Study On The Mechanism Of Methyltransferase METTL3 Mediating Mirna Through M6A Modification In The Process Of Gastric Cancer Induced By N-nitroso-compound

Background and ObjectivesGastric cancer(GC)is one of the most common malignant tumors of the digestive system,which seriously threatens human health.Early diagnosis of GC is difficult,and patients with advanced GC were usually had a poor prognosis.Therefore,there is an urgent need to deeply study the pathogenesis of GC,which could provide evidence for effective biomarkers of GC early diagnosis and treatment.N-Nitroso-compounds(NNCs)exposure is closely associated with the pathogenesis of GC.N6-methyladenosine(m6A)RNA methylation modification is a recent breakthrough in the field of epigenetics research.The present study is focused on the high-risk population living in the western GC high-incidence area of China.Solid-phase extraction and gas chromatography-mass spectrometry(SPE-GC-MS)was used to investigate the environmental levels and inner levels of N-nitrosamine for GC patients,exploring possible triggers for the high incidence of GC in this region(Chapter 1).Subsequently,the Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)large-scale sequencing data were analyzed to depict the aberrant expression profiles of fourteen major m6 A regulators and the association between gene expression with relevant clinical diagnosis,progression,and prognosis.Further combined with the integrated expression verification of western high-risk area GC patients’ tumor tissues,we systematically screened the core gene of m6 A modification Methyltransferase-like 3(METTL3)as the major research target.We selected METTL3 through expanded serum samples verification to further investigate its association with clinicopathological features and potential value as an effective biomarker for high-risk GC populations(Chapter 2).Accordingly,we established a cellular model of MNNG-induced malignant transformation and the long-term MNNG exposure rat model to explore METTL3 expression variation and bio-function in the MNNG-induced carcinogenesis.Then,the METTL3 inhibited cell model was constructed to deeply explore the bio-function and involvement of METTL3 in the process of MNNG-induced GC by affecting the epithelial-mesenchymal transition(EMT)process of cells from multiple perspectives through verified experimentally in vitro and in vivo(Chapter 3).Finally,the m6 A methylated RNA immunoprecipitation(Me-RIP)sequencing method was used to systematically identify the m6A-induced downstream.m RNAs,and pre-mi RNAs significantly affected by METTL3.Subsequent experiments such as m6A-IP-q PCR,q RT-PCR,Western blot accurately identified the target mi RNA and downstream genes regulated by METTL3 through m6 A modification.Biological processes involved in the MNNG-induced malignant transformation of gastric cells were performed(Chapter 4).The research design is progressive,integrating multi-level research data such as cells,animals,and field investigations,and systematically analyzes the biological functions and mechanisms of key molecular regulatory axes of m6 A methylation modification in the process of MNNG-induced GC.The finding of the present study revealed the key role and pathogenesis of m6 A methylation modification,and accurately identified the therapeutic targets of N-nitrosamine exposure-induced GC.It provided a scientific basis for proposing effective prevention and treatment measures for GC patients and enriched the existing molecular biomarkers system in the western GC high-incidence area of China.Methods and ResultsChapter I Investigation of N-nitrosamine exposure level in drinking water and urinary of GC patients in the GC high-incidence areaSection 1 Detection of N-nitrosamine exposure levels in drinking water in in the GC high-incidence areaThe western GC high-incidence area was selected as the investigated field and the adjacent low GC incidence area control field,the natural and social environment information such as the distribution of water system was investigated,and 20 water samples were collected from each area.The concentrations