PLK1 Knockdown Inhibits Cell Proliferation and Cell Apoptosis,and PLK1 Is Negatively Regulated by miR-4779 in Osteosarcoma Cells

Abstract

Polo-like kinase 1 (PLK1) is a ubiquitous serine/threonine protein kinase. It is reported to be involved in the occurrence and progression of various human cancers. In the present study, we explored the role and molecular mechanism of PLK1 in the proliferation of osteosarcoma (OS) cells. We found that PLK1 expression was higher in MG63/Dox cells than in MG63 cells, while inhibiting or interfering with the level of PLK1 suppressed cell proliferation of MG63/Dox cells. TargetScan analysis predicted that miR-4779 would interact with the 3′-UTR of PLK1 mRNAs and also inhibit cell autophagy of MG63/Dox cells. The data demonstrated that miR-4779 negatively regulates the expression of PLK1, and both miR-4779 and PLK1 regulate cell proliferation and cell apoptosis of MG63/Dox cells, processes that are involved in the drug resistance of OS cells.

Introduction

Osteosarcoma (OS) is a commonly seen cancer with bone malignancies that occurs in teenagers and adolescents (Misaghi et al., 2018), accounting for about 5% of pediatric tumors (Durfee et al., 2016; Omer et al., 2017). OS is derived from mesenchymal cells, and its rapid tumor growth is due to the direct or indirect formation of tumor-like tissue and bone tissue (Hu et al., 2016; Liu et al., 2017). However, the molecular biological mechanism of OS still has not been clearly described.

Reversible protein phosphorylation regulated by protein kinases is the most basic molecular regulation mechanism in eukaryotes. Polo-like kinase 1 (PLK1) is a ubiquitous serine/threonine protein kinase with highly conserved structure and function (Lu et al., 2013). It plays an important role in cell cycle progression (Yamaguchi et al., 2009), centromere maturation, cytoplasmic separation in mitosis, and maintenance of genomic stability (Elez et al., 2003; Bucur et al., 2014). It has been reported that PLK1 plays an important role in the occurrence and development of various tumors, and it is also recognized as an important target for tumor treatment (Driscoll et al., 2014; Raab et al., 2018). In OS treatment, PLK1 has been proposed as a new prognostic marker for malignancies (Duan et al., 2010; Cheng et al., 2015). Sero et al. (2014) reported that PLK1 expression was higher in OS clinical samples than in normal human tissue; moreover, PLK1 inhibitor NMS-P937 decreased clonogenic and migration ability of human OS cell lines either as a single agent or in combination with OS chemotherapy drugs. Inhibiting PLK1 expression induced the suppression of OS cell proliferation in vitro and in vivo (Liu et al., 2011). It is regarded as a promising treatment target in human OS (Yen et al., 2014; Chou et al., 2016). However, the molecular mechanism of tumorigenesis and progression of OS has not been fully clarified.

PLK1 participates in the cell growth and invasion of renal cancer cells (Zhang et al., 2013), colorectal cancer cells (Han et al., 2012), and human esophageal cancer cells (Bu et al., 2008). PLK1 overexpression is detected in various human cancer cells, and the pathogenic mechanism has also been initially discussed in various cancers. For instance, downregulation of PLK1 increased drug sensitivity of breast cancer cells in vitro and in vivo (Spankuch et al., 2006). In nonsmall cell lung cancer, PLK1 inhibitor BI-6727, in combination with radiation, significantly decreased tumor growth. Interference with PLK1 enhanced radiosensitization by modulating DNA repair proteins, including DNA-dependent protein kinase (DNAPK) and topoisomerase II alpha (TOPO2A) (Yao et al., 2018). Phosphorylation of PLK1-Ser99 is dependent on the PI3K/Akt pathway, which is required for metaphase–anaphase transition. Moreover, cell apoptosis and proliferation of pancreatic cancer cells were regulated by the PI3K/Akt pathway through PLK1 (Mao et al., 2016).

Several PLK1 inhibitors are widely used in the study and clinical treatment of human cancers. For example, the PLK inhibitor TAK-960 is reported to possess antitumor activity against colorectal cancer (Klauck et al., 2018). BI2563, a potent and selective inhibitor of PLK1, is a widely used dihydropteridinone compound in preclinical and clinical studies. It inhibits PLK1 by binding to the ATP-binding site and cross-suppresses PLK2 and PLK3 simultaneously. It has been found that BI2536 and cisplatin synergistically suppress cell proliferation of gastric cancer cells (Lian et al., 2018). In the present study, we chose BI2563 as the positive control PLK1 inhibitor to treat OS cells. We investigated the role of PLK1 and explored its regulating mechanism in the progression of OS cancer cells, which may provide a theoretical basis for clinical treatment of OS.

