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Bioinformatics identification and validation of m6A/m1A/m5C/m7G/ac4 C-modified genes in oral squamous cell carcinoma
Cheng-Hui Lu1,2, Xue-Lai Yin3, Zhuo-Deng Huang4,5
1Department of Stomatology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Core Unit of National Clinical Research Center for Oral Diseases, Shanghai, 200092, China.
Background:
RNA modifications, including m6A, m1A, m5C, m7G, and ac4C, may play a role in the occurrence and development of cancer, such as proliferation. However, the effects of RNA modification-related genes (RRGs) in the development of oral squamous cell carcinoma (OSCC) have not been fully elucidated. The present study aimed to evaluate the effects and mechanisms of RRGs on OSCC development progression.
Methods:
RNA-seq transcriptome data, along with clinical and prognostic information, were extracted for 328 patients with OSCC from the TCGA database. A total of 49 RRGs were analyzed for differential expression. We then performed Lasso analysis, as well as univariate and multivariate Cox regression analyses, followed by Kaplan-Meier survival analysis to identify relevant prognostic genes and establish a risk-prognosis model. Patients were categorized into high-risk and low-risk groups, and gene set enrichment analysis (GSEA) was conducted to analyze differences in gene signatures between these two groups, using data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. RT-PCR was employed to validate the expression levels of differentially expressed genes in OSCC samples. The four most significantly differentially expressed genes were selected for further functional analysis, and small interfering RNA (siRNA) vectors targeting these genes were transfected into OSCC CAL27 cells. The Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Additionally, a subcutaneous CAL27 xenograft model transfected with short hairpin RNA (shRNA), combined with Ki-67 immunohistochemical (IHC) staining and TUNEL assay, was used to investigate their underlying molecular mechanisms in vivo.
Results:
Among the 49 RRGs, four genes (IGF2BP2, HNRNPC, NAT10, and TRMT61B) were found to be associated with the development of OSCC. Based on various methodological validations, a risk score model was constructed using these four genes. The high-risk and low-risk groups of OSCC patients exhibited significantly different survival outcomes and clinicopathological characteristics. Patients in the low-risk group had longer overall survival (OS) and lower mortality rates compared to those in the high-risk group. The nomogram and decision curve analysis (DCA) demonstrated that our risk model accurately and reliably predicted the impact of risk factors on OS at 1-, 3-, and 5-year. Additionally, risk scores correlated with the infiltration of several immune cells, particularly CD8+ T cells and B cells, which showed significant negative correlations. Furthermore, the results of the CCK-8 assay indicated that inhibition of NAT10 and IGF2BP2 expression using siRNA inhibited the proliferation of OSCC cell lines in vitro. Meanwhile, inhibition of NAT10 and IGF2BP2 expression using shRNA influenced proliferation of tumorigenicity in vivo.
Conclusion:
In this study, we established a risk model and nomogram based on four RRGs, which can be used for risk stratification and predicting survival outcomes in patients with OSCC. This provides a reliable reference for individualized therapy in OSCC patients.
Insights
This study identifies four RNA modification-related genes (RRGs) that predict oral squamous cell carcinoma (OSCC) patient outcomes. A novel risk model using these RRGs aids in stratifying patients and guiding personalized OSCC treatment strategies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- RNA modifications are implicated in cancer development, including proliferation.
- The specific roles of RNA modification-related genes (RRGs) in oral squamous cell carcinoma (OSCC) remain unclear.
- This study investigates the impact of RRGs on OSCC progression.
Purpose of the Study:
- To identify key RRGs involved in OSCC development.
- To establish a prognostic risk model for OSCC patients based on RRGs.
- To explore the underlying mechanisms of RRGs in OSCC progression.
Main Methods:
- Utilized TCGA RNA-seq data from 328 OSCC patients.
- Performed differential expression analysis, Lasso, and Cox regression to identify prognostic RRGs.
- Constructed a risk model, validated through Kaplan-Meier survival analysis, GSEA, RT-PCR, in vitro (siRNA, CCK-8), and in vivo (shRNA, IHC, TUNEL) assays.
Main Results:
- Identified four RRGs (IGF2BP2, HNRNPC, NAT10, TRMT61B) associated with OSCC.
- Developed a risk score model demonstrating significant differences in survival and clinicopathological characteristics between high-risk and low-risk groups.
- Risk scores correlated negatively with CD8+ T cell and B cell infiltration; inhibition of NAT10 and IGF2BP2 suppressed OSCC cell proliferation in vitro and in vivo.
Conclusions:
- A risk model and nomogram based on four RRGs effectively stratify OSCC patients and predict survival outcomes.
- This provides a valuable tool for personalized therapy in OSCC.
- The identified RRGs offer potential therapeutic targets for OSCC treatment.
