The regulation network consisting of microRNAs (miRNAs) and their target genes remains largely elusive in hepatocellular carcinoma (HCC), especially the reciprocal loop between specific miRNAs and the miRNA processing machinery. In this study, we found that miR-99a was remarkably decreased in 111 of 152 (73.03%) primary HCC tissues and low-level expression of miR-99a was correlated with low tumor differentiation (P=0.001), liver cirrhosis (P=0.015), poor tumor-free survival (P=0.004) and overall survival (P=0.006) for HCC patients. By restoration of miR-99a, the HCC growth could be considerably inhibited both in vitro and in vivo. Subsequently, Argonaute-2 (Ago2), a central component of RNA-induced silencing complex, was found to be directly regulated by miR-99a via translational repression. Overexpression of Ago2 could partly impair the inhibitory effect of miR-99a on HCC cells in vitro. Then, we demonstrated that Ago2 was upregulated in HCC tissues at both RNA and protein levels and the expression of AGO2 protein and miR-99a was negatively correlated within detected HCC tissues (r=−0.727, P=0.004). Interestingly, the tumorigenicity of Ago2-knockdown HCC cells was severely impaired (4/10 vs 10/10, P<0.05), and this was in contrast to the miR-99a-overexpressing HCC cells. Functionally, the increased AGO2 protein could specifically facilitate oncogenic miR-21 to repress its targeted gene phosphatase and tensin homolog (Pten) in HCC, whereas leave the regulatory capacity of let-7a on its targeted oncogenes almost unaltered. In summary, our study has revealed a novel pathway for the tumor suppressor miR-99a to control tumor growth in HCC, via its downstream signaling of AGO2/miR-21/PTEN. In addition, this study provides potential strategies for HCC therapy by reintroduction of miRNA suppressors.

No abstract

Highly selective therapy for hepatocellular carcinoma (HCC) remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4%) and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011let7T, by introducing eight copies of let-7 target sites (let7T) into the 3′ untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels) of the SG7011let7T was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011let7T was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68) expressing high level of let-7 (>300 folds), whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5) with low level of let-7. Consequently, the cytotoxicity of SG7011let7T to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011let7T in vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011let7T may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.

No abstract

Chimeric antigen receptor modified T cell-based immunotherapy is revolutionizing the field of cancer treatment. However, its potential in treating bile duct carcinoma has not been fully explored. Herein, we developed the second-generation mesothelin-targeting chimeric antigen receptor-modified T cells with the 4-1BB co-stimulatory module by the piggyBac transposon system. Mesothelin-targeting chimeric antigen receptor was expressed by 66.0% of mesothelin-targeting chimeric antigen receptor-modified T cells post electrophoretic transfection and stimulation with K562-meso cells; the expressions of activation markers were tested by flow cytometry assay and showed greater activation of mesothelin-targeting chimeric antigen receptor-modified T cells than control T cells (CD107α: 71.9% vs 48.6%; CD27: 92.1% vs 61.8%; CD137: 55.5% vs 8.4%; CD28: 98.0% vs 82.1%; CD134: 37.5% vs 10.4%). Furthermore, mesothelin-targeting chimeric antigen receptor-modified T cells exerted cytotoxicity toward mesothelin-expressing EH-CA1b and EH-CA1a cells in an effector-to-target ratio-dependent manner, while leaving mesothelin-negative GSC-SD and EH-GB1 cells and normal liver L02 cells almost unharmed. Mesothelin-targeting chimeric antigen receptor-modified T cells secreted cytokines at higher levels when co-cultured with mesothelin-positive EH-CA1a and EH-CA1b cells than with mesothelin-negative GSC-SD and EH-GB1 cells. Enhanced cytotoxicity and cytokine secretion of mesothelin-targeting chimeric antigen receptor-modified T cells compared to control T cells were also observed when co-cultured with 293-meso cells (interferon γ: 85.1% ± 1.47% vs 8.3% ± 2.50%, p = 0.000; tumor necrosis factor α: 90.9% ± 4.67% vs 18.5% ± 3.62%, p = 0.0004; interleukin 2: 60.8% ± 2.00% vs 15.6% ± 2.06%, p = 0.002; interleukin 6: 6.4% ± 2.95% vs 1.7% ± 0.63%, p = 0.055). In addition, mesothelin-targeting chimeric antigen receptor-modified T cells showed greater inhibitory and proliferative capability than control T cells within EH-CA1a cell xenografts. This study shows the potential of mesothelin-targeting chimeric antigen receptor-modified T cells in treating bile duct carcinoma.

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.