Materials and Methods

Cell lines

The human OS MG63 cell line was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The doxorubicin-resistant OS MG63/DOX cell line was selected and screened with increasing doses of doxorubicin by exposing drug-sensitive MG63 cells in a stepwise manner. Cells were maintained and cultured in vitro in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and streptomycin and penicillin (1 × ) in a cell incubator under a humid atmosphere with 5% CO₂ at 37°C.

Reagents

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) reagent was obtained from Sigma, Inc. (Saint Louis, MO). Doxorubicin (Cat. No.: D1515) was obtained from Sigma-Aldrich Corporation. The PLK1 inhibitor BI2536 was purchased from Selleck (Cat. No.: S1109). The following were obtained from Sigma: human kinase PLK1 siRNA1 (Cat. No.: SIHK1741), PLK1 siRNA2 (Cat. No.: SIHK1742), MISSION^® siRNA universal negative control #1 (Cat. No.: SIC001), MISSION microRNA mimic hsa-miR-4779 (Cat. No.: HMI1866), MISSION miRNA negative control 1 (Cat. No.: HMC0002), MISSION synthetic microRNA inhibitor hsa-miR-4779 (Cat. No.: HSTUD1866), MISSION synthetic microRNA inhibitor-negative control (NCSTUD001), and MISSION lenti-microRNA inhibitor hsa-miR-4779 (Cat. No.: HLTUD1866). PLK1 cDNA ORF Clone (Cat. No.: HG10676-ACG) and the pCMV3-N-GFPSpark Control Vector (Cat. No.: CV027) were purchased from Sino Biological Corporation.

MTT assay

Cell viability was determined by MTT assay. In one experiment, MG63/Dox cells were transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 24, 48, and 72 h. Cell viability was determined by MTT assay. In another experiment, MG63/Dox cells were treated with increasing concentrations of BI2536 for 24, 48, or 72 h. The concentrations of BI2536 were 5, 25, and 100 nM. Four hours before treatment, 10 μL of 5 mg/mL MTT reagent was added to the culture medium, and the absorbance of each well in a 96-well plate was read at 490 nm.

Cell transfection

MG63 cells and MG63/Dox cells at a concentration of 1 × 10⁵ cells per well were plated into a 24-well plate and cultured overnight for adherence to the plate. Cells were then transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for an indicated time, such as 24, 48, and 72 h. The PLK1-specific siRNAs were transfected using Lipofectamine 2000 (ThermoFisher, MA) according to the manufacturer's protocol.

Flow cytometry assay

MG63/Dox cells were plated into six-well plates and transfected with PLK1 siRNA1 and negative control siRNA for 48 h. The cell cycle was analyzed by flow cytometry using the propidium iodide staining method. In brief, the single-cell suspension of 1 mL at a density of 2 × 10⁶ cells/mL was prepared. Cells were fixed with 75% ethanol at 4°C for 1 h, centrifuged at 1000 rpm for 5 min, and resuspended with 1 mL phosphate-buffered saline (PBS). Then, 100 μL of 200 μg/mL RNase A was added and cultures were incubated for 30 min at 37°C. Finally, 100 μL of 1 mg/mL propidium iodide was added and incubated for 5 min at room temperature.

β-Galactosidase assay

Cell senescence was detected by β-galactosidase (senescence-associated β-galactosidase [SA-β-gal]) activity as described (Chou et al., 2016). In brief, MG63 cells were transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 72 h. MG63 cells were fixed and stained with X-gal and washed three times with PBS buffer. Cells were photographed using an Olympus IX51 inverted microscope.

Antibodies

The following primary antibodies were used for immunoblotting: PLK1 (Cat. No.: 101119-T02, 1:1000; Sino Biological); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. No.: ab8245, 1:1000; Abcam); β-actin (Cat. No.: sc-8432, 1:1000; Santa Cruz Biotechnology); caspase-3 (Cat. No.: ab13585, 1:2000; Abcam); and poly ADP-ribose polymerase (PARP) (Cat. No.: 9542, 1:5000; Cell Signaling Technology). Secondary antibodies used were as follows: goat anti-rabbit IgG H&L HR (Cat. No.: ab205718, 1:10,000; Abcam) and goat anti-mouse IgG-HRP (Cat. No.: sc-2005, 1:10,000; Santa Cruz Biotechnology).