of 9 N-nitrosamines including NDMA,NDEA,NDPA,NDBA and NEMA in water samples were detected by solid-phase extraction-gas chromatography-mass spectrometry(SPE-GC-MS),and the N-nitrosamines exposure levels in drinking water of the two regions were evaluated.The results showed that four N-nitrosamine compounds were detected in the investigation area.The average concentration of NDPA was 9.22±7.64 ng/L;the average concentration of NDph A was 5.73±5.49 ng/L;the average concentration of NMEA was 18.62±2.87 ng/L;the detected concentration of NDMA was 0.55 ng/L.Four N-nitrosamine compounds were also detected in the control area.The average concentration of NDMA was 11.27±4.51 ng/L;the average concentration of NPyr was 6.78±4.79 ng/L;the detected concentration of NDPA was 1.97 ng/L;and the detected concentration of NDph A was 0.93 ng/L.In addition,the detected concentration of total N-nitrosamines(133.00 ng/L)in the drinking water in the investigation area was significantly higher than that in the control area(36.83 ng/L,P<0.05).Section 2 Investigation of urinary N-nitrosamine exposure level of GC patients in the GC high-incidence areaUrine samples were collected from 24 GC patients and paired healthy controls,and a questionnaire was used to collect the exposure data and related clinical information of GC patients.SPE-GC-MS was used to detect the concentrations of the nine N-nitrosamines in urine samples,and the internal exposure levels of nitrosamines in GC patients and healthy controls were evaluated.The questionnaire results showed that high consumption of fresh vegetables(>5 catties/week)may be a protective factor for GC and was negatively correlated with the risk of GC(OR=0.25,95% CI 0.07-0.83).Compared to occasional consumption of salted vegetables,long-term consumption of salted vegetables(>5 catties/week)was positively correlated with the occurrence of GC(OR=5.00,95%CI 1.22-20.46);Consumption of salted meat(including bacon,cured meat,etc.,>5 catties/week)was positively correlated with the occurrence of GC(OR=4.41,95% CI 1.21-13.59).The results of urine samples showed that 9 N-nitrosamines were detected in the urine samples of GC patients and healthy controls,among which NDPA,NDBA,NPip,NPyr,NMor had higher detection rates.The average detection levels of NMor(18.67±14.94 ng/m L),NPyr(7.03±12.49 ng/m L),NDPA(6.11±6.50 ng/m L)and NDph A(5.52±2.86 ng/m L)in the urine samples of GC patients were significantly higher than that in control urine NMor(8.91±16.14 ng/m L)(P<0.01),NPyr(3.78±8.34 ng/m L)(P<0.01),NDPA(3.75±3.32 ng/m L)(P<0.05),NDph A(3.98±3.03 ng/m L)(P<0.05).In addition,the average detection level of total N-nitrosamines in the urine samples of GC patients(44.34±33.59 ng/m L)was significantly higher than that in the urine samples of healthy controls(26.34±17.54 ng/m L)(P<0.05).Chapter II Screening of GC-related m6 A key molecules and analysis of METTL3 expressionSection 1 Screening of GC-related m6 A key molecules and analysis of its clinical significanceThe TCGA and GEO large-scale sequencing data were analyzed to depict the aberrant expression profiles of fourteen major m6 A regulators and the association between gene expression with relevant clinical diagnosis,progression,and prognosis.q RT-PCR was used to verify the expression level of target m6 A regulator in 40 pairs of GC and adjacent tissues in the high incidence area of GC in western China.Functional analysis revealed the biological process and related important transcription factor.TCGA silicon analysis results revealed that m6 A Writer: METTL3,METTL14,METTL16,WTAP,KIAA1429,RBM15 and ZC3H13;Reader: YTHDF1,YTHDF2,YTHDC1,YTHDC2 and HNRNPC were significantly up-regulated in GC.Survival analysis from GEO data showed that METTL3,METTL16,YTHDF1,YTHDC2,WTAP,RBM15,YTHDF2,HNRNPC were significantly associated with OS(Overall survival)time with GC patients.The average survival time of GC patients in the METTL3 low expression group(33.6 months)was significantly longer than that in the high expression group(19.8 months)(HR 1.81 95% CI: 1.53-2.15 P<0.01).The results of ROC curve analysis showed that