| ObjectivePrimary liver cancer is one of the most common clinical malignant tumors. Its global mortality is very high. But there is curative therapy for the early liver cancer. So it is particularly urgent to seek a new marker which can be used to diagnose liver cancer. Exosome and miRNA have attracted more and more attention in the diagnosis of liver cancer. Exosome is nanometer membranous vesicle secreted by various cells. The study showed that it contained unique protein and nucleic acid. Exosome has the characteristic of certain biological function. Thereby, the material having biological function in exosome can be used as a marker for diagnosis of some diseases. This study based on the exosome and plasma miRNA chip analysis in preliminary experiment and used relative quantitative PCR method. The unique biological function of exosome combined with its miRNA was used as a marker for the diagnosis of liver cancer to verify. And its sensitivity and specificity as the markers were compared with those of plasma miRNA. The possibility of exosomal miRNA panel as a potential diagnostic marker of liver cancer was investigated.Methods:Plasma samples were from the hospital and randomly collected plasma of liver cancer patients (hepatitis B-liver cirrhosis-liver cancer) in this experiment, including the patients undergoing palliative care (including palliative radiotherapy, palliative chemotherapy, targeted therapy and supportive treatment etc.) and early liver cancer patients without any treatment. There were a total of 50 cases. All specimens were confirmed by pathological examination. Meanwhile the plasma of liver cirrhosis patients was randomly collected during the same period as experimental group in the hospital. There were a total of 50 cases.20 cases of normal human plasma were set as control group. The miRNA chip analysis was conducted by Affymetrix; The expression content of miRNA in plasma was detected by relative quantitative PCR using tapman probe method. Statistical analysis:The sensitivity and specificity of each miRNA were measured using prism software and ROC curve analysis; The data were analyzed by SPSS 13.0 statistical software. And the sensitivity and specificity of miRNA panel were calculated, P< 0.05 showed the result had statistical significance.Results1. The miRNA chip analysis on exosome solution, plasma of 3 cases of liver cancer patient and 3 cases of liver cirrhosis patients and 3 cases of nomal people were conducted. The results displayed that 204 differentially expressed miRNAs could be measured in exosome group, which was much higher than 92 in plasma group. The chip result also showed that there were 43 miRNAs contained in plasma group in exosome group.2. Comparison of miRNA26a in 36 patients with liver cancer and 38 patients in control group (18 patients with liver cirrhosis,20 normal people):(1) Compared with patients with liver cirrhosis, the miRNA26a in patients with liver cancer has sensitivity of 82%, specificity of 100%, they have significant difference (P<0.01); when compared with patients with cirrhosis peripheral blood, the miRNA26a in patients with liver cancer has sensitivity of 68%, specificity of 100%, they have significant difference (P<0.01)(2) Compared with health people, the miRNA26a in patients with liver cancer has sensitivity of 100%, specificity of 100%, they have significant difference (P <0.01); when compared with patients with cirrhosis peripheral blood, the miRNA26a in patients with liver cancer has sensitivity of 50%, specificity of 95%, they have significant difference (P<0.01)(3) Compared with health people, the miRNA26a in patients patients with liver cirrhosis has sensitivity of 90%, specificity of 89%, they have significant difference (P<0.01); when compared with patients with cirrhosis peripheral blood, the miRNA26a in patients with liver cancer has sensitivity of 88%, specificity of 50%, they have no significant difference (P=0.51)3. Comparison of miRNA199a in 20 patients with liver cancer and 31 patients in control group (11 patients with liver cirrhosis,20 normal people):(1) Compared with patients with liver cirrhosis, the miRNA199a in patients with liver cancer has sensitivity of 59%, specificity of 89%, they have significant difference (P<0.05); when compared with patients with cirrhosis peripheral blood, the miRNA199a in patients with liver cancer has sensitivity of 67%, specificity of 83%, they have significant difference (P<0.01)(2) Compared with health people, the miRNA199a in patients with liver cancer has sensitivity of 100%, specificity of 100%, they have significant difference (P <0.01); when compared with patients with cirrhosis peripheral blood, the miRNA199a in patients with liver cancer has sensitivity of 67%, specificity of 89%, they have significant difference (P<0.01)(3) Compared with health people, the miRNA199a in patients patients with liver cirrhosis has sensitivity of 100%, specificity of 100%, they have significant difference (P<0.01); when compared with patients with cirrhosis peripheral blood, the miRNA199a in patients with liver cancer has sensitivity of 67%, specificity of 61%, they have no significant difference (P=0.25)4. Comparison of miRNA29c in20 patients with liver cancer and 40 patients in control group (20 patients with liver cirrhosis,20 normal people):(1) Compared with patients with liver cirrhosis, the miRNA29c in patients with liver cancer has sensitivity of 79%, specificity of 86%, they have significant difference (P<0.01); when compared with patients with cirrhosis peripheral blood, the miRNA29c in patients with liver cancer has sensitivity of 83%, specificity of 45%, they have no significant difference (P=0.59)(2) Compared with health people, the miRNA29c in patients with liver cancer has sensitivity of 100%, specificity of 100%, they have significant difference (P <0.01); when compared with patients with cirrhosis peripheral blood, the miRNA29c in patients with liver cancer has sensitivity of 50%, specificity of 90%, they have significant difference (P<0.01)(3) Compared with health people, the miRNA29c in patients patients with liver cirrhosis has sensitivity of 100%, specificity of 100%, they have significant difference (P<0.01); when compared with patients with cirrhosis peripheral blood, the miRNA29c in patients with liver cancer has sensitivity of 55%, specificity of 95%, they have no significant difference (P<0.01)5. Among the peripheral exosome in 20 cases of liver cancer and 30 cases of control group which contains 10 of liver cirrhosis and 20 of normal people, the multivariate logistic regression analysis of miRNA26a, miRNA29c and miRNA199a were conducted by SPSS software. It was measured that the sensitivity of miRNA panel was 94% and the specificity was 90%. The difference showed statistical significance between two (P<0.01).6. The relative quantitative PCR was conducted on miRNA 122 in exosome in 20 cases of liver cancer patients and 10 cases of normal patients. It was measured that the sensitivity was 89% and the specificity was 100%. The difference showed statistical significance between two (P<0.01). The sensitivity was 83% and the specificity was 100% in plasma group. The difference showed statistical significance between two (P<0.01).Conclusion1. The expression levels of miRNA-26a, miRNA-29c and miRNA-199a in control group were higher than those of liver cancer group.2. In the diagnosis of liver cancer and cirrhosis, the miRNA-26a in Exosome has higher sensitivity and specificity than that in blood plasma, and exosomal miRNA26a can better reflect the development of the disease.3. In the diagnosis of liver cancer and cirrhosis, the miRNA-29c in Exosome has higher sensitivity and specificity than that in blood plasma, and exosomal miRNA29c can better reflect the development of the disease.4. In the diagnosis of liver cancer, the miRNA-199a in Exosome has higher sensitivity than that in blood plasma. Furthermore, in the diagnosis of liver cirrhosis, the miRNA-199a in Exosome has higher sensitivity than that in blood plasma, and exosomal miRNA199a can better reflect the development of the disease.5. The exosome miRNA panel had higher sensitivity and specificity than those of miRNA-26a, miRNA-29c and miRNA-199a.6. The expression level of miRNA-122 in liver cancer group was higher than that of normal group.7. The miRNA-122 had higher sensitivity in exosome. |
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