1. Volume 23, Issue 1, Pages 79-88 (January 2015)
Baculovirus-Mediated miRNA Regulation to Suppress Hepatocellular Carcinoma Tumorigenicity and Metastasis 
Chiu-Ling Chen, Jaw-Ching Wu, Guan-Yu Chen, Pei-Hsiang Yuan, Yen-Wen Tseng, Kuei-Chang Li, Shiaw-Min Hwang, Yu-Chen Hu 
Molecular Therapy 
Volume 23, Issue 1, Pages (January 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Expression profiles of miR-122 and miR-151. (a) Endogenous miR-122 levels in primary human hepatocytes (HH), HCC and non-HCC cell lines. (b) Endogenous miR-151 levels. (c) Schematic illustrations of BV vectors, the expressed miR-151 sponge and target miRNA. (d) miR-122 levels after BV transduction. (e) miR-151 levels after BV transduction. The miR-122/miR-151 levels were determined by TaqMan qRT-PCR using U6 small nuclear RNA as an internal control. For endogenous expression, the data were normalized to those in HH. For miRNA levels after BV transduction, the data were normalized to those of mock-transduced cells. All data represent the means ± SD of three independent culture experiments. CMV, CMV promoter; W, WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence; pA, polyadenylation signal; d2EGFP, destabilized green fluorescent protein.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Sustained miR-122/miR-151 modulation in Mahlavu cells by the hybrid BV vectors. (a) Illustrations of BV vectors. (b) miR-122 expression. (c) miR-151 suppression. The miR-122/miR-151 levels at different times were measured by qRT-PCR as in Figure 1 and normalized to the levels at 1 dpt. All data represent the means ± SD of three independent culture experiments.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Effects of miR-122/miR-151 modulation on downstream protein levels in Mahlavu cells. (a) Protein levels of ADAM10, ADAM17, Bcl-w, cyclin G1 (CCNG1), IGF-1R, and SRF. (b) Protein levels of RhoGDIA, FAK/PTK2, Cdc42-GTP, Rac1-GTP, and RhoA-GTP. Mahlavu cells were transduced with the BV vectors and analyzed by western blot at 1 dpt. The band intensity in each group was normalized to that in the mock-transduced cells to yield the relative quantity. All data represent the means ± SD of three independent culture experiments.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Effects of miR-122/miR-151 modulation on Mahlavu cell behavior. (a) Proliferation rates. (b) Anchorage-independent growth. (c) Apoptosis. (d) Motility. (e) Migration and invasion. For proliferation rate analysis, the cells were cultured and passaged at 6 and 11 dpt. Cell densities were measured by the BrdU proliferation assay and are expressed as OD450. For the anchorage-independent growth determined by colony formation assay, the colony number of each group was counted and normalized to that of the Mock group. Cell apoptosis was analyzed by Annexin V-PE/7-AAD staining coupled with flow cytometry. Percentages of apoptotic (lower right) and late apoptotic/dead (upper right) cells are shown. The cell motility, as judged by F-actin polymerization and hence stress fiber assembly, was determined by rhodamine-phalloidin staining. Images representative of three independent culture experiments are shown. Migration/invasion was evaluated using transwell assays at 1 dpt. All quantitative data are means ± SD of three independent culture experiments.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Effects of miR-122/miR-151 modulation on HCC tumorigenesis. (a) Tumor growth (left panel) and tumor volume at 30 days after BV injection (right panel). (b) In vivo cell proliferation analysis by Ki-67 immunostaining. (c) In vivo apoptosis analysis by caspase-3 immunostaining. (d) MMP-2 immunostaining. (e) MMP-9 immunostaining. Mahlavu cells were injected subcutaneously into nude mice and tumor volume reached ≈10 mm3 in 7 days. The mice received intratumoral injection of 100 μl PBS or BV vectors (n = 5–8) at this time (defined as day 0). The tumor volume was recorded for another 30 days and the data represented means ± SD. All mice were sacrificed at day 30 and the tumor sections were subjected to immunohistochemical staining and DAPI counterstaining. The images representative of sections from five animals are shown. The numbers of Ki-67+, caspase-3+, MMP-2+, or MMP-9+ cells were quantified from 15 fields (from five tumors each group) and divided by the total number of cells (DAPI+ cells) to yield the percentage of specific protein-positive cells.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Gross and microscopic views of metastatic nodules in nude mice. (a) Pictures of metastatic nodules (indicated by arrows) in the lung of nude mice. (b) H&E staining of lung sections. CT26 or Mahlavu cells were injected into nude mice via tail vein and the lungs were removed at week 8 for visual observation and sectioning/H&E staining.
Molecular Therapy  , 79-88DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions