| Gastric cancer is one of the malignant tumors that pose a serious threat to human health and quality of life.For patients with early gastric cancer,surgical resection is the best treatment option.However,the process of gastric cancer is very insidious,and the early clinical symptoms are not typical.Most patients have local or distant metastasis at the time of diagnosis and are already in the progressive stage of gastric cancer,losing the opportunity for radical surgical treatment.In addition,tumor recurrence after surgery is also one of the reasons for the high mortality rate of gastric cancer.Despite the developments and advances in neoadjuvant chemotherapy,targeted therapy and immunotherapy,the various treatment options for gastric cancer currently have limitations,so it is critical to further search for potential prognostic and precise treatment markers for gastric cancer.RNA methylation is a common epigenetic modification whose dysregulation is closely associated with the development of human cancers.RNA 5-methylcytosine(m5C)methylation refers to the modification of the fifth C of RNA cytosine by methylation and is one of the major post-transcriptional modifications of RNA.With the advancement of high-throughput sequencing technology,the distribution and biological functions of RNA m5 C methylation modifications on m RNAs and non-coding RNAs are gradually being discovered,which play important roles in regulating RNA translation,stability,exiting the nucleus,and a variety of biological processes.Long noncoding RNAs(lnc RNAs)are non-coding RNAs greater than 200 nucleotides in length and do not function as coding proteins due to the lack of meaningful open reading frames.However,lnc RNAs can regulate gene expression through various pathways such as epigenetic,transcriptional and post-transcriptional modifications,playing an important regulatory role in biological processes such as proliferation,invasion and apoptosis of tumor cells,and they can be involved in tumor development as oncogenic or oncogenic factors.The tumor immune microenvironment(TIME)is particularly important in mediating pro-and anti-tumor immune responses.lnc RNAs can influence tumorigenesis and development by regulating biological processes such as differentiation,proliferation,and secretion factors of immune cells in the TIME.lnc RNAs are expected to be novel tumor markers and therapeutic targets for tumor diagnosis,treatment,and prognosis monitoring.In this study,we propose to use information from The Cancer Genome Atlas(TCGA)database to screen m5C-related lnc RNAs with prognostic value in gastric cancer and construct risk models by bioinformatics methods.The association of risk score with immune cell infiltration,immune cell function,immune checkpoint gene expression,and chemotherapeutic drug sensitivity was assessed.The expression of m5C-related lnc RNAs in patients’ gastric cancer tissues and paracancerous tissues was detected by quantitative real-time PCR(q RT-PCR),and the association of these lnc RNAs with patients’ clinicopathological characteristics and the prognosis was systematically assessed.The specific study was divided into three parts as follows.Part one Construction and validation of the prognostic risk model of m5C-related lnc RNAs in gastric cancerObjective: To screen prognostic m5C-related lnc RNAs biomarkers for gastric cancer,construct a prognostic risk model and clinical prognostic nomogram.Methods: Clinical information and RNA-sequencing data of normal samples and stomach adenocarcinoma patients were downloaded from the TCGA database.The differential expression of m5 C methylation-regulated genes in gastric cancer and normal samples were analyzed.Co-expression relationships between m5 C methylation regulatory genes and lnc RNAs were constructed by Pearson correlation analysis to identify m5C-related lnc RNAs in gastric cancer.Prognostic m5C-related lnc RNAs signature(m5C-RLSig)was determined by univariate Cox regression analysis,Kaplan-Meier analysis and multifactorial Cox regression analysis,based on which a prognostic risk model was constructed.Kaplan-Meier method were used to compare the overall survival of different groups of patients.Univariate and multifactorial Cox regression analyses were performed to verify the independence of the model by combining clinicopathological information and patients’ risk scores.The clinical prognostic nomogram was constructed,and calibration curves were used to compare the fit between the survival predicted by the nomogram and the actual survival.Results: Eleven of the 13 m5 C methylation-regulated genes were highly expressed in gastric cancer tissues compared with normal controls.By Pearson correlation analysis,565 m5C-related lnc RNAs were obtained in gastric cancer,from which six prognostic m5C-related lnc RNAs required for the construction of risk models were identified.The risk model constructed based on these could effectively predict the prognosis of gastric cancer patients,and the overall survival was lower and the survival status was worse in the high-risk group.The m5C-RLSig risk score was an independent risk factor for prognosis in patients with gastric cancer.Risk score was significantly correlated with clinical stage,pathological grade,N stage and survival status,and patients with high risk score were more likely to have advanced clinicopathological features.A prognostic nomogram constructed using the m5C-RLSig risk score and clinicopathological factors could accurately predict the 1-,3-,and 5-year survival probabilities of patients with gastric cancer.Conclusion:1.The risk model constructed based on HAGLR,AC009948.1,AC005586.1,AL590666.2,AP001271.1,and IPO5P1,which are six m5C-related lnc RNAs,can effectively