Use of MicroRNA Let-7 to Control the Replication Specificity of Oncolytic Adenovirus in Hepatocellular Carcinoma Cells

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

The regulation effect of let-7 target sites on upstream gene, analyzed by dual-luciferase assay system.

<p>The relative Renilla/Firefly ratio of cells transfected with the control plasmid, psiCHECK2 was set to 100%. The Renilla/Firefly values of cells transfected with psiCHECK2-Let7T or psiCHECK2-Let7MT was normalized to its corresponding value of cells transfected with psiCHECK2 in a same independent experiment, respectively. Duplicated experiments were conducted from the procedure of cell culture. Error bars correspond to mean ± SD. n = 3. (^** P<0.01).</p

Univariate Analysis of let-7a Expression and Clinicopathological Risk Factors in Patients with HCC.

a<p>The let-7a expression of primary HCC tissues relative to normal liver tissues.</p>b<p>The correlation coefficient between types of each clinicopathological risk factors and the three status of let-7a expression (i.e. decreased, increased, or unaltered).</p

The cytotoxicity of recombinant adenovirus to normal liver cells and HCC cells <i>in vitro</i>, analyzed by MTT assay.

<p>Cell viability of L-02, WRL-68, Hep3B, and PLC/PRF/5 were infected with WAd5, SG7011^let7T, and SG7011^let7MT at various MOIs were measured on day 7 postinfection by MTT assay. Let7T, SG7011^let7T; Let7MT, SG7011^let7MT. Error bars correspond to mean ± SD. n = 3.</p

The capacity of let-7 target sites to regulate E1A expression of the recombinant adenovirus, SG7011^let7T.

<p>(A) Virus construction. Eight copies of let-7 target sites were inserted into the 3′UTR of E1A gene and the Ad5 fiber was replaced by Ad5/11 chimeric fiber. ITR, inverted terminal repeats. (B) Detection of E1A protein expression by Western Blotting. The 1, 2, 3 lane represented cells infected with WAd5, SG7011^let7T and SG7011^let7MT, respectively. PLC, PLC/PRF/5. (C) Detection of E1A mRNA expression by quantitative RT-PCR. The gene of GAPDH was used as an endogenous control and HEK293 cell uninfected with adenovirus was used as a reference sample. Let7T, SG7011^let7T; Let7MT, SG7011^let7MT. Error bars correspond to mean ± SD. n = 3. (^** P<0.01).</p

Quantitative measurement of let-7a expression by SYBR Green Real-time PCR.

<p>The U6 was used as an endogenous control and L-02 was used as a reference sample. Error bars correspond to mean ± SD. n = 3.</p

The capacity of let-7 target sites in regulation of virus replication.

<p>(A) Viral replication assay. Cells were planted in 6-well dishes (10⁶ cells/well) and infected with indicated viruses at a MOI of 5 pfu/cell, and the cell lysates and supernatant at 48 h post infection were tittered by the TCID50 method in HEK293 cells and normalized to those at the beginning of infection. Let7T, SG7011^let7T; Let7MT, SG7011^let7MT. (B) Fluorescence analysis. The L-02 and Hep3B cells were infected with test or control virus at an MOI of 0.01 and 0.001, respectively. Three days (3D), 7 days (7D), and 10 days (10D) post infection, cells were observed under fluorescent microscope and photos were taken. The WAd5, let7T, and let7MT represented WAd5-EGFP, SG7011^let7T, and SG7011^let7MT, respectively. Bar = 10 µm.</p