Western blot analysis

Cell lysates were prepared using RIPA buffer (Cat. No.: P0013K; Beyotime, Shanghai, China), and 30 μg/lane of total proteins were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All proteins were transferred into polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked with 5% solution of skimmed milk powder for 1 h at room temperature. The membrane was incubated with the indicated primary antibodies at 1:1000 and incubated at 4°C overnight. The membrane was then washed three times with 1 × Tris-buffered saline +0.1% Tween 20 buffer, each time for 10 min. The corresponding secondary antibody (at 1:10,000 dilution) was incubated for 40 min at room temperature. The membrane was washed with 1 × Tris-buffered saline +0.1% Tween 20 buffer at room temperature three times again. The bands were visualized using enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific, Inc.).

Statistical analysis

The data in the present study were analyzed using the SPSS statistical package. All results are shown as the mean ± standard error of the mean. Results were analyzed using an independent samples t-test. p-values <0.05 was considered to indicate a statistically significant difference.

Results

PLK1 levels increase in Dox-resistant OS cell lines

Drug resistance is usually observed during cancer treatment. In the present study, we used a pair of human OS cell lines as a cell model: doxorubicin-resistant subline MG63/Dox and its parental doxorubicin-sensitive subline MG63. MG63 cells and MG63/Dox cells were treated with increasing concentrations of doxorubicin for 24, 48, and 72 h, and the MTT assay was used to determine cell viability. IC50 values were then determined by GraphPad 5.0. As depicted in Figure 1A, IC50 values of MG63 cells were 182.4 nM, 114.2 nM, and 23.24 nM at 24 h, 48 h, and 72 h; and IC50 values of MG63/Dox cells were 452.6 nM, 362.2 nM, and 92.74 nM at 24 h, 48 h, and 72 h, respectively. There was a 2.48-fold, 3.17-fold, and 3.99-fold increase in the IC50 values of MG63/Dox cells relative to MG63 cells at 24, 48, and 72 h. Thus, MG63/Dox cells showed higher drug resistance to doxorubicin than that by the parental drug-sensitive MG63 cell line.

FIG. 1.

PLK1 levels are increased in Dox-resistant OS cell lines. (A) The Dox-sensitive OS cell line MG63 and Dox-resistant OS cell line MG63/Dox were treated with increasing concentrations of doxorubicin for 24, 48, and 72 h. Cell viability was determined by MTT assay, and inhibition rate was calculated by GraphPad 5.0. (B) MG63 cells and MG63/Dox cells were cultured for 24 and 48 h, and PLK1 expression was verified by western blotting analysis. In this study, GAPDH was used as an internal reference gene. The PLK1 level is also shown in the histogram on the right (**p < 0.01, between MG63 and MG63/Dox cell groups). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; OS, osteosarcoma; PLK1, polo-like kinase 1.

To identify the role of PLK1 in chemotherapy of OS cells, we measured the expression of PLK1 in MG63 cells and MG63/Dox cells. As shown in Figure 1B, the level of PLK1 was significantly higher in doxorubicin-resistant OS cells than in doxorubicin-sensitive OS cells (**p < 0.01). The data demonstrate that PLK1 expression may contribute to chemotherapeutic drug resistance to doxorubicin.

Interference with PLK1 expression suppresses cell viability of Dox-resistant OS cells

To identify the role of PLK1 in proliferation of MG63/Dox cells, PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA were used to transfect MG63 cells and MG63/Dox cells for 48 h. As depicted in Figure 2A, PLK1 was successfully suppressed by PLK1-specific siRNA1 or PLK1-specific siRNA2. Cell viability was determined by MTT assay, and the results demonstrated that cell proliferation was significantly suppressed more by PLK1 siRNA1- or PLK1 siRNA2-transfected MG63/Dox cells for 48 or 72 h than by negative control siRNA-transfected cells.

FIG. 2.

