| Background: Oral squamous cell carcinoma(OSCC)is the most common malignant tumor in the oral cavity,with a high lymph node metastasis rate.Tissue biopsy is currently the gold standard for diagnosis of OSCC.Most OSCC have reached the middle,advanced stage of the disease or the tumor has metastasized when they are found and diagnosed.Metastasis is the leading cause of death in patients with OSCC.When patients have lymph node metastasis,their five-year survival rate dropped sharply from 80% to less than 40%.In recent years,studies have found that exosome may play an important role in explaining tumor metastasis and recurrence,and microRNA(miRNA)is one of the content of exosomes.More and more studies have shown that miRNA is related to the occurrence and development of cancer.MiRNAs of serum-derived exosomes have unique expression spectrums,which can manifest the characteristics of malignant tumor.To our knowledge,there are currently no reports on the significance of circulating exosomal miRNA in OSCC metastasis and recurrence.Objective: The purpose of this study was to detect the expression of circulating exosomal miRNA in human OSCC and to explore the disease diagnostic capabilities of differential miRNA in OSCC,so as to obtain a new and effective diagnostic marker.The effect of differential miRNA in the OSCC metastasis group on the function of the tumor was also investigated,so as to provide a new theoretical basis for the occurrence and development of OSCC.Methods:(1)Serum exosomes of OSCC patients(including patients with lymph node metastasis,patients without lymph node metastasis and patients with recurrence),patients with oral leukoplakia,patients with other oral tumors and healthy control group were collected by ultra-high speed centrifugation,and identified by Western Blot(WB)and transmission electron microscopy.Each group had 30 samples.(2)The high-throughput sequencing method was used to explore the expression profile of miRNA in OSCC serum exosomes,and the differential miRNAs in different comparison groups were screened by bioinformatics analysis.GO analysis and KEGG analysis were performed.(3)Compared with other groups,we screened out the differential miRNAs in the OSCC lymph node metastasis group and performed cluster analysis on the results.(4)The differential miRNA between the OSCC lymph node metastasis group and other groups was selected as mi R-548ah-5p.After extracting totalRNA from serum exosomes and performing reverse transcription of miRNA,the expression of mi R-548ah-5p in serum exosomes of each group was detected by quantitative real-time PCR(q PCR)to further verify the sequencing results.(5)According to the relative expression of miRNA-548ah-5p in serum exosomes of OSCC patients,we analyzed whether it was related to clinical pathological parameters.And the disease diagnostic ability of miRNA-548ah-5p was determined by ROC curve.(6)Establishing stable strains of mi R-548ah-5p overexpression group,silent expression group and control group in oral cancer cell,and exploring the effects of mi R-548ah-5p on the biological functions of oral cancer cells,such as CAL27 and SCC9.Results:(1)The extracted serum exosomes were observed under transmission electron microscopy,and the exosomes were seen as round or Suborbicular vesicle-like bodies with double-layer membrane structures at the edges,they were about 30 to 150 nm in diameters.WB showed positive expression of exosomal marker proteins HSP70,CD9 and CD63.(2)According to the results of bioinformatics analysis,we compared the OSCC with lymph node metastasis(OSCC-LNM)group with the OSCC without lymph node metastasis(OSCC-NLNM)group and the recurrent OSCC(ROSCC)group,it was found that the expression levels of 7 miRNAs in the OSCC-LNM group were significantly increased,and the expression levels of 4 miRNAs were obviously reduced;when the ROSCC group was compared with OSCC-LNM and OSCC-NLNM,one miRNA was significantly up-regulated and 29 miRNAs were significantly down-regulated in the ROSCC group;when the ROSCC group was compared with the healthy controls(HCs)group,oral leukoplakia(OL)group,and other oral tumors(OOTs)group,there were a total of 42 differentially expressed miRNAs,including 16 up-regulated and 26 down-regulated expressions;when the OSCC-LNM group was compared with the HCs group,OL group,and OOTs group,the expression of 4 miRNAs in the OSCC-LNM group was significantly up-regulated;when the ROSCC group was compared with the HCs group,the OL group,the OSCC-LNM group,the OSCC-NLNM group and the OOTs group,the ROSCC group had 14 obviously down-regulated miRNAs;When the OSCC-LNM group was compared with the HCs group,OL group,ROSCC group,OSCC-NLNM group and OOTs group,we found that only mi R-548ah-5p in the OSCC-LNM group was significantly increased,and the difference was statistically significant.(3)GO analysis showed that the differential miRNAs in each comparison group were significantly enriched in terms of cell metabolism,cells and cell components,binding and protein binding;KEGG analysis results showed that the target genes in different comparison groups were enriched in pathways in cancer,microRNAs in cancer and so on.(4)Compared to the other 5 groups,we found that mi R-548ah-5p was the only significantly up-regulated miRNA in the OSCC lymph node metastasis group.The q PCR method verified that its expression was consistent with the sequencing results.(5)By analyzing the correlation between the expression of mi R-548ah-5p in OSCC serum exosomes and clinical pathological parameters,we know that the relative expression of mi R-548ah-5p in serum exosomes in poorly differentiated oral squamous cell carcinoma was higher than highly differentiated oral squamous cells carcinoma,and the difference was statistically significant(P <0.05).There were no significant differences in other clinical pathological parameters such as gender,ethnicity,clinical stage,smoking and drinking.(6)Over time,the mi R-548ah-5p overexpression group in transfected SCC9 and CAL27 cells grew faster than the negative control group and the silent expression group,the growth rate of the silent expression group was only slightly slower than the negative control group,and the difference was statistically significant(P<0.05).SCC9 and CAL27 cells of mi R-548ah-5p overexpression and silent expression group had no significant difference in migration and invasion ability.Conclusion:(1)Serum exosome miRNA was differentially expressed in OSCC patients and control population.(2)Serum exosomes mi R-548ah-5p may be related to the degree of differentiation of OSCC,which can be used as an auxiliary diagnostic tool for patients with OSCC lymph node metastasis,and it may also guide the prognosis of patients with OSCC.(3)Mi R-548ah-5p can promote the proliferation of CAL27 and SCC9 cells to a certain extent,but the target gene has no obvious effect on the migration and invasion of OSCC cells. |
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