MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells

Abstract

Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin.

Fig 1. Endogenous expression of miR-21 in osteosarcoma-derived cell lines.

Logarithmically growing cells were harvested to perform a miRNA Northern blot. (A) One representative Northern blot measuring miR-21 levels of six osteosarcoma-derived cell lines is shown. U6 served as loading control. (B) Results of two to three Northern blots were quantified using Image Quant 5.0. The miR-21/U6 ratios were determined and depicted as a block diagram. Means ± SEM are shown.

Fig 2. Influence of reduced miR-21 activity on cell proliferation and migration.

U2OS were transfected with either pBabepuro (pBp), pBabepuro luciferase (pBluc) or a miR-21 sponge. (A) Cell proliferation was measured by counting the cells daily. A representative growth curve is depicted (Means ± SEM). Using exponential growth equation, doubling times were calculated using Graph Pad Prism 5 (Means ± SEM). Values from six experiments of three clones are depicted and significance was calculated. **p < 0.01 (B) A scratch assay with U2OS cells transfected with the indicated plasmids was performed. Representative pictures of cells 1 and 12 hours after scratch was set are shown (upper panel). Migration velocity was calculated using linear regression. Means ± SEM of three independent experiments are shown (lower panel). (C) Transfection efficiency was verified by measuring luciferase activity of the selected mixed clones (MC1-3) (Means ± SEM).

Fig 3. Influence of reduced miR-21 activity on the sensitivity of U2OS cells towards chemotherapeutic drugs.

Logarithmically growing MCs were incubated for 48 hours to the indicated increasing concentration of cisplatin (A), methotrexate (B) or doxorubicin (C) and a cell viability assay was performed (left panel). Means ± SEM of a representative experiment are presented as curves. The IC50 values of three to four experiments are depicted as means ± SEM and an unpaired t-test was performed; *p < 0.05 (right panel).

Fig 4. Influence of ectopic miR-21 expression on cell proliferation and cisplatin sensitivity of MG63 cells.

Cells were transfected with pBp or primiR-21 and single clones were selected. (A) A representative growth curve of one clone is depicted as means ± SEM. Doubling times of six experiments with three different clones were calculated, the means ± SEM are presented in a column graph and analyzed using unpaired t-test. **p < 0.01. (B) A cell viability assay of a representative single clone obtained after transfection with the indicated plasmid is presented as means ± SEM. IC50 values of three different clones were calculated. Means ± SEM summarize the data. The significance was determined using an unpaired t-test. **p < 0.01 (C) MiRNA Northern blots of the indicated clones obtained after transfection with primiR-21or pBp are shown.

Fig 5. Influence of ectopic miR-21 expression on potential target proteins.

Protein lysates of cell clones obtained after transfection and selection of MG63 cells with either pBp or primiR-21 were analyzed using an immunoblot. Protein expression was calculated using Image quant 5.0 software. The highest value was arbitrarily set as 1. GAPDH was used as reference value. (A) Means ± SEM of two to three independent experiments are depicted. The analyzed protein is indicated as title. (B) Calculated levels of the indicated proteins in the clones overexpressing miR-21 were compared to the one in the corresponding control clones. Means ± SEM are presented. Significance was calculated using unpaired t-test. *p < 0.05; **p < 0.01.

Fig 6. Influence of Spry2 and Spry4 expression on proliferation of miR-21 overexpressing cells.

Prior to growth curve analysis, MG63 empty vector or primiR-21 expressing clones were infected with either an adenovirus expressing lacZ (control), Spry2 or Spry4. (A) Representative curves depicting means ± SEM are presented. The ectopically expressed genes are indicated as legend. (B) Doubling times of three to six independent experiments were calculated and depicted as means ± SEM. Significance was determined by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001 (C) Protein lysates of the infected clones were analyzed by performing an immunoblot with the indicated antibodies.

Fig 7. Influence of ectopic Spry protein expression on cisplatin-sensitivity of MG63 cells with different miR-21 expression levels.

MG63 clones expressing an empty vector (pBp) or a primiR-21 were infected with adenoviruses expressing the indicated proteins. LacZ was used as control protein. (A) The cell viability assays of representative clones infected with the indicated proteins (see legend) are shown as curves (Means ± SEM). (B) IC50 values of three different clones are summarized as means ± SEM and a t-test was performed. *p < 0.05.