predict the prognosis of gastric cancer patients.2.The nomogram constructed using the m5C-RLSig risk score can be used as a valid quantitative tool to predict the prognosis of gastric cancer patients.Patr two Immune infiltration characteristics and therapeutic value of m5C-related lnc RNAs in gastric cancerObjective: To explore the biological functions and potential signaling pathways involved in m5C-related lnc RNAs,and to assess the immune infiltration characteristics and individualized therapeutic value of m5C-RLSig in gastric cancer.Methods: Gene set enrichment analysis(GSEA)was used to perform Gene Ontology(GO)functional annotation and to explore the Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway in which m5C-related lnc RNAs are involved.The expression matrix of immune cells was analyzed and the percentage of immune cell infiltration was quantified using the CIBERSORT algorithm(Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts,CIBERSORT).The association of risk scores with immune cells and immune function was assessed using single-sample gene set enrichment analysis(ss GSEA).Immune checkpoint gene expression levels and chemotherapeutic drug sensitivity were analyzed in the high-and low-risk groups to assess the therapeutic value of m5C-RLSig.Results: GO enrichment analysis showed that immunoglobulin binding,collagen trimer and other biological functions were enriched in the high-risk group.KEGG enrichment analysis showed that glycosphingolipid biosynthesis-ganglio series,neuroactive ligand-receptor interaction and other pathways were enriched in the high-risk group.There was a difference in the proportion of immune cell infiltration between the high-risk and low-risk groups of gastric cancer,with an increased proportion of NK cells activated,dendritic cells resting and mast cells resting infiltrating in the high-risk group and a decreased proportion of NK cells resting.The ss GSEA results revealed that the enrichment scores of multiple immune cell(a DCs,B cells,DCs,i DCs,macrophages,mast cells,neutrophils,p DCs,T helper cells,TIL,Tregs)were elevated in the high-risk group.Furthermore,immune function scores such as APC co-stimulation,CCR,HLA,parainflammation,T cell co-stimulation,Type I IFN response,and Type II IFN response were also significantly elevated in the high-risk group.The immune checkpoint genes TNFRSF14 and TNFRSF25 were highly expressed in the low-risk group.The sensitivity of the low-risk group to the chemotherapeutic drugs cisplatin and paclitaxel was higher than that of the high-risk group.Conclusion:1.Biological functions and signaling pathways such as immunoglobulin binding,collagen trimer,and glycosphingolipid biosynthesis-ganglio series were enriched in the high-risk group.2.m5C-RLSig risk score can distinguish the immune infiltration characteristics of gastric cancer.3.The immune checkpoint genes TNFRSF14 and TNFRSF25 were highly expressed in the low-risk group,and the low-risk group was more sensitive to the chemotherapeutic drugs cisplatin and paclitaxel.m5C-RLSig could be a biomarker for immunotherapy and chemotherapy.Part three Expression and clinical significance of m5C-related lnc RNAs in gastric cancer tissuesObjective: The expression levels of the six m5C-related lnc RNAs obtained from the first part of the study were detected in clinical tissue specimens,and the relationships between the expression levels of these lnc RNAs and the clinicopathological factors and the prognosis of gastric cancer patients were analyzed.Methods: Specimens of gastric cancer tissues and paracancerous tissues from patients were collected,and the expression levels of HAGLR,AC009948.1,AC005586.1,AL590666.2,AP001271.1,and IPO5P1 were detected by q RT-PCR.The correlation between the expression levels of the above lnc RNAs in gastric cancer tissues and the clinicopathological characteristics of patients(gender,age,tumor size,degree of pathological differentiation,vascular infiltration,infiltration depth,lymphatic metastasis,TNM stage)was analyzed using chi-square test.Kaplan-Meier method and Log-rank test were used to analyze the relationship between the expression levels of lnc RNAs and the prognosis of gastric cancer patients.Results: A total of 52 gastric cancer patients were included,and the expression of HAGLR and AC009948.1 was upregulated in gastric cancer tissues and the expression of AC005586.1,AL590666.2,AP001271.1 and IPO5P1 was downregulated in gastric cancer tissues compared with paracancerous tissues.HAGLR expression correlated with lymph node metastasis,TNM stage;AC009948.1 expression correlated with tumor size,lymph node metastasis,depth of infiltration;AC005586.1 expression correlated with tumor size,lymph node metastasis,TNM stage;AL590666.2expression correlated with lymph node metastasis,depth of infiltration,TNM stage;AP001271.1 expression correlated with expression correlated with the degree of pathological differentiation and lymph node metastasis;IPO5P1expression correlated with the degree of pathological differentiation,vascular infiltration and lymph node metastasis.Overall survival was lower in patients with high expression of HAGLR,AC009948.1 and lower in patients with low expression of AC005586.1,AL590666.2,AP001271.1,and IPO5P1.Conclusion:1.The expression of HAGLR and AC009948.1 was upregulated in gastric cancer tissues,and the overall survival was lower in patients with high expression.2.AC005586.1,AL590666.2,AP001271.1,and IPO5P1 were expressed down-regulated in gastric cancer tissues,and the overall survival was lower in patients with their low expression.m5C-RLSig is expected to be a potential biomarker for the prognostic assessment of gastric cancer. |
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