| Objective The expression levels of serum exosome mi RNAs were compared between patients with non-small cell lung cancer and healthy controls,as well as between lung adenocarcinoma patients and lung squamous cell carcinoma patients with non-small cell lung cancer.The serum exosome mi RNAs that can be used for early diagnosis of non-small cell lung cancer,as well as the differentiation diagnosis of lung adenocarcinoma and lung squamous cell carcinoma were screened,so as to explore biomarkers conducive to the clinical study of non-small cell lung cancer.Methods From July 2020 to June 2022,47 patients with non-small cell lung cancer(26cases of lung adenocarcinoma and 21 cases of lung squamous cell carcinoma)and 47 healthy controls who came to Affiliated Hospital of Qingdao University for physical examination were selected as the subjects.(1)Two sets of mi RNAs expression profile data and corresponding clinical data from TCGA database were download,to analyze the difference of mi RNAs expression data of non-small cell lung cancer and healthy control group,lung adenocarcinoma and lung squamous cell carcinoma with the Limma package of R language and screen out five mi RNAs with different expression.(2)The serum exosomes of non-small cell lung cancer patients and healthy controls were separated by ultracentrifugation,and the appearance of exosomes was observed by transmission electron microscope(TEM).(3)Western blot method was used to detect the marker surface protein molecules CD9 and CD63 of exosomes.(4)Quantitative PCR(RT-q PCR)was used to quantitatively analyze 10 mi RNAs selected from TCGA database.Three serum exosomes mi RNAs were preliminary screened from non-small cell lung cancer and healthy control group,as well as lung adenocarcinoma and lungs squamous cell carcinoma groups.(5)Six serum exosome mi RNAs with significant differences were selected as the test objects and tested in all subjects included in the study.The accuracy of selected serum exosome mi RNAs was further verified by RT-q PCR quantitative detection.(6)The six serum exosome mi RNAs verified were analyzed by the receiver operating characteristic curve(ROC curve)to determine their value in the diagnosis of non-small cell lung cancer and the differentiation of lung adenocarcinoma and lung squamous cell carcinoma.Results(1)Compare the non-small cell lung cancer patients downloaded from TCGA database with healthy controls.306 differentially expressed mi RNAs were observed,including 118 up-regulated mi RNAs and 188 down-regulated mi RNAs.Five mi RNAs with significant differential expression were screened,mi R-1269 a,mi R-6788-3p,mi R-210-3p,mi R-31-5p and mi R-4732-3p(| log2 FC |>1,P<0.05).Compared with lung adenocarcinoma patients and lung squamous cell carcinoma patients,77 mi RNAs were observed to be upregulated in both lung adenocarcinoma and lung squamous cell carcinoma,and 83 mi RNAs were down-regulated in both lung adenocarcinoma and lung squamous cell carcinoma.Five mi RNAs,mi R-205-5p(↑),mi R-147b(↑),mi R-210-3p(↑),mi R-1269a(↑)and mi R-139-3p(↓),which were up-regulated or down-regulated at the same time and were significantly differentially expressed in the two groups were screened(| log2 FC |>1,P<0.05).(2)The appearance and morphology of exosome were identified by transmission electron microscopy(TEM).The results indicated that exosome was an irregular oval structure,and the membrane boundary could be observed at its outer edge.(3)Western blot was used to detect the marker protein molecules CD9 and CD63 of exosome membrane surface,and both CD9 and CD63 were found to be positive,indicating that the collected samples were exosome.(4)Preliminary screening of serum exosomes mi RNAs in nonsmall cell lung cancer patients and healthy controls through RT-q PCR quantitative analysis.Three of the five mi RNAs were found to have statistically significant differences in mi R-1269 a,mi R-6788-3p and mi R-4732-3p,respectively(P<0.05),while there was no significant difference in mi R-210-3p and mi R-31-5p.Preliminary screen the serum exosome of mi RNAs in patients with lung adenocarcinoma and lung squamous cell carcinoma.The expression levels of mi R-205-5p,mi R-147 b and mi R-139-3p were significantly different(P<0.05),but there was no significant difference between mi R-210-3p and mi R-1269 a.(5)RT-q PCR quantitative analysis results of all samples were verified.The expression of serum exosome mi R-1269 a,mi R-6788-3p and mi R-4732-3p in patients with non-small cell lung cancer and healthy controls was significantly different(P<0.05).The expression of serum exosome mi R-205-5p,mi R-147 b and mi R-139-3p in patients with lung adenocarcinoma and lung squamous cell carcinoma was significantly different(P<0.05).The results of both groups are consistent with the results of preliminary screening.(6)By calculating the AUC results of ROC curve,it was found that the diagnosis of nonsmall cell lung cancer with mi R-1269 a was AUC=0.942 and 95% CI was 0.894-0.990.The diagnostic value of mi R-4732-3p for non-small cell lung cancer was AUC=0.888 and 95%CI was 0.825-0.951.The differential diagnosis of lung adenocarcinoma and lung squamous cell carcinoma by mi R-205-5p was AUC=0.914 and 95% CI was0.837-0.990.Conclusion(1)The differential expression of mi RNAs in exosomes between NSCLC patients and healthy controls was verified through the extraction and identification of exosomes.(2)Serum exosomes mi R-1269 a and mi R-4732-3p can serve as potential biomarkers for early diagnosis of non-small cell lung cancer.(3)The serum exosomes mi R-205-5p can serve as potential biomarkers for the differential diagnosis of lung adenocarcinoma and squamous cell carcinoma. |
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