| Objective:Malignant tumor metastasis is the main cause of death in patients with advanced tumors,and it is also a problem that cannot be completely solved at present.In the past ten years or more,with the development of next-generation sequencing technology,the metastasis mechanism at the cancer genome level has been studied in depth.However,with the deepening of research,it is found that a considerable part of cancer metastasis cannot be explained by existing genetic-level mechanisms,which has prompted people to try to clarify the mechanism of cancer metastasis from a new perspective such as epigenetics.In recent years,epigenetics,represented by non-coding RNA,chromatin recombination,and DNA methylation,has provided new ideas for the study of cancer metastasis mechanisms.Some of these recent studies have shown that,at the level of post-transcriptional regulation,the role of RNA methylation and its function in the genesis and development of cancer cannot be ignored.However,the current research on the role of RNA methylation in cancer is just a tip of the iceberg,and the research on metastasis in particular still needs further exploration.m6A methylation modification(N6-methyladenosine)is one of the most widely distributed RNA methylation modifications.It is a reversible RNA modification and is simultaneously regulated by m6A methyltransferase and m6A demethylase.The m6A methyltransferase mediates the formation of m6A methylation modifications,and the m6A demethylase can selectively remove m6A modifications and binding proteins can mediate the effect of m6A.Current studies have shown that m6A is involved in multiple biological processes,such as stem cell differentiation and pluripotency,circadian rhythm,embryogenesis,and DNA damage response.Therefore,the possible role of m6A modification and m6A modulators in various diseases,especially malignant tumors,has drawn great attention.Recent studies have shown that abnormal expression of m6A modulators can affect cancer development by altering the level of m6A modification in RNA.However,the abnormal levels of m6A modulators play a complex and variable role in various types of cancer.They may play a role of promotion and suppression in different types of cancer by regulating different specific target genes.However,this effect has not been systematically analyzed in most tumors,especially in two cancers that are more easily metastatic,triple negative breast cancer and gastric cancer.Breast cancer is one of the most common malignant tumors in women.Its incidence is the highest among female malignancies,and it has been increasing in recent years.In breast cancer-related deaths,most patients do not die from primary breast cancer but from distant metastases.In the four types of luminal molecular typing of breast cancer,triple negative breast cancer has a poor prognosis and is prone to early distant metastases Therefore,an in-depth discussion of the metastatic mechanism of triple negative breast cancer is of great significance for improving the long-term survival and quality of life of patients with triple negative breast cancer.Gastric cancer is one of the most common types of malignant tumors in China.The incidence of gastric cancer is the second highest among malignant tumors,and the number of deaths ranks third among all cancers.Among gastric cancer-related deaths,most patients die from distant metastases.However,the early detection rate of gastric cancer in China is low.A large proportion of patients are already in the advanced stage when diagnosed.Therefore,the metastasis of gastric cancer is a great threat to people's life and health.Therefore,an in-depth study of the metastasis mechanism of gastric cancer is of great significance to improve the long-term survival and quality of life of gastric cancer patients.This study explored the differences in the roles of the five major m6A modulators in overall breast cancer and triple negative breast cancer,and explored the mechanism of action.All m6A modulators currently found in gastric cancer were included in the study,and a model for predicting prognosis was established.The different effects of the same modulators in gastric cancer and triple negative breast cancer were compared.This provides the basis for future treatment and prediction.Methods:1.The KM-plotter online database was used to analyze the association between the distant-free metastatic survival of breast cancer patients and the expression of m6A modulators(METTL3,METTL14,WTAP,FTO and ALKBH5)and COL3A1.The TCGA database and GTEx database were obtained from the GEPIA website for the transcription levels of the above five m6A modulators in breast cancer tissue and normal breast tissue.The TCGA database was used to analyze the correlation between m6A modulators and target genes.The GSE62254 data set from the GEO database was selected to analyze the correlation between m6A modulators and pathological parameters and prognosis of gastric cancer,and the survival curve was verified using GSE15459.2.The qRT-PCR assay was used to detect the mRNA expression levels of METTL3 and COL3A1.3.The protein expression levels of METTL3,ALKBH5,and COL3A1 were detected by Western blot.4.ELISA assay was used to detect the supernatant