Regular paper

MicroRNA-15 suppresses viability, migration and invasion of the human MG-63 osteosarcoma cells via inhibition of cyclin dependent kinase 6 (CDK6)

Zhao Shen, Chen Wang, Bin Liu, Jieli Du, Zhiqiang Li and Jitang Zhao✉

Department of Orthopedics, Cangzhou People’s Hospital, Cangzhou, Hebei, China, 061000

MicroRNA-15a-3p (miR-15) acts as tumor-suppressor in different human cancers including osteosarcoma. Nonetheless, the molecular function of miR-15 in osteosarcoma via suppression of cyclin dependent kinase 6 (CDK6) is yet to be studied. The results showed significant downregulation of miR-15 in osteosarcoma tissues and cell lines. Overexpression of miR-15 inhibited the proliferation and colony formation of the MG-63 osteosarcoma cells via induction of apoptosis. Moreover,
miR-15 inhibited the migration and invasion of MG-63 osteosarcoma cells. The tumor-suppressive functional role of miR-15 was shown to be exerted via suppression of CDK6. The expression of CDK6 was upregulated in osteosarcoma and its silencing could exert growth inhibitory effects on human osteosarcoma cells. However, overexpression of CDK6 could nullify the tumor-suppressive effects of miR-15 on the MG-63 osteosarcoma cells. Taken together, miR-15 negatively regulates growth, migration and invasion of osteosarcoma cells by targeting CDK6 at post-transcriptional level. These findings suggest the therapeutic potential of miR-15/CDK6 in human osteosarcoma.

Keywords: Osteosarcoma, micro-RNA, miR-15, CDK6, epithelial to mesenchymal transition

Received: 04 November, 2021; revised: 26 January, 2022; accepted: 13 February, 2022; available on-line: 22 October, 2022

✉e-mail: 49852275@qq.com

Abbreviations: CDK6, cyclin dependent kinase 6; EMT, epithelialto mesenchymal transition; qRT-PCR, quantitative real-time polymerase chainreaction; PI, propidium iodide

Introduction

Osteosarcoma is highly aggressive and lethal type of bone cancer with mesenchymal origin resulting from the osteoid producing cells (Lindsey et al., 2017). This malignancy is comparatively more prevalent among the children and young adolescents (Rojas et al., 2021). It is the most dominant type of primary bone cancer and accounting for approximately 10% of the solid tumors in young human individuals below 20 years of age (Mirabello et al., 2009). Moreover, the incidence of osteosarcoma is slightly higher in males than females (Mustafa et al., 2018). Osteosarcoma treatment generally involves surgery combined with chemotherapy and parallel adjuvant therapeutic procedures (Zhang et al., 2018). Although, this disease has an overall 5-year survival rate ranging between 60 to 70%, nevertheless in more than 20% cases, osteosarcoma exhibits metastasis to lung tissues and therefore the 5-year survival rate goes down to less than 20% (Shaikh et al., 2016; Tsiambas et al., 2017). With the advancement of molecular biology, several genetic alterations and chromosomal abnormalities have been shown to be associated with the pathogenesis of osteosarcoma (Czarnecka et al., 2020). Characterization of such molecular irregularities might thus be fruitful in devising more efficient therapeutic measures against this deadly disorder.

Small non-coding RNAs, in particular the micro-RNAs (miRs), have been shown to act as potential prognostic and therapeutic targets in osteosarcoma (Czarnecka et al., 2020; Evola et al., 2017; Zhao et al., 2019). For instance, miR-210-5p has recently been reported for its oncogenic role in osteosarcoma growth and epithelial to mesenchymal transition (EMT) via PIK3R5 (Liu et al., 2020). In addition, the regulation of NOTCH1 by miR-139 has been shown to be crucial for inhibition of osteosarcoma progression by resveratrol (Xiao et al., 2020). MicroRNA-15a-3p (now onwards referred as miR-15), since its identification in chronic lymphocytic leukemia, has been deduced to act as tumor-suppressor in different human cancers like ovarian cancer, breast cancer, pancreatic cancer and non-small cell lung cancer (Calin et al., 2002; Bhattacharya et al., 2009; Luo et al., 2013; Zhang et al., 2010; Bandi et al., 2009). The negative regulatory role of miR-15 in osteosarcoma growth and proliferation through multiple regulatory targets has also been reflected by a number of studies (Cai et al., 2012; Tian et al., 2015; Leng et al., 2018; Shi et al., 2018). Additionally, the oncogenic role of cyclin dependent kinase 6 (CDK6) has been reported in osteosarcoma (Zhu et al., 2016; Yuan et al., 2017). However, the characterization of miR-15 functional role via cyclin dependent kinase 6 (CDK6) in osteosarcoma is yet to be studied. Against this backdrop, the present study was designed to study the role of miR-15/CDK6 axis in osteosarcoma proliferation and epithelial to mesenchymal transition.

