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1.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 1. Expression of miR-93 in human breast carcinoma specimens. (A) RNAs were isolated from paraffin blocks of human breast carcinoma specimens and the benign breast tissues, followed by real-time PCR analysis of miR-93 levels. The breast carcinoma tissues expressed significantly higher levels of miR-93 than the benign tissues. (B) RNAs were isolated from paraffin blocks of human breast carcinoma specimens with lymph-positive (metastasis) and the benign breast tissues followed by analysis of miR-93 levels. The tumor tissues expressed significantly higher levels of miR-93.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
2.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 3. MiR-93 enhanced breast cancer metastasis to the lung. (A) Mock- or miR-93-transfected MT-1 cells (2 × 105) were injected into the tail vein of CD-1 nude mice (n = 20). Six weeks after the injection, six mice in the miR-93 group developed visible tumors in the lungs, but only one in the control group. Typical metastatic lesions in the lungs are shown (arrows). (B) H&E staining of lungs from mock and miR-93 mice showed metastasis lesions in the miR-93 lungs (arrows). (C) The sections were immunohistochemically stained with antibody against Ki67. The miR-93 tumor sections showed higher levels of Ki67 staining than the control group. (D) The sections were also probed with antibody against E2F4. The miR-93 tumor sections showed E2F4 staining, which was not detected in the control group. (E) DNA was isolated from lung tissues and subjected to PCR to amplify the CMV promoter to indicate metastasis of the tissues. Expression of miR-93 promoted metastasis.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
3.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 7. LATS2 mediates the effects of miR-93. (A) The MT-1 cells (1 × 105 cells/ml/well) were transfected with siRNA oligos targeting LATS2 or a control oligo. The cells were seeded onto a 12-well plate and incubated in serum-free medium at 37°C for 7 days. The survived cells were counted. Transfection with siRNAs increased cell survival. Data are mean ± SD (n = 4). (B) The MT-1 cells transfected with siRNA oligos targeting LATS2 or a control oligo were subjected to invasion assays at 37°C for 48 h. Transfection with anti-miR-93 inhibited cell invasion. Transfection with siRNAs increased invasion. Error bars, SD (n = 4). (C) Ectopic expression of LATS2 in the miR-93 cells reversed miR-93 effect on cell survival. (D) The miR-93-expressing cells were transfected with LATS2 and a control vector. The cells were suspended in 100 ml serum-free medium and loaded in the transwell insert containing Matrigel, followed by incubation at 37°C for 60 h for invasion assays. Transfection with LATS2 partially reversed the effect of miR-93 on cell invasion. (E) Ectopic expression of LATS2 in the miR-93 cells reversed miR-93 effect on tube formation.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
4.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 6. Repression of LATS2 expression in the miR-93 tumors. (A) Sections of tumor from mice injected subcutaneously with miR-93- or mock-MT-1 cells were probed with LATS2 primary goat antibody in 10% goat serum, followed by probing with anti-goat IgG. There were higher levels of LATS2 staining (arrows) in the mock tumor section compared with the miR-93 tumor sections. (B) To examine the metastatic tumors, sections from the lungs were probed with anti-LATS2 antibody. The levels of LATS2 were lower in the miR-93 group than in the control group. (C) Sections from human breast carcinoma and normal tissues (N) were subjected to immunohistochemistry for LATS2 expression. LATS2 was detected in the duct structure (open arrows) but not in the tumor mass (closed arrows). (D) Top: Cell lysates prepared from different human breast cancer cell lines were subjected to protein gel blot analysis probed with anti-LATS2 and anti-actin antibodies. LATS2 was detected in the benign breast cell line MCF-7. Right: the levels of miR-93 were analyzed by real-time PCR. The MCF-7 cells expressed significantly lower levels of miR-93 than the other breast cancer cell lines.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
5.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 4. MiR-93 promotes cell survival and invasion. (A) The MT-1 cells transfected with miR-93 or mock (2.5 x 105 cells per 1 mL) were seeded onto Petri dishes and tissue culture dishes and were maintained in a serum-free medium at 37°C and 5% CO2 for 7 d. The cells were counted at the end of seventh day. The miR-93 transfected cells were seen to survive better. Error bars indicated standard deviation (SD) with n = 5. (B) The miR-93 and mock cells (1 x 105) suspended in 100 μl serum-free medium were loaded onto the insert and incubated at 37°C for 48 h. The invasive cells were stained blue and were counted in six fields of views/membrane using a light microscope. Error bars indicate SD (n = 6). (C) MT-1 cells were transiently transfected with anti-miR-93 oligos or control oligos with random sequence. The cultures were maintained in tissue culture dishes in serum-free DMEM for 5 d, followed by microscopic examination and photographed. The number of cells was counted for statistical analysis. Error bars indicate SD (n = 6). (D) MT-1 cells were transiently transfected with anti-miR-93 oligos or control oligos with random sequence or with anti-miR-93 plasmid or control vector. The cells (1 x 105) were subjected to invasion assays at 37°C for 64 h. Transfection with anti-miR-93 inhibited cell invasion.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
6.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 5. Targeting analysis of LATS2 by miR-93. (A) Computational analysis indicated that miR-93 potentially targeted LATS2 located at nucleotides 3955–3977 and nucleotides 4058–4078. (B) Cell lysate prepared from miR-93- or mock-transfected MT-1 cells was analyzed on protein gel blot for LATS2 expression. LATS2 expression was repressed by miR-93 transfection. Staining for β-actin from the same membrane confirmed equal loading. (C) Top: two luciferase constructs were generated, each containing a fragment harboring the target site of miR-93, producing Luc-Lats-3955 and Luc-Lats-4057. Mutations were also generated on the potential target sequence (red color), resulting in two mutant constructs Luc-Lats-3955-mut and Luc-Lats-4057-mut. Bottom: MT-1 cells were co-transfected with miR-93 and a luciferase reporter construct. The luciferase reporter vector was used as a control. Asterisks indicate significant differences. Error bars, SD (n = 3). (D) Top: alignment of the miR-93 target sites on LATS2 located at nucleotides 3955–3977 across Homo sapiens (NM_014572), Pan troglodytes (XM_509566), Bos taurus (XM_584953), Mus musculus (NM_015771), Gallus gallus (XM_417143), Xenopus (Silurana) tropicalis (NM_001102704), Rattus norvegicus (NM_001107267) and Taeniopygia guttata (XM_002191071). The seed regions for miR-93-LATS2 interactions are boxed. Bottom: conservation of the sequences is shown across all species. (E) Top: alignment of the miR-93 targeting LATS2 located at nucleotides 4058–4078 across the same species. The seed regions for miR-93-LATS2 interactions are boxed. Bottom: conservation of the sequences is shown across all species.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.
7.

