The Evaluation On Stability, Individual Differences And Gender Correlation With Liver Cancer Related MicroRNAs

MicroRNA (miRNA) is non-coding, single-stranded RNAs of ~22 nt and constitute a novel class of gene regulators that is found in both plants and animals. Recent evidences have shown that miRNA aberrant expression correlate with various human cancers and indicate that miRNA can function as tumour suppressors and oncogenes. While many studies have focused on miRNA expression in physiological and pathological processes, variables related to miRNA for new serum biomarkers have simultaneously emerged. Now miRNA has been applied to early detection of cancer and monitoring of cancer recovery by using detection of peripheral blood. Several studies have demonstrated that serum miRNA could serve as potential biomarkers for the detection of various cancers and other diseases. However, few documents regarding the stability of liver cancer related miRNA in serum are available. A systemic analysis of the stability of miRNA in serum is quite necessary.The purpose of this study was to evaluate the stability of miRNA from three different sources, cultured liver cancer Huh-7 cell-line, clinical liver tumor and serum under different experimental conditions, including a) same time period of 3 h treatment under different temperatures (-80°C, -20°C, 4°C, room temperature and 37°C); b) under room temperature treatment for 0, 1, 3, 6, 12, 24 h; c) RNase A treated for 0, 3, 6, 12 h incubation in 37°C; d) DNase I treated for 0, 3, 6, 12 h incubation in 37°C; e) treatment under various free-thaw cycles (0, 2, 5, 7, 10 cycles); f) different pH value (control (pH = 7), pH = 1, 6, 9, 13) of solution treated for 3 h incubation in 37°C. The Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis with 40 cycles is conducted on the data collected from the systemic experiment. We used 18S rRNA as control gene. All the results demonstrated that liver cancer related miRNAs were detectable under each of the test conditions, indicating that miRNA was extremely stable and resistant to destruction and degradation under harsh environmental conditions. However, ribosomal RNA was fragile and easily degraded by demonstrating sharp decrease of relative expression under the non-physiological test conditions. The repeatability of the data was significant at p < 0.05 from Student's t-test.We also use the Pearson's correlation coefficient to evaluated the relationship between the liver cancer and miRNA expression of 22 healthy human subjects by qRT-PCR. Expression level of serum miRNA was reproducible and consistent among 22 healthy human subjects for the R value was access to 1, p-value < 0.05. The result was considered to be significant. Pearson correlation scatter plot of the relative serum miRNA expression between male and female of R~2 was 0.0953. Results suggested that miRNA expression is not correlated between genders.Simultaneously, we compared the expression of 7 miRNAs (miR-21, -25, -29c, -93, -198, -221, -222) in liver cancer serum with which in normal serum, and found that they were different. The expression of miRNA in liver cancer serum is significant higher than that in normal serum. That means the expression of 7 miRNAs can specifically distinguish liver serum from normal serum. They were liver cancer biomarkers.On the other hand, it is difficult to obtain sufficient and high-quality of RNA in serum. Our tests have shown that pre-heating procedure is a robust serum RNA extraction method and is efficient for RNA isolation. This method is also essential for further serum source miRNA study. In fact, after qRT-PCR, the CT (threshold cycle) value decreased at least 5. In conclusion, we established a robust procedure for serum RNA extraction, which is of great importance not only for the miRNA profiling studies but also for the disease prognosis based on abnormal miRNA expression.Taking together, these results implied that liver cancer related miRNAs (miR-21, -25, -29c, -93, -198, -221, -222) expression levels in serum were quite stable, present and detectable, and also were consistent reproducibly among individuals of the same species in serum. The miRNA expression levels were not correlated between genders, and can specifically distinguish liver serum from normal serum. They may be the potential biomarkers for liver cancer serum in future.