| Background:In recent years,the diagnosis and treatment of oral squamous cell carcinoma(OSCC)has gradually improved,however,the 5-year survival rate of OSCC patients was still less than 60%.Studies have shown that the early diagnosis and prompt treatment can improve the prognosis of patients with OSCC.Micro RNA(miRNA),a non-coding RNA with a length of about 20-24 nucleotides,has high sensitivity and stability in the early diagnosis of OSCC.In addition,some miRNAs were related to the survival time,lymph node metastasis,and distant metastasis of OSCC patients,which may have certain value in the prognosis prediction of OSCC.Screening and identification of diagnostic and prognostic miRNA biomarkers for OSCC is one of the hot spots in oral cancer research nowadays.In previous studies,screening miRNAs has typically been performed using differential expression analysis.However,differential expression analysis has certain limitations.Only differentially expressed miRNAs can be screened,but miRNAs related to clinical traits cannot be screened using differential expression analysis.Different from differential expression analysis,weighted gene co-expression network analysis(WGCNA)method can construct miRNA co-expression modules,analyze the correlation between the modules and OSCC clinical characteristics,and then screen out miRNAs related to OSCC clinical characteristics.In this study,based on the WGCNA analysis method,we screened out the hub miRNAs associated with the occurrence of OSCC,namely miR-30e-5p.Although the research showed that miR-30e-5p acted as a tumor suppressor in liver cancer and colon cancer,the role and mechanism of miR-30e-5p in OSCC were unclear.The value of miR-30e-5p in OSCC diagnosis and prognostic prediction was similarly poorly supported by the literature.Part 1:Evaluating the value of Micro RNA-30e-5p in the diagnosis and prognostic prediction of OSCC based on bioinformatics methodsObjective:This study aimed to screen diagnostic and prognostic biomarkers for OSCC based on bioinformatics methods,and evaluate the value of miR-30e-5p in the diagnosis and prognostic prediction of OSCC,in order to provide new options for the diagnosis and treatment of OSCC.Methods:(1)Screening of target molecules:1)miRNA expression data from the cancer genome atlas(TCGA)database were extracted.Hub miRNAs related to the occurrence of OSCC were identified using WGCNA and difference analysis;2)Subsequently,Cox regression analysis was used to evaluate the relationship between the hub miRNAs and the prognosis of OSCC patients,and screen the miRNAs related to the prognosis of OSCC patients.(2)Evaluation of the diagnostic value of miR-30e-5p:1)Differential expression analysis was performed to identify the expression level of miR-30e-5p in the OSCC tissues and normal oral tissues;2)The receiver operating characteristic curve(ROC)was performed to evaluate the diagnostic value of miR-30e-5p in OSCC.(3)Evaluation of the prognostic value of miR-30e-5p:1)Kaplan-Meier survival analysis was carried out to identify the correlation between the expression level of miR-30e-5p and the prognosis of patients with OSCC in the TCGA database.Firstly,OSCC patients were divided into 1-year survival,3-year survival,5-year and overall survival groups based on the survival time of OSCC patients.Then,OSCC patients in each group were also divided into high miR-30e-5p expression group and low miR-30e-5p expression group based on the median value of miR-30e-5p expression level.Then,the difference in survival rates between the high miR-30e-5p expression group and the low miR-30e-5p expression group was compared;2)Cox regression analysis was performed to identify the correlation between miR-30e-5p expression levels and the prognosis of patients with OSCC.(4)Exploration of the potential mechanism of miR-30e-5p regulating the occurrence and development of OSCC:1)Clinical correlation analysis was performed to evaluate the correlation between miR-30e-5p expression level and clinical characteristics(T stage,N stage,and clinical stage)of OSCC patients,and to analyze the potential role of miR-30e-5p in the progression of OSCC;2)The correlation between the expression level of miR-30e-5p and immune cell infiltration was evaluated using immune infiltration analysis;3)Target genes of miR-30e-5p were predicted through the target gene prediction website,and then the Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of the target genes was performed in order to identify the signal pathways that the target gene of miR-30e-5p may participate in;4)Protein Protein Interaction Networks(PPI)was used to further screen hub target genes of miR-30e-5p;5)Subsequently,correlation analysis was performed to evaluate the correlation between the expression level of hub target genes and the expression level of miR-30e-5p.The immune infiltration analysis was performed to evaluate the correlation between expression level of hub target genes and immune cell infiltration level.Results:(1)Screening of target molecules:1)Five hub miRNAs related to the occurrence of OSCC were identified using weighted gene co expression network analysis and difference analysis,namely miR-100,miR-205,miR-29a,miR-29c,and miR-30e-5p;2)Among the five miRNAs,only the expression level of miR-30e-5p was associated with the prognosis of OSCC patients(P<0.05).Therefore,miR-30e-5p was selected as the research objective.