MicroRNA-145 regulates oncolytic herpes simplex virus-1 for selective killing of human non-small cell lung cancer cells

Abstract

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, and novel treatment modalities to improve the prognosis of patients with advanced disease are highly desirable. Oncolytic virotherapy is a promising approach for the treatment of advanced NSCLC. MicroRNAs (miRNAs) may be a factor in the regulation of tumor-specific viral replication. The purpose of this study was to investigate whether miRNA-145 regulated oncolytic herpes simplex virus-1 (HSV-1) can selectively kill NSCLC cells with reduced collateral damage to normal cells.

Methods: We incorporated 4 copies of miRNA-145 target sequences into the 3'-untranslated region of an HSV-1 essential viral gene, ICP27, to create AP27i145 amplicon viruses and tested their target specificity and toxicity on normal cells and lung cancer cells in vitro.

Results: miRNA-145 expression in normal cells was higher than that in NSCLC cells. AP27i145 replication was inversely correlated with the expression of miRNA-145 in infected cells. This oncolytic HSV-1 selectively reduced cell proliferation and prevented the colony formation of NSCLC cells. The combination of radiotherapy and AP27i145 infection was significantly more potent in killing cancer cells than each therapy alone.

Conclusions: miRNA-145-regulated oncolytic HSV-1 is a promising agent for the treatment of NSCLC.

Figure 1

Expression levels of microRNA (miRNA)-145 in normal cells and non-small cell lung cancer (NSCLC) cells. Expression levels of miRNA-145 in various cell lines were determined using quantitative reverse transcription polymerase chain reaction assay. Expression levels of miRNA-145 were normalized to an internal control (miRNA-93) to obtain ΔCT values, and then all ΔCT values were compared with cells obtained from pneumonia/heart failure associated pleural effusions (PL1) to give -ΔΔCT. HUVECs, human umbilical vein endothelial cells.

Figure 2

Expression levels of ICP27 in normal cells and non-small cell lung cancer cells after infection with AP27i145. The mRNA and protein expression levels of ICP27 in AP27i145-infected cells was determined using quantitative reverse transcription polymerase chain reaction assay and Western blot assays. (a) The expression of ICP27 mRNA in AP27i145-infected cells. The mRNA expression level of ICP27 was normalized to an internal control (actin) to obtain ΔCT values, and then all ΔCT values were compared with PL1 cells to give -ΔΔCT. (b) The expression of ICP27 protein in AP27i145-infected cells. The protein expressions of ICP27 in normal cells and non-small cell lung cancer cells after infection with AP27i145 were examined by Western blotting with antibodies specifically against ICP27 and β-actin.

Figure 3

Cytotoxicity of viral infection in normal and NSCLC cells. Cells were treated with varying doses of AP27i145 or 5dl1.2 herpes simplex virus-1 (HSV-1). Culture medium alone was used for mock infection. Triplet cultures were performed for each treatment, and viable cells were counted on day 5. The results are expressed as a percentage of the mock-infected cells (survival ratio). Data are expressed as means ± standard error (SE). MOI, multiplicity of infection.

Figure 4

Correlation of miRNA-145 expression and virus replication in normal and NSCLC cells. Cells were treated with AP27i145 at an MOI of 0.1. The media were collected 5 days after treatment, and viral titer was determined using 7B cells. The correlation of AP27i145 replication and miRNA-145 expression was normalized at log 10 and calculated using SPSS software.

Figure 5

Effects of AP27i145 infection on cell growth in vitro. HSV-1 infection was performed at an MOI of 0.1. Twenty-four hours after infection, cells were seeded onto 6-well culture plates at a concentration of 10³ cells per well and were cultured for additional 12 days. The colonies were stained with methylene blue for photography. The experiments were performed in triplicate, and representative data are shown. (a) H460, (b) H1975, (c) A549, and (d) H838 cells.

Figure 6

Cytotoxicity of combined radiation and AP27i145 HSV-1 treatment. (a) A549, (b)) H460, (c) H838, and (d) H1975 lung cancer cells were treated with AP27i145 or 5dl1.2 HSV-1 at an MOI of 0.01. Culture medium alone was used for mock infection. Cells were subsequently treated with radiation at 0, 2, 4, or 8 Gy 48 h after infection. On day 3 after irradiation, 3 cultures for each combination treatment were counted for viable cells. The results are expressed as a percentage of the mock-infected and mock-irradiated cells (survival ratio). Data are expressed as means ± SE.