Inhibiting the expression of PLK1 suppresses cell viability of Dox-resistant OS cells. (A) The OS cell lines MG63/Dox and MG63 were cultured for 8 h, then transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 48 h. Expression of PLK1 was determined by western blotting analysis. β-actin was used as an internal reference gene. The ratios of PLK1 level are shown in the histogram. **p < 0.01, compared with negative control siRNA-transfected MG63/Dox cells. ^## p < 0.01, compared with negative control siRNA-transfected MG63 cells. (B) MG63/Dox cells were treated with BI2536 at 5, 25, and 100 nM for 48 h. Western blotting was performed to detect the expression level of PLK1. β-actin was used as an internal reference gene. The ratios of PLK1 level are shown in the histogram. **p < 0.01, compared with 0.1% DMSO-treated group. (C) MG63/Dox cells were cultured for 8 h and transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 24, 48, and 72 h. The cell viability was determined by MTT assay to test the OD490 value. (D) MG63/Dox cells were cultured for 8 h and treated with 5 nM, 25 nM, and 100 nM of BI2536 for 24 h, 48 h, and 72 h. Cell viability was determined by MTT assay. Untreated MG63/Dox cells were used as negative controls. **p < 0.01, compared with negative controls.

BI2536, a potent PLK1 inhibitor, was also used to treat MG63/Dox at 5, 25, and 100 nM for 48 h, and the level of PLK1 was determined by western blotting. As shown in Figure 2B, the level of PLK1 clearly decreased as the concentration of BI2536 increased. In addition, MG63/Dox cells were treated with 5 nM, 25 nM, and 100 nM for 24 h, 48 h, and 72 h, and the cell viability was determined by MTT assay. Cell viability was significantly inhibited by BI2536 in a dose-dependent manner.

Knockdown of PLK1 induces G2/M arrest and cell apoptosis in MG63/Dox cells

To examine whether transfection with PLK1 siRNA affected cell cycle progression of MG63/Dox cells, cell cycle analysis was performed by fluorescence-activated cell sorting (FACS) assay with the propidium iodide staining method. As shown in Figure 3A, the cell ratio reached 34.8% at G₂/M phase in PLK1 siRNA-transfected MG63/Dox cells, which is higher than the ratio of 14.2% at G₂/M phase in negative control siRNA-transfected MG63/Dox cells. All results showed that knockdown of PLK1 induced G₂/M arrest in MG63/Dox cells.

FIG. 3.

Knockdown of PLK1 induces G2/M arrest and cell apoptosis in MG63/Dox cells. (A) Cell cycle analysis was performed by FACS assay. The MG63/Dox cells were transfected with PLK1 siRNA1 and negative control siRNA for 48 h. The cell cycle was analyzed by flow cytometry using the propidium iodide staining method. The experiment was repeated twice, in triplicate. (B) MG63/Dox cells were transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 48 h. The levels of caspase-3, cleaved caspase-3, PARP, and cleaved PARP were determined by western blotting analysis. β-actin was used as an internal reference gene. (C) The levels of cleaved caspase-3 and cleaved PARP are shown in the histogram. In this study, untreated cells were used as negative controls. **p < 0.01, compared with untreated control. (D) β-Galactosidase assay. MG63 cells were transfected with PLK1 siRNA1, PLK1 siRNA2, or negative control siRNA for 72 h. The SA-β-gal staining assay was performed to test the senescence of each group of cells. The percentage of SA-β-gal-positive cells is shown in the histogram. Percentage = the number of positive cells/the total number of cells × 100%. Three fields per dish were randomly chosen, and 100 cells were counted. The data are shown as mean value ± SD. **p < 0.01, compared with negative control-transfected cells. FACS, fluorescence-activated cell sorting; PARP, poly ADP-ribose polymerase; SA-β-gal, senescence-associated β-galactosidase.

We then tested whether knockdown of PLK1 induced cell apoptosis of MG63/Dox cells. Interestingly, we found that the apoptosis rate in PLK1 siRNA-transfected MG63/Dox cells reached 15.3%, significantly higher than in negative control siRNA-transfected MG63/Dox cells (4.2%) as measured by cell cycle analysis (Fig. 3B). Levels of caspase-3 and cleaved caspase-3 were detected by western blotting. As shown in Figure 3B, in PLK1 siRNAs-transfected MG63/Dox cells, the levels of cleaved caspase-3 were obviously increased relative to negative control siRNA-transfected MG63/Dox cells. PARP cleavage is considered an important indicator of apoptosis and an indicator of caspase 3 activation. We also found that levels of PARP in PLK1 siRNA-transfected MG63/Dox cells were clearly increased compared to negative control siRNA-transfected MG63/Dox cells. The data suggest that knockdown of PLK1 induced cell apoptosis of MG63/Dox cells.

