Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer

MicroRNAs are a novel family of small non-coding RNAs that regulate the expression of several genes involved in normal development as well as human disorders including cancer. Here we show that miR-874 plays a tumor suppressor role in non-small cell lung cancer (NSCLC) in vitro and in vivo. In silico target prediction analysis revealed numerous genes associated with tumor progression including MMP-2 and uPA as the putative target genes of miR-874. Our preliminary in situ hybridization experiments demonstrated the diminution of miR-874 expression in lung cancer tissues compared to their normal counter parts. Overexpression of miR-874 in CD133-positive cancer stem cell (CSC) population led to a significant loss in CSC-phenotype and enhanced sphere de-differentiation into epithelial-like cells. Restoration of miR-874 expression drastically reduced cell invading ability in comparison to mock and control-miR-treated cells by suppressing the protein levels of MMP-2 and uPA. In in vivo experiments, miR-874 treatment decreased orthotopic tumor growth in nude mice compared to mock and control-miR treatments. Further, the immunoreactivity of human anti-MMP-2 and anti-uPA was significantly reduced in tumor sections from mice that received miR-874 treatment. In conclusion, our study highlights the possible tumor suppressor role of miR-874 in NSCLC-initiating cells and suggests miR-874 as a potential target in the treatment of NSCLC

p-MMP-2 combined with radiation enhances apoptosis <i>in vivo</i>.

<p><b>A,</b> Immunohistochemical analysis of brain sections using anti-MMP-2, anti-VEGF and anti-pFAK antibodies. Sections were photographed (60×). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (insets). <b>B,</b> Tissue sections of mice were evaluated with the TUNEL assay according to manufacturer's instructions and photographed under fluorescent microscopy (60×). For the negative control, samples were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture (insets). <b>C,</b> siRNA against MMP-2 inhibits U251 tumor cell invasion <i>in vivo</i>. H&E staining was performed according to standard protocol, and representative pictures of tumor sections from mock, pSV, p-MMP-2-treated mice are shown (20× and 60×). <b>D</b>, Immunohistochemical analysis of brain sections using anti-human nuclei (HuNu) antibody, a histological marker for identification of human cells (a specific human nuclear antigen). Entire brain sections were photographed (4×; middle row); shown on the top row is a non-tumor region (40×; top row); and shown on the bottom row is tumor and non-tumor overlapping region (40×; bottom row). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (inset).</p

p-MMP-2 combined with radiation inhibits tumor growth <i>in vivo</i>.

<p>U-251 (1×10⁶) cells were injected intracerebrally into athymic mice. After ten days, animals were separated into five groups and were treated on alternate days with intracerebral injections of p-SV or p-MMP-2 for a total of 4 doses (60 µg per dose) and 2 doses of radiation (4 Gy per dose) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020614#s4" target="_blank">Materials and Methods</a>. Six weeks after the experiment was initiated, mice were euthanized with intracardiac perfusion of PBS, followed by formaldehyde. The brains were then removed. <b>A,</b> Six weeks after the experiment was initiated, an intraperitonal injection of 2.5 mg D-luciferin sodium salt diluted in 50 µL of PBS was given, and animals were photographed under the IVIS camera for fluorescent light emission. The brains were removed and fixed in 10% phosphate-buffered formaldehyde, and the fixed tissue samples were then processed into paraffin blocks. Brain sections (5 µM thick) were stained with hematoxylin and eosin (H&E), and photographed under a light microscope (4× and 40×). <b>B,</b> Every fifth or sixth brain section (5 µM thick) was stained with H&E solution, and the tumor masses (H&E-stained) were manually traced on the microscope attached computer screen. Areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Silver Spring, MD). The total tumor volume was calculated as the summed area on all slices, multiplied by the slice separation. <i>Columns</i>: mean of area of tumor portion of all mice in the group (n = 8); <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock, p-SV or irradiated controls.</p

p-MMP-2 inhibits radiation-enhanced tumor culture medium-induced microtubule network formation in endothelial cells and downregulates expression of angiogenesis-associated molecules.

<p><b>A,</b> Human microvascular endothelial cells (5×10⁴) were seeded in 96-well plates and cultured with conditioned medium collected from U-251 and U-87 glioma cells transfected with mock, p-SV, and p-MMP-2, and irradiated as described earlier. 24 h after radiation treatment, the cells were washed, fixed and stained with Hema-3 and photographed. Percentages of branches were quantified by counting five fields in each condition. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or irradiated controls. <b>B,</b> U-251 and U-87 transfection and radiation was carried out as described earlier. 24 h after radiation, whole cell lysates were prepared and analyzed by Western blotting for the angiogenic molecules VEGF, VEGFR-2 and p-VEGFR-2 as well as p-FAK, FAK, p-p38 and p38. GAPDH served as a loading control.</p

Radiation enhances MMP-2 and p-MMP-2 inhibits MMP-2 activity and expression in glioma cell lines.