METTL3(AUC=0.796,P<0.01)had diagnostic value for TCGA GC patients.The results of qRT-PCR showed that the expression of METTL3 was significantly up-regulated in 40 pairs of GC tissues(Fold Change=3.967,P<0.01).The ROC curve analysis showed that METTL3 had better diagnostic efficacy for GC patients(AUC=0.889,P <0.001).The expression levels of METTL3 in 7 GC tissue sequencing datasets(GSE54129,GSE79973,GSE64951,GSE13911,GSE19826,GSE33335,GSE63029)in the GEO database were analyzed,and the results confirmed that METTL3 was significantly up-regulated in GC(P<0.01).Bioinformatics analysis indicated that METTL3 was significantly associated with biological processes including epithelial-mesenchymal transition(EMT),apoptosis(Apoptosis)and early estrogen response(Early Estrogen Response).Section 2 Expression verification of GC Population in high incidence AreaA case-control study was used to collect serum samples of 200 pairs of GC patients with different advanced stages and healthy control in the western GC high-incidence area.Real-time q RT-PCR was used to detect the expression level of METTL3 in the serum samples.We analyzed the association between serum METTL3 level and clinical features(including Age,gender,tumor size,TNM clinical stage,histological type,tumor differentiation grade and Lauren classification)to explore its potential value its potential value as an effective biomarker for GC patients.The results of q RT-PCR showed that the serum METTL3 level of GC patients(ΔCt: 1.601±3.306)was higher than that of healthy controls(ΔCt: 2.138±3.192),while without statistic significant(P>0.05).The clinical characteristics analysis suggested that GC serum METTL3 expression is significantly associated with age,TNM stage,T stage,lymph node metastasis status and tumor grade.The serum METTL3 expression was significantly increased in GC patients over 65 years old(P<0.05);Compared to TNM stage I GC patients,serum METTL3 expression level was significantly increased in stage II,III and IV GC patients(P<0.05);Compared with T1-stage GC patients,the serum METTL3 expression level in T4-stage GC patients was significantly increased(P<0.05);Compared with gastric cancer patients without lymph node metastasis,the serum METTL3 expression level was significantly increased in GC patients with lymph node metastasis(P<0.05);Compared with G1 stage GC patients,serum METTL3 expression levels in G2 and G3 stage GC patients were significantly increased(P<0.05).The results of the serum METTL3 in GC patients were highly consistent with sequencing data from TCGA and GEO analysis,which indicated the potential value of METTL3 as biomarker for GC.Chapter III The function and role of METTL3 in MNNG-induced GC Section 1 Expression variation of METTL3 in the process of MNNG-induced GCHuman normal gastric epithelial cells GES-1 were treated with 5μM MNNG for 40 passages to establish MNNG-induced malignant transformation cell model MC-cells.The cell malignant phenotype of the MC-cell was evaluated by plate and soft agar colony formation assay,wound healing assay,and nude mice tumorigenesis assay.q RTPCR and Western blot were used to detect the expression variation of METTL3 in different generations of MC-cell(MC-20,MC-30,MC-40)and two different type of GC cell(HGC-27、AGS).Immunohistochemistry(IHC)technique was used to detect the expression of METTL3 protein and EMT marker protein in tumor tissue of MC cells and GC cells of nude mice.m6 A detection kit was used to measure the total RNA m6 A modification level in in different generations of MC-cell and GC cell.Long-term MNNG exposure model of Wistar rat was established,and gastric lesions of rats induced by MNNG exposure under different treatment conditions were observed.HE staining was used to observe the pathological changes of rat foregut and glandular stomach,and the expression levels of METTL3 and EMT marker proteins in gastric tissue were detected.The results of the cell malignant phenotype evaluation experiment showed that compared with the control normal GES-1 cells,the morphological changes of the malignantly transformed cells in the 40 th passage(MC-40)were