ABSTRACT
BACKGROUND@#Malignant plural effusion (MPE) is one of the most common specimen for liquid biopsy gene detection. This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection.@*METHODS@#Pleural effusions (>500 mL) from 20 advanced lung cancer patients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll. Cell number and purity were calculated. DNA was extracted from the supernatant (etDNA), total cells and isolated tumor cells of pleural effusion (ETC-DNA) to detect the mutation of tumor-related genes by next-generation sequencing.@*RESULTS@#The median number of cells isolated from malignant pleural effusion was 8.50×10⁴ (interquel range: 9.25×10³-3.75×10⁵), 85.50%±5.80% of the cells were identified as tumor cells. The detection rates of epidermal growth factor receptor (EGFR) gene mutation of etDNA, total cell DNA and ETC-DNA were 70.00%, 50.00% and 70.00%, reseparately, while the median EGFR mutation abundance in 3 components was 16.05% (4.78%-43.06%), 1.09% (0.00%-2.39%), and 33.02% (18.50%-76.70%), respectively. ETC-DNA had good consistency with tissue DNA (P>0.999, kappa=1.000) and etDNA (P>0.999, kappa=1.000). ETC-DNA inclined to have higher EGFR mutation than etDNA, but the result was not statistically significant.@*CONCLUSIONS@#Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity. Using ETC-DNA as specimen improves the efficacy of gene detection, thus is worth further study.
ABSTRACT
Objective miRNA-122 levels may correlate with liver cancer prognosis,and therefore understanding its expression is crucial for future treatment.This study investigates the effect of DNA methylation on the expression of liver specific miRNA-122 and its effects on proliferation and apoptosis of hepatocellular carcinoma cells.Methods Methylation sequencing detected the methylation of the miRNA-122 promoter region,and the level of miRNA-122 expression was measured by using real-time quantitative PCR.The proliferation and apoptosis of hepatocellular cell lines were detected by flow cytometry and CCK8.Results Compared with human primary hepatocytes [(21.9 ± 11.4)％],the level of miRNA-122 promoter methylation in Huh7,HepG2,and QSG-7701 cell lines were (87.6±9.3) ％,(89.0 ± 14.3)％,and (69.5 ±11.5)％,respectively.This represents a significant increase (P=0.000),especially in Huh7 and HepG2 cell lines.Compared with human primary hepatocytes (2.83× 104 ±3746),the levels of miR-122 expression in the above three cell lines were significantly decreased,especially in Huh7 and HepG2 cell lines (P=0.007).After treatment with 5-Aza-dc,the degree of methylation in Huh7 and HepG2 cell lines were significantly lower than that of the blank group (P=0.038,P=0.025),and the levels of miRNA-122 expression were significantly elevated (P=0.008,P=0.003).Also,compared with the control groups,the apoptosis of Huh7 cells and HepG2 cells were significantly increased (P=0.001,0.027).Conclusion The expression of miRNA-122 is regulated by DNA methylation and correlated with the apoptosis of liver cancer cells.Therfore,the methylation regulation of miRNA-122 expression might be involved in the occurrence and development of hepatocellular carcinoma.