and intracellular expression levels of collagen ?1(?).5.Transwell assay and cell adhesion assay were used to detect the cell's metastatic ability.6.The RNA methylation co-immunoprecipitation assay was used to detect the m6A modification level of COL3A1.7.The immunohistochemistry was used to detect METTL3 and COL3A1 levels in tissue microarray sections.8.Statistical processing:Statistical analysis was performed using SPSS(version 16.0)and R(V.3.2.5).The Limma package was used to analyze differential genes.The univariate COX regression and lasso regression were used to analyze the relationship between 26 m6A modulators and gastric cancer OS in the GSE62254 dataset.Chi-square test was used to explore the relationship between riskscore and clinical pathological parameters of gastric cancer.The relationship between gastric cancer OS was analyzed by multivariate COX regression,and Kaplan-Meier survival curves of riskscore were drawn in the GSE62254 data set and test set GSE15459.A prognostic analysis model of riskscore's nomogram was constructed,and a calibration test was performed.One-way ANOVA and Student's t-test were used to determine statistical significance,and statistical significance was determined as a P value of less than 0.05.All experimental data were obtained from 3 independent experimental results,expressed as mean ± standard deviation.Results:1.METTL3 specifically inhibited triple negative breast cancer metastasis.The online database was used to analyze the prognostic effects of five major m6A modulators in triple negative breast cancer.It was found that the low expression of METTL3 was related to the poor prognosis of triple negative breast cancer.The expression of METTL3 in triple negative breast cancer is lower than that in non-triple negative breast cancer tissues.The results of cell experiments proved that the migration,invasion,and adhesion of triple negative breast cancer cells were enhanced after METTL3 knockdown.2.METTL3 decreased the expression level of COL3A1 by increasing the m6A modification level of COL3A1.The silence of METTL3 can reduce the m6A level of COL3A1,and thus up-regulate the expression of collagen type? alpha 1 chain(COL3A1).At the same time,the m6A inhibitor cycloleucine can rescue the decrease of COL3A1 expression and the decrease of metastatic ability caused by METTL3 overexpression.The correlation was confirmed by immunohistochemical results.3.COL3A1 promotes metastatic ability of triple negative breast cancer cells.After knocking down COL3A1,the migration,invasion,and adhesion of triple negative breast cancer cells were reduced.4.There are twelve m6A modulators that make sense in the prognosis of breast cancer for gastric cancer,and riskscore was constructed based on them.Twenty-six m6A-related regulators were included in the study,and the relationship between them and the prognosis of gastric cancer was analyzed using the dataset in the GEO database.The results of the COX regression and lasso regression analysis suggest that the 12 modulators are meaningful in both analyses,and a riskscore is constructed based on them.5.The high score of riskscore is associated with poor prognosis of gastric cancer.Analysis of the relationship between riskscore and the cl ini copath ol ogi cal parameters and prognosis of gastric cancer suggests that a high riskscore score is associated with a higher TNM stage and is more prone to peritoneal metastasis.6.A collinear chart prognostic prediction model was constructed to predict 3-year survival probability.According to the results of riskscore regression analysis,a collinear chart of gastric cancer prognosis prediction model was constructed and adjusted,which showed that the 3-year survival probability prediction of gastric cancer patients was more accurate.7.ALKBH5 promotes the metastatic ability of gastric cancer metastatic cells.In Lasso regression analysis,the coefficient of ALKBH5 was the highest,and knockdown of ALKBH5 weakened the metastasis ability of gastric cancer cells.8.The possible target genes of m6A modulators in gastric cancer was analyzed and predicted.The patients were divided into two groups according to the riskcore,and the differential genes in the two groups were analyzed.The differential genes were selected according to P<0.05,log FC>1.Differential genes were enriched,and enrichment projects with P<0.05 and FDR<0.25 were selected,and many of them were related to extracellular matrix and cell adhesion.These genes may be target genes for m6A regulators to participate in regulating transfer.Conclusions:1.Methyltransferase METTL3 down-regulates the expression of COL3A1 by increasing the m6A level of COL3A1,thereby specifically inhibiting the metastasis of triple negative breast cancer2.Riskscore of m6A modulator is associated with poor prognosis of gastric cancer.A prognostic prediction model for riskscore was constructed through data analysis,which accurately predicted the 3-year survival rate of gastric cancer.3.Demethylase ALKBH5 can promote the metastasis of gastric cancer.4.The potential target genes of m6A modulators were predicted,which provided potential therapeutic targets for the treatment of gastric cancer. |
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