Interference with PLK1 promoted cell senescence of OS cells

In addition, the relationship of PLK1 and senescence of tumor cells was studied. The results showed a significant increase in SA-β-gal activity in PLK1 siRNA-transfected MG63 cells, suggesting that PLK1-KO induced senescence of OS cell lines, which was tested by SA-β-gal assay (Fig. 3D).

Has-miR-4779 regulates the expression of PLK1 in OS cell lines

MiRNAs are a class of short, noncoding RNAs that generally bind to the 3′-UTR of their target mRNAs to regulate gene expression posttranscriptionally. In the present study, TargetScan analysis was performed to predict the miRNAs that regulate the translation of PLK1. As shown in Figure 4A, has-miR-4779 is predicted to bind to the 3′-UTR of PLK1 mRNA at position 27–33. MG63/Dox cells were transfected with miR-4779 mimic for 48 h, and western blotting analysis showed that PLK1 expression was significantly decreased in MG63/Dox cells (Fig. 4B). Conversely, MG63/Dox cells were transfected with miR-4779 inhibitor for 48 h, and the western blotting analysis showed that PLK1 level was significantly increased in MG63/Dox cells. These results demonstrate that has-miR-4779 negatively regulates the expression of PLK1 in MG63/Dox cells.

FIG. 4.

Has-miR-4779 regulates the expression of PLK1. (A) TargetScan prediction. Position 27–33 of PLK1 3′-UTP is possibly regulated by has-miR-4779. (B) MG63/Dox cells were cultured for 8 h and transfected with miR-4779 mimics or mimic-negative control for 48 h. Western blotting analysis was used for detection of PLK1 expression. The gray value of PLK1 level is shown in the histogram (**p < 0.01, compared with mimic-negative control). (C) MG63/Dox cells were cultured for 8 h and transfected with miR-4779 inhibitor or inhibitor-negative control for 48 h. The levels of PLK1 were determined by western blotting analysis, and the gray value is shown in the histogram (**p < 0.01, compared with inhibitor-negative control).

Transfection of miR-4779 mimic or miR-4779 inhibitor affects cell viability of MG63/Dox cells

MiR-4779 mimic and miR-4779 inhibitor are chemically modified, double-stranded miRNA-like RNAs. Both were used to test the function of mature endogenous miRNA upon transfection. To identify the role of miR-4779 in Dox-resistant MG63 cells, miR-4779 mimic or mimic-negative control was used to transfect MG63/Dox cells for 24, 48, and 72 h. Cell viability was determined by MTT assay, and the results showed that OD490 values of MiR-4779 mimic-transfected MG63/Dox cells were significantly lower than those of transfected mimic-negative control, and the decrease varied in a time-dependent manner (**p < 0.01). Conversely, MTT assay results showed that transfection of MG63/Dox cells with MiR-4779 inhibitor resulted in significantly higher cell proliferation than that by transfection with the negative control inhibitor (**p < 0.01) in both 48 and 72 h groups. Moreover, PLK1 vector-transfected MG63/Dox cells were transfected with MiR-4779 mimics for different periods of time. The MTT assay results showed that cell proliferation was not obviously changed compared with that of untreated cells (p > 0.05). Conversely, transfection with miR-4779 inhibitor increased the cell proliferation of MG63/Dox cells; however, cotransfection of PLK1 siRNA clearly resulted in higher inhibition of MG63/Dox cell viability than that in inhibitor control transfected cells (**p < 0.01, ^## p < 0.01). As shown in Figure 5, PLK1 expression appears to promote cell proliferation of drug-resistant OS cells, while miR-4779 negatively regulates PLK1 expression and cell proliferation of MG63/Dox cells in combination with PLK1 siRNAs.

FIG. 5.

Transfection of miR-4779 mimics or miR-4779 inhibitor affects cell viability of MG63/Dox cells. (A) MG63/Dox cells were cultured for 8 h and transfected with miR-4779 mimic or mimic-negative control for 24, 48, and 72 h. Cell viability was determined by MTT, and OD490 value is shown in the line chart. **p < 0.01, compared with mimic-negative control group. (B) MG63/Dox cells were transfected with miR-4779 inhibitor or inhibitor-negative control for 24, 48, and 72 h. OD490 values were determined by MTT assay. **p < 0.01, compared with inhibitor-negative control group. (C) MG63/Dox cells were cultured for 8 h. The cells were transfected with PLK1 vector or control vector for 24 h. Cells were then transfected with miR-4779 mimic for 24, 48, and 72 h. OD490 values were determined by MTT assay. **p < 0.01, compared with untreated group. (D) MG63/Dox cells were transfected with PLK1 siRNA1 or PLK1 siRNA2 for 24 h. Cells were then transfected with miR-4779 inhibitor for 24, 48, and 72 h. OD490 values were determined by MTT assay. **p < 0.01, ^## p < 0.01, compared with inhibitor control group.