(2)Evaluation of the diagnostic value of miR-30e-5p:1)Compared to normal oral tissues,the expression levels of miR-30e-5p in oral squamous cell carcinoma tissues were decreased(P<0.05);2)ROC analysis showed that the AUC values of miR-30e-5p were 0.906,0.870,0.743,and 0.745 in the TCGA,GSE82064,GSE34496,and GSE168227 datasets,respectively(all greater than 0.700).The results showed that miR-30e-5p could accurately distinguish between OSCC patients and healthy people,and was of value in diagnosis of OSCC.(3)Evaluation of the prognostic value of miR-30e-5p:1)Kaplan-Meier survival curves showed that the overall survival rate(Hazard Ratio=0.66;log rank P=0.012),5-year survival rate(Hazard Ratio=0.65;log rank P=0.010),and 3-year survival rate(Hazard Ratio=0.63;log rank P=0.009)of the low miR-30e-5p expression group were lower than those of the high miR-30e-5p expression group.The results suggested that down-regulated expression level of miR-30e-5p was associated with poor prognosis in patients with OSCC;2)Cox regression analysis showed that miR-30e-5p was an independent risk factor for the prognosis of OSCC(HR=0.632;P=0.031).(4)Exploration of the potential mechanism of miR-30e-5p regulating the occurrence and development of OSCC:1)Clinical correlation analysis showed that the expression level of miR-30e-5p in OSCC patients with T2/T3/T4 stage was lower than that in OSCC patients with T1 stage(P<0.05).The expression level of miR-30e-5p in OSCC patients with N3 stage was higher than that in OSCC patients with N0 stage(P<0.05),but there was no statistical difference in the expression level of miR-30e-5p among OSCC patients with different M and clinical stages(P>0.05).The results showed that the expression level of miR-30e-5p was correlated with T stage and N stage in patients with OSCC;2)The infiltration level of immune cell in the low miR-30e-5p expression group was significantly lower than that in the high miR-30e-5p expression group(P<0.05).Further analysis showed that the infiltration levels of naive B cells,CD8~+T cells,and activated CD4~+memory T cells in the low expression group decreased,compared to the high expression group(P<0.05);3)215 target genes of miR-30e-5p were predicted.KEGG analysis showed that these target genes might participate in immune response related signaling pathways such as T cell receptor signaling pathways.4)215 genes were further screened and 10 hub genes were obtained.Among the 10 hub genes,only SNAI2 was negatively correlated with the expression level of miR-30e-5p(r=-0.388;P<0.001),and the increased expression level of SNAI2 suggested poor prognosis in patients with OSCC(HR=1.41;P=0.049).Immune infiltration analysis showed that the high expression level of SNAI2 was negatively correlated with the infiltration level of CD4~+T cells,CD8~+T cells,and other T cells in OSCC patients(P<0.05).Conclusion:miR-30e-5p serves as a potential diagnostic and prognostic biomarker for OSCC.Part II: Validation of Micro RNA-30e-5p expression level at tissular and cellular levelObjective:To verify the expression level of miR-30e-5p at the tissular and cellular levels,and to validate the value of miR-30e-5p in the diagnosis of OSCC and provide the evidence for the clinical application of miR-30e-5p.Methods:Validation of diagnostic value of miR-30e-5p: 1)Validation at the tissular level: 10 pairs of OSCC and paracancerous tissues were collected,and then the expression levels of miR-30e-5p in OSCC and paracancerous tissues were verified via the quantitative real-time polymerase chain reaction(q RT-PCR).2)Validation at the cellular level: Human oral keratinocyte cell lines HOK and OSCC cell lines Cal-27,SCC-4,and SCC-172 were cultured,and then the expression levels of miR-30e-5p in HOK and OSCC cell lines were verified via q RT-PCR.3)Validation of diagnostic value: ROC analysis was performed to verify the diagnostic value of miR-30e-5p in tissue samples,and the AUC value was calculated.Results:Validation of Micro RNA-30e-5p expression level at tissular and cellular level: 1)Validation at the tissular level: Compared with the paracancerous tissues,the expression levels of miR-30e-5p in OSCC tissues decreased(P=0.01).2)Validation at the cellular level: Compared to the human oral keratinocytes HOK cell line,the expression levels of miR-30e-5p in the OSCC cell line Cal-27,SCC-4,and SCC-172 cell line decreased(P<0.05).3)Validation of diagnostic value: The ROC curve showed that the AUC value of miR-30e-5p in tissue samples was 0.890.miR-30e-5p was of value in the diagnosis of OSCC.Conclusion: The expression levels of miR-30e-5p were downregulated in OSCC tissues and OSCC cell lines,and miR-30e-5p was of value in the diagnosis of OSCC. |