significantly changed,and the plate colony formation rate was significantly increased(P<0.05).The nude mouse tumorigenic results showed that tumors could be obtained 21 days after mc-40 cell vaccination.The pathological analysis showed tumor cells in the tumor tissue,suggesting that MC-40 cells have characteristics similar to GC cells.The IHC showed that METTL3 protein and EMT marker proteins N-cadherin(N-cadherin),vimentin (Vimentin),α-smooth muscle moieties Protein(α-SMA),and Snail were positively expressed in MC-40 cells and GC cells in nude mice tumor tissues.The results of q RTPCR and Western blot showed that during the malignant transformation of GES-1 cells induced by MNNG,the METTL3 expression level was progressively up-regulated(P<0.05),and E-cadherin protein was gradually decreased.The expression levels of Ncadherin,Vimentin,and α-SMA proteins gradually increased,and the total RNA m6 A level was significantly increased(P<0.05).The results of long-term exposure to MNNG in rats showed that the gastric mucosal epithelium of the rats exposed to high concentrations of MNNG was uneven,with a small amount of blood vessel congestion,and some gastric mucosal tissue showed obvious neutrophil infiltration.Section 2 Function and role of METTL3 in MNNG malignantly transformed cells and GC cellsSh RNA-lentivirus was used to down-regulate METTL3 expression in 40 passages of MNNG malignantly transformed cells(MC-40)and GC cells(HGC-27,AGS).CCK-8 assay,wound healing assay and Trans Well invasion and migration assay were used to evaluate the effect of down-regulation of METTL3 on biological functions such as cell proliferation,migration,and invasion.The effects of down-regulation of METTL3 on tumorigenesis and EMT marker protein expression in nude mice were investigated by using Western blot,and immunohistochemistry in vitro and in vivo.The results of CCK8 assay showed that down-regulation of METTL3 significantly inhibited the proliferation of MC-40 cells and GC cells(AGS,HGC-27)(P<0.05);Wound healing assay and Trans Well migration assay re-verified that down-regulation of METTL3 significantly inhibited the migration of MC-40 and GC cells(AGS,HGC-27)(P<0.05);The results of the Trans Well invasion assay showed that down-regulation of METTL3 significantly inhibited the invasion ability of MC-40 and GC cells(AGS,HGC-27)(P<0.05).Malignant phenotype evaluation results showed that downregulation of METTL3 significantly inhibited the colony formation in the plate and soft agar of MC-40 and GC cell(AGS).The nude mice showed that down-regulation of METTL3 significantly affected the tumorigenesis of GC cells(HGC-27,AGS).Western blot results confirmed that down-regulation of METTL3 significantly affected the expression of EMT marker proteins N-cadherin,Vimentin,snail,and α-SMA in MC-40 and GC cells(AGS,HGC-27),indicating that METTL3 was closely related to cellular EMT processes.Chapter IV The mechanism of METTL3 regulating mi RNA in the process of MNNG-induced GC through m6 A modificationSection 1 Screening of key mi RNAs in GC mediated by METTL3 through m6 A modificationThe RNA methylation co-immunoprecipitation(Me-RIP)combined with highthroughput sequencing technology was used to detect the differences in the distribution of m6 A modifications caused by down-regulation of METTL3 in MC-40 and GC cells(HGC-27).Bioinformatics analysis were used to analyze the biological process of down-regulation of METTL3 in MNNG-induced GC.The mi RNA expression profile was constructed by taking the intersection of the sequencing results,and systematically screened the key mi RNAs that METTL3 regulates downstream through m6 A modification.The expression levels of key mi RNAs were verified by q RT-PCR in different generations of MNNG malignant transformed cells,GC cells,GC tissue samples,down-regulated METTL3 MC-40 and GC cells(AGS,HGC-27).Me-RIP-seq results of down-regulated METTL3 MC-40 cell showed that m6 A modification levels of 616 m RNAs were significantly up-regulated,and m6 A modification levels of 1464 m RNAs were significantly down-regulated(FC>2,P<0.001).The main involved molecular