Identification of microRNAs specific for epithelial cell adhesion molecule-positive tumor cells in hepatocellular carcinoma

Abstract

Therapies that target cancer stem cells (CSCs) hold promise in eliminating cancer burden. However, normal stem cells are likely to be targeted owing to their similarities to CSCs. It is established that epithelial cell adhesion molecule (EpCAM) is a biomarker for normal hepatic stem cells (HpSCs), and EpCAM(+) AFP(+) hepatocellular carcinoma (HCC) cells have enriched hepatic CSCs. We sought to determine whether specific microRNAs (miRNAs) exist in hepatic CSCs that are not expressed in normal HpSCs. We performed a pair-wise comparison of the miRNA transcriptome of EpCAM(+) and corresponding EpCAM(-) cells isolated from two primary HCC specimens, as well as from two fetal livers and three healthy adult liver donors by small RNA deep sequencing. We found that miR-150, miR-155, and miR-223 were preferentially highly expressed in EpCAM(+) HCC cells, which was further validated. Their gene surrogates, identified using miRNA and messenger RNA profiling in a cohort of 292 HCC patients, were associated with patient prognosis. We further demonstrated that miR-155 was highly expressed in EpCAM(+) HCC cells, compared to corresponding EpCAM(-) HCC cells, fetal livers with enriched normal hepatic progenitors, and normal adult livers with enriched mature hepatocytes. Suppressing miR-155 resulted in a decreased EpCAM(+) fraction in HCC cells and reduced HCC cell colony formation, migration, and invasion in vitro. The reduced levels of identified miR-155 targets predicted the shortened overall survival and time to recurrence of HCC patients.

Conclusion: miR-155 is highly elevated in EpCAM(+) HCC cells and might serve as a molecular target to eradicate the EpCAM(+) CSC population in human HCCs.

Figure 1. The raw sequencing data of small RNA transcriptome in 16 samples

(A) Histogram of all the reads from each output file of 16 sequenced samples. FL refers to fetal liver. (B). Read length distribution of reads aligned to miRBase (Black) and reads aligned to Genome (Orange) in 16 sequenced samples. The results are shown as mean ± standard deviation (SD). (C) The total number of identified miRNAs (grey bars) and the total raw reads number for identified miRNAs (Black dots) in each sample. An in-house pipeline was used to process the aligned reads. Details are shown in Materials and Methods.

Figure 2. Aberrant high expression of three miRNAs in EpCAM⁺ HCC cells

(A) Unsupervised clustering of 16 sequenced samples based on the expression of 600 miRNAs with relative high abundance. (B) Venn-diagram of miRNAs with ≥ 2-fold alteration in the comparison of EpCAM⁺ and EpCAM⁻ HCC cells and miRNAs from the comparison of EpCAM⁺ and EpCAM⁻ normal liver cells. The right panel shows the bar graph of 99 miRNAs with ≥ 2-fold changes in the comparison of EpCAM⁺ and EpCAM⁻ HCC cells. Red bars: n=21, ≥5-fold of EpCAM⁺ HCC cells vs. EpCAM⁺ HCC cells. Blue bars: n=7, ≤0.2-fold of EpCAM⁺ HCC cells vs. EpCAM⁻ HCC cells. (C) Scatter plot of 21 miRNAs (left channel, red spots) and 7 miRNAs (right channel, blue spots) from the right panel of Fig 2B to display their expression in EpCAM⁺ hepatic stem cells and hepatoblasts compared to EpCAM⁻ hepatocytes. The red spots shadowed in red refer to miRNAs with ≥ 5-fold of EpCAM⁺ HCC cells vs. EpCAM⁻ HCC cells and ≤ 2-fold of EpCAM⁺ hepatic progenitors vs. EpCAM⁻ hepatocytes. The blue spots shadowed in blue refer to miRNAs with ≤ 0.2-fold of EpCAM⁺ HCC cells vs. EpCAM⁻ HCC cells and ≥ 0.5-fold of EpCAM⁺ hepatic progenitors vs. EpCAM⁻ hepatocytes. (D) Pearson correlation analysis of EpCAM⁺ HCC cells-related miRNAs expression data (log2) from small RNA sequencing and from qRT-PCR validation in all 16 samples. The EpCAM⁺ HCC cells - related miRNAs were listed in (C). (E) miRNA array data for three miRNAs in extreme EpCAM⁺AFP⁺-HCCs and EpCAM⁻AFP⁻-HCCs. Y-axis refers to the log2 intensity. Student t-test was performed.