Discussion

In the present study, we explored the role of PLK1 in drug-resistant OS cancer cells. We found that doxorubicin-resistant OS cells had higher levels of PLK1 than doxorubicin-sensitive OS cells. Moreover, interference with PLK1 expression inhibited cell proliferation of MG63/Dox cells, and this inhibition occurred in a time- and dose-dependent manner. Meanwhile, MG63/Dox cells were also treated with an increasing concentration of BI2536 for different times, and the results similarly showed that the PLK1 inhibitor significantly suppressed cell proliferation of MG63/Dox cells. Thus, we concluded that PLK1 expression was likely associated with drug resistance of OS cells. In other human cancers, there have been studies on PLK1 expression and drug resistance: Jeong et al. (2018) reported that PLK1 was selectively overexpressed in tamoxifen-resistant MCF-7 breast cancer cells, as was its downstream phosphatase Cdc25c, which regulates the tumor growth and metastasis of tamoxifen-resistant breast cancer. Moreover, PLK1 inhibition significantly increased the efficacy of olaparib, an FDA-approved PARP inhibitor, in the presence of p53 inhibitor in castration-resistant prostate cancer cells (Li et al., 2017).

MiRNAs are a class of short, non-coding RNAs that bind to the 3′-UTR mRNAs of the target genes to negatively regulate gene expression posttranscriptionally. In the present study, we predicted the possible miRNA targeted to the 3′-UTR of PLK1 mRNA using TargetScan software. The results showed that has-miR-4779 binds to the 3′-UTR of PLK1 mRNAs at positions 27–33. Moreover, western blotting data revealed that miR-4779 negatively regulates the expression of PLK1 in MG63/Dox cells. Furthermore, miR-4779 mimic/inhibitor or mimic/inhibitor-negative controls were transfected into MG63/Dox cells for 24, 48, and 72 h. Cell viability was determined by MTT assay, and the results showed that miR-4779 mimics suppressed the MG63/Dox cells, while miR-4779 inhibitor significantly promoted higher cell proliferation of MG63/Dox cells than that in the negative control group. Thus, we concluded that miR-4779 negatively regulates PLK1 expression in MG63/Dox cells. At the same time, overexpression of miR-4779 or interference with the expression of PLK1 inhibits cell proliferation of drug-resistant OS cells.

Duan et al. (2010) optimized a systematic screen of known kinases in OS cell lines and confirmed the effectiveness of PLK1 as a potential drug target for OS patients. Our results are consistent with the results of Yamaguchi et al. (2009) who screened a panel of siRNAs that targeted 691 kinases and related proteins and found different siRNAs that could specifically target PLK1 and induce cell apoptosis by interfering with cell cycle arrest. Both studies are important clues to establish the role of PLK1 as a key treatment target in OS, based on functional siRNA or lentiviral shRNA screening. In the present study, we used a pair of cell lines (drug-resistant OS cell line MG63/Dox and its paired drug-sensitive cell line MG63) as our model.

Because it was inconvenient to collect clinical samples, we have not previously checked the expression of PLK1 and MiR-4779 in clinical samples. Measuring these expression levels is a meaningful way to test the relationship of PLK1 and MiR-4779 in patients with OS. Going forward, we will attempt to collect clinical samples and check the expression of PLK1 and MiR-4779.

Cell autophagy is a highly conserved biological process, but its role in promoting chemosensitivity or chemoresistance has not been described in OS. In the near future, we plan to explore how miR-4779 regulates autophagy in the proliferation of MG63/Dox cells and how cell autophagy regulates chemosensitivity in the proliferation of OS cells.

In conclusion, we found that miR-4779 negatively regulates the expression of PLK1 and possesses a cancer-inhibiting role, while PLK1 works as an oncogene in drug-resistant OS cells. This provides new clues to clarify the molecular mechanism of drug-resistant OS.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received.

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