functions(MF)mainly include protein/enzyme binding,ATP binding,and adenyl ribonucleotide binding.The annotations analysis of down-regulated METTL3 MC-40 cell showed that m6 A modification levels of 107 premi RNAs were significantly changed(FC>2,P<0.001),including 92 pre-mi RNAs were significantly downregulated,and 15 pre-mi RNAs were significantly upregulated.MeRIP-seq results of down-regulated METTL3 HGC-27 cell showed that m6 A modification levels of 1722 m RNAs were significantly up-regulated,and m6 A modification levels of 2196 m RNAs were significantly down-regulated(FC>2,P<0.001).The main involved molecular functions(MF)mainly include ion binding,carbohydrate derivative binding,and GTPase binding.The annotations analysis ofdown-regulated METTL3 HGC-27 cell showed that m6 A modification levels of 159 pre-mi RNAs were significantly changed(FC>2,P<0.001),including 96 pre-mi RNAs were significantly downregulated,and 63 pre-mi RNAs were significantly upregulated.The results of q RT-PCR verification showed that the expressions of mi R-1184 and mi R-1825 gradually increased during the malignant transformation of GES-1 cells induced by MNNG(P<0.05).The expression of mi R-1184 in MC-40 was significantly upregulated by 5.39 times(P<0.05),and the expression of mi R-1825 was significantly upregulated by 3.69 times(P<0.05).In GC AGS cells,the expression of mi R-1184 was significantly up-regulated by 5.76 times(P<0.05),and the expression of mi R-1825 was significantly up-regulated by 3.77 times(P<0.05).However,down-regulation of METTL3 significantly inhibited the expression of mi R-1184 and mi R-1825 in MC-40 and GC AGS cells.Combined with relevant literature and bioinformatics analysis,mi R-1184 and mi R-1825 were finally identified as candidate key mi RNAs regulated by METTL3.Section 2 The function and mechanism of METTL3 regulating key mi RNAs in GC induced by MNNGq RT-PCR was used to validate target pre-mi RNA and pri-mi RNA expression level(mi R-1184,mi R-1825)to identify the target mi RNAs regulated by METTL3 through m6 A modification ultimately.The target mi RNA was overexpressed in down-regulated METTL3 MC-40 and GC cells(AGS-sh METTL3,HGC-27-sh METTL3)using mimic,and the transfection efficiency was detected by q RT-PCR.The CCK8 assay and Trans Well migration and invasion assay were used to evaluate the recovery effect of overexpression of target mi RNA on the biological functions such as cell proliferation,migration and invasion.m6A-IP-q PCR was used to measure the effect of downregulation of METTL3 on the binding of target mi RNA primary pri-mi RNA to m6 Aspecific antibody in MC-40 and GC cell(HGC-27).Through bioinformatics analysis combined with Me-RIP-seq m RNA sequencing results we screen downstream protein regulated by target mi RNA.The effect of overexpression of target mi RNA on the expression levels of downstream target protein and EMT marker proteins in downregulated METTL3 MC-40 and HGC-27 cell was detected by Western blot assay.The results of q RT-PCR verification showed that the expression level of pre-mi R-1184 was significantly down-regulated by 4.43 times(P<0.01),and the expression level of pri-mi R-1184 was down-regulated by 1.31 times in down-regulated METTL3 MC-40 cell.The expression level of pre-mi R-1184 was significantly down-regulated by 2.90 times(P<0.05),and the expression level of pre-mi R-1184 was up-regulated by 1.19 times in down-regulated METTL3 AGS cell.The expression level of pre-mi R-1184 decreased,and the expression level of pri-mi R-1184 was significantly increased by 2.21 times(P<0.05)in down-regulated METTL3 HGC-27 cell.Based on the literature and experimental verification results,mi R-1184 was identified as the target mi RNA for subsequent functional mechanism research.The results of cell function experiments showed that overexpression of mi R-1184 significantly enhanced cell proliferation,migration,and invasion(P<0.05),also overexpression of mi R-1184 partially reversed cell proliferation,migration,and invasion affected by downregulated METTL3.The results of m6A-IP-q PCR showed that the level of pri-mi R-1184,which