Figure 3. Gene surrogates of EpCAM⁺ HCC cells-related miRNAs were associated with HCC prognosis

(A) We have used two HCC cohorts, i.e., HCC cohort 1 (n= 292) and HCC cohort 2 (n=139). In cohort 1, 196 HCC cases had the available miRNA and mRNA array data, while 45 patients have miRNA array data only and 51 patients have mRNA array data only. All patients in HCC cohort 2 have mRNA array data only. (B) Pearson correlation was performed between three EpCAM⁺ HCC cells-miRNAs and ~13,000 genes in 196 HCC cases with available miRNA and mRNA array from cohort 1. 511 gene surrogates of EpCAM⁺ HCC cells-related miRNAs were identified with the criteria described in Materials and Methods. (C) Unsupervised hierarchical clustering based on these gene surrogates for extreme EpCAM⁺AFP⁺ and EpCAM⁻AFP⁻HCCs with both miRNA and mRNA expression data (n=57). (D) Kaplan-Meier analysis of overall survival and recurrence in HCC cases from Cohort 1 (n=196, cases used for detecting gene surrogates) based on the classification of cluster 1 and cluster 2 by 511 genes. (E) Kaplan-Meier analysis of overall survival and recurrence in HCC cases from cohort 2 (n=139) based on the classification of cluster 1 and cluster 2 by 511 genes. Log-rank test was performed.

Figure 4. miR-155 was specifically highly expressed in EpCAM⁺ HCC cells

(A) The sequencing data of miR-155 in EpCAM⁺ and EpCAM⁻ cells from HCC primary samples and normal livers. (B) qRT-PCR data of miR-155 in enriched EpCAM⁺ and EpCAM⁻ cells from HuH7 HCC cell line. EpCAM⁺ cells were enriched via KnockOut medium/Serum Replacement culture and cells under regular media were used as control. EpCAM⁻ cells were enriched via EpCAM silencing technology and control siRNA was used as controls. MiR-155 expression in these cells was compared to the corresponding controls. The top channel is the FACS data showing the enriched EpCAM⁺ and EpCAM⁻ population. APC-conjugated EpCAM antibody was used. (C) EpCAM⁺ cell distribution (upper channel, FACS) and miR-155 expression (lower channel, qRT-PCR) in HuH7 cells under normoxia and hypoxia condition for two days. For FACS analysis, APC-conjugated EpCAM antibody was used. (D) qRT-PCR data of miR-155 in human fetal livers and normal livers. EpCAM⁺ HCC cells enriched from KnockOut medium/Serun Replacement culture were used as a control. (E) miR-155 expression during the development of mouse liver from embryonic stage to adult stage. MiR-181d was also examined as a positive control. (B,C,D) Student t-test was performed.

Figure 5. Suppressing miR-155 reduced HCC malignancy features in vitro

(A) The duplex between anti-miR-155 expressed from miRZip-155 and miR-155, and the reporter plasmid for miRZip-155 with mature miR-155 in the 3’UTR region of luciferase (left channel). The lower cases in mature miR-155 were mutated to ATCCCC in reporter plasmid with mutant miR-155. Luciferase activities of reporter plasmids with wild type and mutant miR-155 in 3’UTR region was measured in HuH7 cells co-transfected with miRZip-000 or miRZip-155 (right channel). The luciferase activity was shown as the mean ± SD. (B) FACS analysis of HuH7 infected with miRZip-155 and control virus for 2 days under hypoxia condition. APC-conjugated EpCAM antibody was used. (C) HuH7 cells infected with miRZip-155 and control virus for 2 days under hypoxia condition were seeded in ultra-low attachment plates to assay spheroid formation. (D) Assays on colony formation, migration and invasion were performed in HuH7 infected with miRZip-155 and control virus for 2 days under hypoxia condition. (A,C,D) Student t-test was performed.

Figure 6. The reduced level of miR-155 potential targets was associated with poor overall survival and short time to recurrence in HCC patients

(A) Venn-diagram of down-regulated genes in extreme EpCAM⁺ HCC cases and predicted miR-155 targets. 27 genes were overlapped, in which three genes were reported as miR-155 targets. (B) The expression levels of CEBPB and Actin in HuH7 and HuH1 cells infected with miRZip-155, as determined by Western blotting. (C) Hierarchical clustering of 27 genes in cohort 1 (n=247) predicted two HCC subgroups, i.e., group 1 (n=121) and group 2 (n=126). The reduction of these genes was noticed in group 2. (D) Kaplan-Meier analysis of overall survival and recurrence in HCC cases from cohort 1 based on the classification of group 1 and group 2 by 27 genes. (E) Hierarchical clustering of 27 genes in cohort 2 (n=139) could predict two HCC subgroups, i.e., group 1 (n=73) and group 2 (n=66). The reduction of these genes was noticed in group 2. (F) Kaplan-Meier analysis of overall survival and recurrence in HCC cases from cohort 2 (n=139) based on the classification of group 1 and group 2 by 27 genes. Log-rank test was performed.