miR-361-3p is upregulated in liver T-ICs.
It was well accepted that CD24, CD133 and EpCAM were liver T-ICs markers (18–20). Therefore, we isolated CD24, CD133 and EpCAM positive HCC cells by flow cytometry. As expected, the expression of miR-361-3p was dramatically increased in CD24, CD133 and EpCAM positive HCC cells compared with their negative HCC cells (Figure. 1A-C). Spheroid culture of cancer cells is a routine approach to enrich T-ICs. We observed that expression of miR-361-3p was significantly upregulated in the self-renewing spheroids compared with the attached cells (Figure. 1D). In serial passages of Huh7 or HepG2 spheroids, miR-361-3p expression was gradually increased (Figure. 1E). Consistently, miR-361-3p expression was also upregulated in CD24, CD133 and EpCAM positive primary HCC cells compared with negative primary HCC cells (Figure. 1F-H). The level of miR-361-3p was increased in primary HCC spheroids compared with the attached cells (Figure. 1I). Taken together, our data demonstrated that miR-361-3p was upregulated in liver T-ICs.
miR-361-3p promotes liver T-ICs self-renew and tumorigenesis.
To evaluate the potential role of miR-361-3p in liver T-ICs, Huh7 and HepG2 cells were infected with miR-361-3p overexpression virus. The overexpression effect of miR-361-3p was determined by real-time PCR (Figure. 2A). Spheroids formation is a routine approach to assess self-renewal ability. We found that Huh7/HepG2 miR-361-3p mimic cells formed much more spheres than control cells (Figure. 2B). Moreover, the expression of liver T-ICs markers was upregulated in miR-361-3p mimic HCC cells compared with control cells (Figure. 2C). Consistently, the expression of stemness associated transcription factors was increased in miR-361-3p mimic HCC cells compared with control cells (Figure. 2D). Furthermore, the in vitro and in vivo limiting dilution assay indicated that the proportion of liver T-ICs and tumorigenic ability were enhanced in miR-361-3p mimic HCC cells (Figure. 2E༆F).
miR-361-3p knockdown inhibits liver T-ICs self-renew and tumorigenesis.
To further explore the role of miR-361-3p in liver T-ICs, Huh7 and HepG2 cells were infected with miR-361-3p interference virus. The knockdown effect of miR-361-3p was determined by real-time PCR (Figure. 3A). Spheroids formation is a routine approach to assess self-renewal ability. We found that Huh7/HepG2 miR-361-3p knockdown cells formed much less spheres than control cells (Figure. 3B). Moreover, the expression of liver T-ICs markers was downregulated in miR-361-3p knockdown HCC cells compared with control cells (Figure. 3C). Consistently, the expression of stemness associated transcription factors was decreased in miR-361-3p knockdown HCC cells compared with control cells (Figure. 3D). Furthermore, the in vitro and in vivo limiting dilution assay indicated that the proportion of liver T-ICs and tumorigenic ability were impaired in miR-361-3p knockdown HCC cells (Figure. 3E༆F). Collectively, our results demonstrated that miR-361-3p promoted liver T-ICs expansion.
SOX1 is required in miR-361-3p-mediated liver T-ICs expansion.
To elucidate mechanism underlying miR-361-3p-mediated liver T-ICs expansion, TargetScan and miRbase were used to predicted the potential targets of miR-361-3p in liver T-ICs. Bioinformatics analysis found that miR-361-3p has a putative binding site in SOX1 mRNA 3’-UTR (Figure. 4A). As expected, both the mRNA and protein expression of SOX1 were upregulated in miR-361-3p knockdown HCC cells (Figure. 4B༆C). Luciferase reporter assays with a vector that included the wild-type (WT) 3'-UTR or mutant-type (MUT) 3'-UTR of SOX1 were then performed to determine whether miR-361-3p could directly regulate SOX1. The results showed that miR-361-3p knockdown significantly enhanced the relative luciferase activity compared with control group. However, such effects were diminished when the predicted binding site was mutated (Figure. 4D). These results indicated that miR-361-3p directly suppressed SOX1 expression by binding to its 3’-UTR. In addition, a significant negative correlation was identified between SOX1 and miR-361-3p expression in clinical samples of HCC (R2 = 0.734, P < 0.05, n = 50) (Figure. 4E). To further confirm whether SOX1 was required for miR-361-3p mediated liver T-ICs expansion, the special SOX1 siRNA was used (Figure. 4F). SOX1 siRNA abrogated the self-renewal ability and liver T-ICs frequency between miR-361-3p knockdown HCC cells and control cells (Figure. 4G༆H).
miR-361-3p knockdown HCC cells are sensitive to cisplatin and sorafenib treatment.
We next explored whether miR-361-3p was involved in the regulation of chemo-resistance of HCC. As expected, miR-361-3p expression was markedly upregulated in cisplatin-resistance or sorafenib-resistance xenograft (Figure. 5A). Consistently, miR-361-3p expression was also significantly increased in cisplatin-resistance or sorafenib-resistance HCC cell lines (Figure. 5B༆C). Moreover, the sensitivity of cisplatin or sorafenib was increased in miR-361-3p knockdown HCC cells compared with control HCC cells (Figure. 5D). We also observed that interference of miR-361-3p sensitized HCC cells to undergo cisplatin-induced or sorafenib-induced cell apoptosis (Figure. 5E-H). Furthermore, Kaplan-Meier analysis revealed the survival benefits in adjuvant TACE-treated or sorafenib-treated HCC patients with low miR-361-3p levels (Figure. 5I༆J).