specifically binds to m6 A antibody,decreased in down-regulated METTL3 MC-40 cell,but there was no statistical difference(P>0.05).The level of pri-mi R-1184,which specifically binds to m6 A antibody,was significantly decreased in down-regulated METTL3 HGC-27 cell(P<0.05).By comparing the sequencing results of Me-RIP-seq m RNA,combined with bioinformatics prediction,it was further screened that METTL3 regulates the downstream target gene TRPM2 mediated by mi R-1184 through m6 A modification.Western blot results showed that the expression of TRPM2 protein was up-regulated,the expression of JNK protein was up-regulated,and the expression of EMT-related marker protein,Vimentin and α-SMA was increased after mi R-1184 was overexpressed in down-regulated METTL3 MC-40 and HGC-27 cells.Overexpression of mi R-1184 could partially reverse the effects of METTL3 on the expression levels of TRPM2,JNK protein,EMT marker protein Vimentin and α-SMA protein.Conclusion1.Four N-nitrosamines were mainly detected in drinking water in the western GC high-incidence area and control area.The average detected concentrations of NDPA,NDph A,NMEA and total N-nitrosamines in areas with high incidence of GC were significantly higher than those in control areas.9 kinds of N-nitrosamines were detected in the urine samples of GC patients and healthy controls,and the average detected contents of NMor,NPyr,NDPA,NDph A and total N-nitrosamines in GC patients were significantly higher than those in the urine samples of healthy controls.Dietary intake is one of the important reasons for the high exposure of local residents to N-nitrosamines,and the long-term high-level exposure of Nnitrosamines may be an important factor causing the high incidence of GC in the local area.2.Studies shown that the expression of m6 A methyltransferase METTL3 is significantly up-regulated in GC tissues.The expression of METTL3 in GC tissue is significantly associated with the clinical diagnosis,progression,and prognosis of GC patients.METTL3 is significantly up-regulated in patients with advanced GC.The serum METTL3 expression level of GC patients was higher than that of healthy controls,and the serum METTL3 expression level of GC patients was closely related to the malignant progression of GC.The expression of m6 A methyltransferase METTL3 gradually increased with the malignant progression of GC patients.METTL3 has potential value as an effective biomarker for clinical progression of GC patients.3.For the first time,we found that the m6 A methyltransferase METTL3 is closely related to the progression of GC induced by MNNG exposure.METTL3 is a key gene in the malignant transformation of gastric mucosal epithelial cell line GES-1 induced by MNNG.METTL3 is progressively up-regulated in the process of malignant transformation induced by MNNG,and down-regulation of METTL3 significantly inhibits the biological functions and malignant phenotypes of MNNG malignantly transformed cells and GC cells such as proliferation,migration,invasion.Epithelial-mesenchymal transition(EMT)is a key biological process in MNNG-induced GC.4.Through Me-RIP high-throughput sequencing,we observed for the first time that the m6 A modification levels of 92 pre-mi RNAs were significantly down-regulated in MNNG malignantly transformed cells,and the m6 A modification levels of 15 pre-mi RNAs were significantly up-regulated.The m6 A modification levels of 96 pre-mi RNAs were significantly down-regulated,and the m6 A modification levels of 63 pre-mi RNAs were significantly up-regulated in GC cell.The expression levels of 11 key mi RNAs potentially regulated by METTL3 were significantly altered.Our findings reveal for the first time that mi R-1184 is a key downstream mi RNA regulated by METTL3 through m6 A modification.In the process of MNNGinduced malignant transformation,METTL3 regulates the processing and maturation of mi R-1184 by affecting the level of m6 A modification at the methylation site of pri-mi R1184,thereby mediating protein expression of the downstream target gene TRPM2,and promoting the process of epithelialmesenchymal transformation(EMT).