MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1

Abstract

Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

Keywords: CCND1; metastatic; miR-195; microRNA; osteosarcoma.

Figure 1. Different microRNA expression between F4 (Labeling Dye: Cy3) and F5M2 (Labeling Dye: Cy5) by the MicroRNA microarray

The images are displayed in pseudo colors so as to expand visual dynamic range. In the Cy3 and Cy5 intensity images, as signal intensity increases from 1 to 65,535 the corresponding color changes from blue to green, to yellow, and to red. In the Cy3/Cy5 ratio image, when Cy3 level is higher than Cy5 level the color is green; when Cy3 level is equal to Cy5 level the color is yellow; and when Cy5 level is higher than Cy3 level the color is red.

Figure 2. miR-195 expression and miR-195 oligonucleotides transfection in osteosarcoma cells

(A) The expression of miR-195 in F4 and F5M2 cells was analyzed by real-time RT-PCR (P = 0.001).(B) SOSP-9607 cells (top) and U2-OS cells (bottom) were observed with white bright (left) and green fluorescence assay (right) in the same vision using fluorescence microscopy (100×;400×).(C) miR-195 expression levels were evaluated by real-time RT-PCR analysis in transfected SOSP-9607 cells. (D) miR-195 expression levels were assayed in transfected U2-OS cells. U6 was used as an internal loading control to normalize the results. The data were presented as the means ±SD, Columns, mean of four independent experiments; bars, SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3. miR-195 inhibits migration and invasion of osteosarcoma in vitro

(A) Representative photographs of migrated and invaded SOSP-9607 or U2-OS cells on the membrane at a magnification of 200×.(B) Quantitative results for the migration and invasion ability of each group of SOSP-9607 or U2-OS cells were shown as migrated and invaded cell number, 16 h after incubation. (C,D) SOSP-9607 or U2-OS cells were seeded in 6-well plates and wounds were created on the next day. Photographs were taken at hour 0, 24, and 48 h, respectively, after the wound was made. The data were presented as the means ±SD, Columns, mean of four independent experiments; bars, SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4. MiR-195 inhibits tumor growth and metastasis of osteosarcoma in vivo

(A)I, Representative photographs of tumors on the right leg of mouse; II, Representative photographs of orthotopic tumors harvested 42 days after inoculation. III, Representative macroscopic pictures of mouse lungs, 42 days after inoculation. (B) Representative photographs of H&E stained spontaneous orthotopic tumors at a magnification of 400×. (C) Representative photographs of H&E stained spontaneous lung metastases at a magnification of 400×. (D) Tumor growth curves measured after the inoculation. The length (L) and width (W) of tumor measured every 7days after inoculation, and the volume of tumor was calculated according to the formula: volume = 1/2×L×W². (E) Orthotopic tumor weights 42 days after inoculation. Data are presented as means±SD. (F) Graph displaying the total number of tumor nodules per lung in three groups. Data are presented as means±SD. (G) 42 days after inoculation, miR-195 expression levels in orthotopic tumors were tested and showed in relative miR-195 levels. The data were presented as the means ±SD, Columns, mean of four independent experiments; bars, SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5. Expression level of miR-195 in 107 OS frozen samples and 99 osteosarcoma formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues

(A) Relative levels of miR-195 in 107 surgical specimens of osteosarcoma and matched adjacent noncancerous tissues(NAT) was quantified by real-time RT-PCR. Data were presented as log2 fold change (ΔΔCt values, Tumor/noncancerous tissues, T/N). (B) Means of miR-195 relative levels for 107 surgical specimens of osteosarcoma and the matched adjacent noncancerous tissues. Data were presented as 2^−ΔΔCt values (p=0.291>0.05). (C) Decreased expression of miR-195 was correlated with poor survival in osteosarcoma patients. Log rank tests show that patients with high miR-195 expression survived statistically significantly longer (p = 0.0012) than those with low miR-195 expression. The median miR-195 expression level (0.92479) in the tumor samples was chosen as the cut-off point. (D) Decreased expression of miR-195 was correlated with poor survival in osteosarcoma FFPE patients. Log rank tests show that patients with high miR-195 expression survived statistically significantly longer (p = 0.019) than those with low miR-195 expression. The median miR-195 expression level (5.0214) in the tumor samples was chosen as the cut-off point. The data were presented as the means ±SD, Columns, mean of four independent experiments; bars, SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6. MiR-195 may target CCND1 in osteosarcoma

(A) Sites of complementarity sequences between microRNAs and CCND1 mRNA. (B) The results of Western blot showed that miR-195 interacted with CCND1 and negatively regulated its expression at the translational level. (C) The results of real-time revealed that miR-195 had no effect on CCND1 in mRNA level. (D) Luciferase assays indicated that miR-195down-regulates the expression of CCND1. Relative expression of the firefly Luciferase expression was standardized for transfection control, Renilla luciferase. PMIR-REPORT™ luciferase (pMIR-R-L, Promega) was used as empty vector. All experiments were repeated three times in triplicate. Columns denotes mean of three independent experiments and bars stands for SD value. (E) Expression of Cyclin D1 was analyzed in osteosarcoma tissues with immunohistochemistry staining. The data were presented as the means ±SD, Columns, mean of four independent experiments; bars, SD; * P < 0.05, ** P < 0.01, *** P < 0.001.