Figure. From: MiR-93 enhances angiogenesis and metastasis by targeting LATS2.

Figure 2. Expression of miR-93 enhanced breast cancer angiogenesis. (A) Top: sections of tumor from mice injected with miR-93- or mock-MT-1 cells were probed with CD34 primary rat antibody in 10% goat serum with TBS, followed by probing with anti-rat IgG (positive). The negative or control tumor sections were probed with anti-rat polyclonal IgG antibody only. There were increased levels of tumor-associated vascularization (as shown with arrows), where the miR-93 expression in MT-1 cells was enhanced. Scale bars, 50 µm. Bottom: the average count of the number of the blood vessels found in the tumor sections at four randomly selected fields. Error bars, SD (n = 4), **p < 0.0001. (B) Mock- and miR-93-transfected cells were co-cultured with endothelial cells YPEN or EOMA or lung cells BEAS-2B at the ratio of 2:1. After 2 d of culture, the co-cultured cells were photographed. The miR-93 cells displayed higher capacities in expansion than the vector-transfected cells. As a result, the endothelial cells were squeezed into small islands by the miR-93 cells. The miR-93 cells could mix well with the lung cells compared with the mock control. (C) MT-1 cells stably transfected with miR-93 or mock were seeded on tissue culture plates at a cell density of 1.5 x 105 cells/well on 6-well plates. After overnight culture, endothelial cells YPEN were inoculated on top of the existing cultures (6 x 104 cells/well). After an additional overnight culture, the endothelial cells were able to spread over the miR-93 cultures, but could not spread over the mock cultures. (D) The miR-93- and mock-transfected cells were mixed with YPEN cells and inoculated in Matrigel, followed by examination of tube formation. The YPEN cells formed larger complexes and longer tubes when mixed with the miR-93 expressing cells compared with the mock-transfected cells.

Ling Fang, et al. Cell Cycle. 2012 December 1;11(23):4352-4365.

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