The heterogeneity of plasma miRNA profiles in hepatocellular carcinoma patients and the exploration of diagnostic circulating miRNAs for hepatocellular carcinoma

Abstract

Heterogeneity is prevalent in cancer both between and within individuals. Although a few studies have identified several circulating microRNAs (miRNAs) for cancer diagnosis, the complete plasma miRNA profile for hepatocellular carcinoma (HCC) remains undefined, and whether the plasma miRNA profiles are heterogeneous is unknown. Here, we obtained individualized plasma miRNA profiles of both healthy subjects and HCC patients via genome-wide deep sequencing. Compared with the highly stable miRNA profile of the healthy subjects, the profile of the HCC patients was highly variable. Seven miRNAs were optimized as potential plasma-based biomarkers for HCC diagnosis. Combined with the clinical data of The Cancer Genome Atlas (TCGA) cohort, three out of the seven miRNAs were correlated with the survival of the HCC patients. To investigate the effect of cancer cells on the plasma miRNAs profile, we compared the most differentially expressed miRNAs between plasma and tissues. Furthermore, miRNAseq data of HCC patients from TCGA were recruited for comparisons. We found that the differences between plasma and tissue were inconsistent, suggesting that other cells in addition to cancer cells also contribute to plasma miRNAs. Using two HCC cancer cell lines, we examined the levels of seven differentially expressed miRNAs. The reverse direction of certain miRNAs alterations between cancer cells and media further confirmed that miRNAs may be selectively pump out by cancer cells.

Fig 1. The plasma miRNA profiles are much more variable for HCC patients than those for heathy subjects.

A. The correlation coefficients for the pairwise comparison of healthy subjects are close to 1.0 between individuals. F indicates female, and M indicates male. B. The plasma miRNAs have minor gender bias among healthy subjects. miRNAs with an RPM more than 10 were used to analyse the types of miRNAs in the plasma. The types of differential miRNAs between genders are comparable to those within gender. C. Comparison of the Pearson’s correlation coefficient (R) comparison between healthy subjects and HCC patients. The differentiation between these two populations is significant. The P value is less than 0.001.

Fig 2. Most abundant miRNAs in plasma.

A. miR-486-5p is more abundant in the plasma of healthy subjects. B. The collected dataset of the top twenty most abundant plasma miRNAs in healthy subjects. C. Collected dataset of the top twenty most abundant plasma miRNAs in HCC patients. D. Distribution of the ratio of the miRNAs shared by all the individuals. Four individuals among ten HCC patients were randomly sampled for two hundred and ten times. The red arrow indicates the ratio of the healthy subjects.

Fig 3. Differentially expressed miRNA in plasma between HCC patients and healthy subjects.

A. The plasma miRNA profiles of HCC patients and healthy subjects are different. B. The twenty-nine most differentially expressed miRNAs can differentiate HCC patients from healthy subjects. C. The seven miRNAs predicted to be the biomarkers for HCC diagnosis can differentiate HCC patients from healthy subjects. D. Kaplan-Meier plots for patient stratification based on the expression of three of the seven miRNAs (the same as C). The gene names of each miRNA are annotated above each plot.

Fig 4. Comparisons of miRNAs between plasma and tissue and between cells and media.

A. Fold change of miRNAs that are most differentiated in plasma (in yellow) and that can be detected in tissues of TCGA dataset (in blue). B. Fold change of miRNAs that are most differentiated in tissues of TCGA dataset and that can be detected in plasma. C. Expression of the seven biomarkers in plasma and tissue within the same individuals. D. Expression of the seven biomarkers in the HCC cell lines (HepG2 and Huh7) and media.