Western blot to explore the mechanism of METTL3 affecting malignant behavior of EC cells.2.BEZ235,a dual inhibitor of PI3K and phosphorylated mTOR(p-mTOR),was applied to Eca-109 and KY-SE150 esophageal cancer cell lines overexpressing METTL3.First,the expression changes of AKT?p-AKT and METTL3 in Eca-109 and KY-SE150 esophageal cancer cell lines after overexpression of METTL3 were detected by Western blot.Then,the effects of BEZ235 on the proliferation of EC cell lines after overexpression of METTL3 were detected by CCK-8 assay.Finally,the changes of BEZ235 on the migration rate of EC cell lines after overexpression of METTL3 were tested by wound healing assay.3.In previous studies,it has been found that METTL3 can enhance the translation of EGFR,and EGFR expression is abnormal in EC.We speculate that METTL3 promotes the progression of EC by affecting EGFR and then activating the AKT signaling pathway.Therefore,western blot was performed to detect the effect of METTL3 on EGFR expression.By interfering with the expression of EGFR in overexpressed METTL3 EC cell lines,Western blot was performed.Next,we observe its effects on the proliferation and migration of EC cells.Results1.METTL3 plays an anti-apoptotic role in EC cells by inhibiting mitochondrial apoptosisWestern blot shows that the expressions of pro-apoptotic protein Bax,Cleaved-caspase3 and Cleaved-caspase9 were up-regulated,and Bcl-2 was decreased in the METTL3-KD group.While an opposite regulation effect was observed in the METTL3-OV group.These data indicated that METTL3 inhibited the mitochondrial apoptotic pathway in Eca-109 and KY-SE150 cells.2.METTL3 promotes the proliferation and migration of EC cells by activating the Wnt3/?-catenin and promoting EMTIn order to explore the molecular mechanism by which METTL3 promotes the migration of ECr cells,we explored the association of the Wnt3/?-catenin pathway and EMT with the function of METTL3 by detecting the expression changes of some proteins in Wnt3/?-catenin signaling pathway and EMT after interference or overexpression of METTL3,and further verified in tumor-formation tissue.As a result,knockdown of METTL3 resulted inreduction of Wnt3 and ?-catenin.In addition,METTL3 knockdown also caused a significant up-regulation of E-cadherin,and a down-regulation of N-cadherin and Vimentin.In contrast,overexpression of METTL3 resulted in down-regulation of E-cadherin,and up-regulation of Wnt3,?-catenin,N-cadherin and vimentin.These experiments in vitro revealed that over-expression of METTL3 was responsible for Wnt3/?-catenin activation,EMT and metastasis.Experiments in vivo also suggested that METTL3 could activate the Wnt3/?-catenin signaling pathway.3.By activaing AKT signaling patyway,METTL3 can promote the effects of cell proliferation and migration.In order to further explore the molecular mechanism by which METTL3 promotes the proliferation and migration of EC cells,we explored the relationship between AKT signaling pathway and the function of METTL3 by detecting the changes in the expression of some proteins in the AKT signaling pathway after interference or overexpression of METTL3.It was found,METTL3 knockdown can decrease the phosphorylation level of AKT and mTOR,and decrease the expression of p70 and Cyclin D1.In contrast,the opposite result was found in METTL3-OV group.These data indicated that over-expression of METTL3 can promote the activation of AKT signal pathway.Similarly,the expression of p-AKT in the Eca-109-shMETTL3 group was lower than that in NC group.Experiments in vivo also suggested that METTL3 could activate the AKT signaling pathway.To identify the AKT signaling pathway role activating by METTL3 on EC cells proliferation and migration,a double-effect inhibitor(BEZ235)which inhibits PI3K and p-mTOR was used for next experiments.We found that,the treatment of BEZ235 inhibited the phosphorylation of AKT on cells which transfected with pcDNA3.1-METTL3 plasmid(METTL3-OV+BEZ235),suggesting that BEZ235 can inhibit activation of AKT caused by METTL3.Furthermore,CCK-8 showed that the proliferation of Eca-109 and KY-SE150 cells in the METTL3-OV group was decreased compared with METTL3-OV+BEZ235 group,suggesting that BEZ235 can reverse the pro-proliferation effect of METTL3-OV on EC cells.The wound healing assay showed that the migration rate of cells in the METTL3-OV+BEZ235 group was significantly lower than that in the METTL3-OV group.The results suggested that BEZ23 5 can reverse the migration of METTL3-OV on EC cells,and the differences were significant.4.EGFR is a key factor in METTL3 mediated EC proliferation and invasion.Previous studies have found that METTL3 can enhance the translation of EGFR,and EGFR is usually highly expressed in esophageal cancer.We speculate that METTL3 promotes the progression of esophageal cancer by affecting EGFR.Western blot was used to detect the effect of METTL3 on EGFR expression.Western blot results showed that EGFR expression in OV-METTL3 group was increased compared with OV-NC group,and EGFR expression in sh-METTL3 group was decreased compared with sh-NC group.Furthermore,compared with the overexpressed METTL3 group(OV-METTL3+sh-NC),the expression of EGFR and p-AKT decreased in the cells transfected with overexpressed METTL3 and sh-EGFR(OV-METTL3+sh-EGFR)group,while the expression of AKT did not change,and the proliferation ability of cells decreased,and the migration rate in scratches slowed down,the differences were significant.Conclusion1.METTL3 plays an anti-apoptotic role in EC cells by inhibiting mitochondrial apoptosis pathway.2.METTL3 can promote the invasion and migration of EC cells by activating the Wnt3/?-catenin signaling pathway and promoting EMT.3.METTL3 can activate the AKT pathway by increasing the expression of EGFR.It can affect the malignant behavior of EC cells and promote tumor progression.So interference of METTL3 can provide theoretical basis for clinical treatment of EC. |
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