<p><b>A,</b> U-251 and U-87 cells were irradiated with 0–12 Gy X-ray, incubated for 24 h, and conditioned medium collected. MMP-2 activity was determined by gelatin zymography. The band intensities of MMP-2 activity were quantified by densitometry. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from non-irradiated (0 Gy) conditioned medium. <b>B,</b> U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (1, 2 or 3 µg). After 72 h of incubation, conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 and MMP-9 levels by Western blotting. The band intensities of MMP-2 activity as well as MMP-2 and MMP-9 protein levels were quantified by densitometry and normalized with the intensity of the mock bands. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or p-SV.</p

p-MMP-2 transfection inhibits radiation-enhanced MMP-2 activity and expression levels as well as cell viability.

<p><b>A,</b> U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (2 µg), and after 72 h of incubation, cells were irradiated with 0, 2, 4, 6 or 8 Gy and incubated for a further 24 h. Conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 levels by Western blotting. <b>B,</b> Total RNA was used to determine MMP-2 mRNA transcription levels by RT-PCR with gene-specific primers. GAPDH served as a loading control. <b>C,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2 and irradiated as described above. 24 h after radiation, the cells were fixed and processed to visualize MMP-2 expression. The cells were mounted using mounting media with DAPI to visualize the nucleus. <b>D,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated for 72 h after transfection. After a another 24 h of incubation, cell viability was analyzed by MTT assay (absorbance read at 550 nm). <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, significant difference from mock, p-SV or irradiated controls.</p

p-MMP-2 transfection in combination with radiation inhibits glioma cell migration.

<p>U-251 and U-87 cells were cultured for formation of spheroids as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020614#s4" target="_blank">Materials and Methods</a>. Spheroids were then transfected with mock, p-SV or p-MMP-2, and followed by irradiation as described earlier. At the end of the migration assay, spheroids were fixed and stained with Hema-3. Migration of cells from spheroids to monolayers was measured using a microscope calibrated with a stage and ocular micrometer. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock, p-SV or irradiated controls.</p

p-MMP-2 transfection inhibits radiation-enhanced glioma cell invasion.

<p>U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated as described earlier. Cells were trypsinized and counted, and 5×10⁵ cells from each treatment condition were allowed to invade transwell inserts containing 12-µm-pore polycarbonate membranes pre-coated with Matrigel for 24 h at 37°C. Afterwards, cells were fixed and stained with Hema-3. Cells that had migrated to the lower side of the membrane were photographed under a light microscope at 20× magnification. Percentages of invading cells were quantified by counting five fields from each treatment condition. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or irradiated controls.</p

Effect of treatment with bicistronic constructs on the brain tumors in nude mice pre-injected with xenograft cells.

<p>(A) shRNA mediated regression of pre-established tumor growth. Hematoxylin and eosin staining performed on the brain sections obtained from various groups of animals revealed a prominent tumor reduction after MMP-9/uPAR (MU-sh) or MMP-9/cathepsin B (MC-sh) treatments. Each group consisted of 6 animals. Yellow curved line in 4910 and 5310 control brain sections indicate the tumor area. (B) Normal nude mice brain section stained with hematoxylin and eosin. (C) Immunohistochemical comparison of control, MU-sh and MC-sh-treated nude mice which are pre-injected with 5310 cells (and sacrificed 2–3 weeks prior to the end of the treatment), to analyze the expression of αVβ3, α6β1 and α9β1 integrin heterodimers. Brown staining indicative of αVβ3, α6β1 or α9β1 integrin expression was reduced in MU-sh and MC-sh treated sections compared to controls. n = 6. Scale bar = 50 µm. (D) Untreated 4910 and 5310 <i>in vivo</i> tumors were prominently stained for platelet endothelial cell adhesion molecule (PECAM-1) whereas the intensity of brown staining was reduced in 5310 <i>in vivo</i> tumors after MU-sh and MC-sh treatments.</p

Involvement of α9β1 integrin on the migration potential of xenograft cells and the effect of bicistronic constructs on the glioma xenograft cell proliferation.

<p>(A) RT-PCR of 5310 and 4910 cells transfected with uPAR (U-fl), MMP-9 (M-fl), and cathepsin B (C-fl) expressing plasmid DNAs was performed as per standard protocols. Prominent increase in respective mRNA expressions noticed after U-fl, M-fl, and C-fl treatments. Further, quantification of the RT-PCR data (B) revealed significant increases in uPAR mRNA and MMP-9 mRNA expression after treatments with M-fl and C-fl, respectively in both the xenograft cells. (C) Wound healing indicative of increased migration potential noticed after treatments with U-fl, M-fl, and C-fl in 5310 cells was reduced with the same treatments in presence of α9β1 antibody. Photographs are the representative images obtained from three independent experiments. (D) Quantification of wound healing assay. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments. Error bars indicate SEM. *<i>p</i><0.05 vs. control. ^#<i>p</i><0.05. (E) FACS analysis showing reduced α9β1 integrin levels in 4910 cells after MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) treatments. (F) Clonogeneic assay depicting the reduced proliferation of 4910 and 5310 cells after MU-sh and MC-sh treatments. After 14 days of incubation, the colonies containing more than 50 cells were counted. Images shown are the representatives obtained from three independent experiments. (G) MTT assay showing significant reduction in the proliferation of 4910 cells from Day 4 to Day 6 after MU-sh and MC-sh treatments. n = 3. *<i>p</i><0.05 vs. control.</p