MATERIALS AND METHODS

Human tissues

35 pairs of osteosarcoma and normal adjacent osteoid clinical tissue specimens were collected from the Cangzhou People’s Hospital, Cangzhou, Hebei, China. The study procedures involving human tissues were approved by the Institutional Ethics Committee. The tissue collection was made prior to application of chemotherapy and all the participants were informed in advance and written consents were obtained from them. Sterilized cold phosphate-buffered saline (PBS) was used for washing the tissues collected which were then frozen using liquid N2 and –80°C temperature conditions were used for their long-term storage.

Cell lines

Four osteosarcoma cell lines (HOS, 143B, MG-63 and U2OS) as well as the normal human foetal osteoblastic cell line (hFOB 1.19) were procured from ATCC (American Type Culture Collection, Manassas, VA). RPMI 1640 (Sigma-Aldrich, MO, USA) carrying 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts, United States) with 1% penicillin/streptomycin (Sigma-Aldrich) as supplementation was used for propagation and maintenance of cell lines at 37°C with 5% CO2 in humidified CO2 incubators.

Cell transfection

The MG-63 osteosarcoma cell line was transfected with miR-15 mimics (25 nM) for miR-15 overexpression with miR-NC as negative control. For the silencing of CDK6, small interfering RNA targeting CDK6 (si-CDK6) was used while si-NC served as a negative control. The miR-15 mimics/miR-NC and si-CDK6/si-NC were ordered from GenePharma (Shanghai, China). The overexpression vector construct of CDK6 (pcDNA-CDK6) was synthesized by RiboBio, Gunagzhou China. The pcDNA3.1 empty vector served as a corresponding negative control. Cell line transfection was performed with the help of Lipofectamine 2000 reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Expression analysis

Total RNA from tissue and cell lines was extracted using the RNeasy Mini Kit (Qiagen, Hiden, Germany) following the manufacturer’s procedure. RevertAid First starnd cDNA synthesis kit (Thermo Fisher Scientific) was used for the generation of cDNA from isolated RNA. The transcript levels of miR-15 and CDK6 were analyzed with quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR was performed using Power SYBRTM Green PCR Master Mix (Thermo Fisher Scientific). The cycling conditions were: 3 min at 95°C, 45 cycles of 30 sec at 95°C and 60 sec at 60°C. GAPDH, snRNA U6 and Rn18s were used for normalizing the expression levels of target genes which were quantified with 2−ΔΔCt method. The sequences of the primers used in the present study are mentioned in Table 1.

CCK-8 proliferation assay

Cell counting kit-8 (CCK-8, Sigma-Aldrich) was used for the determination of cell viability. In brief, the transiently transfected MG-63 cancer cells were placed at a density of 103 cells per well into a 96-well plate. After culturing the cells for 0, 12, 24, 48, 72 or 96 h at 37°C, 10 µL of CCK-8 solution was added to each well and cells were again incubated at 37°C for 3 h. Finally, the absorbance was obtained for each well at 450 nm with the help of a micro-plate spectrophotometer.

Colony formation assay

For the analysis of colony formation, 5×103 transfected MG-63 cells were plated into each well of a 6-well plate. The cells were cultured for 16 days at 37°C till colony formation. At this stage, PBS was used for washing the colonies which were then fixed with 70% ethanol and stained with 0.25% crystal violet for 35 min. Finally, the colonies were photographed and counted under microscope.

Annexin V/PI staining assay

A total of 3×104 transfected MG-63 cells/well were plated into 96-well plates and were cultured for 24 h at 37°C. The cells were subsequently stained with propidium iodide (PI) and Annexin Vfluroescein isothiocyanate (FITC) kit (Multisciences (Lianke) Biotech Co., Ltd.) and analyzed by flow cytometry. The percentage of live cells, apoptotic cells and dead cells were analyzed using FlowJo software (version 10; FlowJo LLC).

Migration and invasion assays

Transwell chambers (Corning, NY, USA) pre-coated without and with Matrigel (bd Biosciences, Franklin Lakes, NJ, USA) were employed for the analysis of migration and invasion of transfected MG-63 osteosarcoma cells, respectively. Briefly, 2×105 transfected cells were seeded into an upper chamber of a 24-well Transwell insert plate. Lower chamber received 750 µL of serum-free culture medium only. After 24 h incubation at 37°C, cells from the upper chamber were swabbed away while those migrating/invading into the lower chamber by passing through the membrane were PBS washed, methanol fixed, crystal violet stained and imagined and counted under an inverted light microscope (Olympus, Tokyo, Japan).

Western blotting

The extraction of total proteins from the transfected MG-63 cells was performed with the help of RIPA lysis buffer (Thermo Fisher Scientific) containing protease inhibitors cocktail (KeyGen Biotech). The protein concentrations were measured with a BCA Protein Assay kit (Beyotime Biotechnology). Around 40 µg protein from each sample were subjected to electrophoresis on 10-12% sodium dodecyl sulfate-polyacrylamide (SGS-PAGE) gels. Proteins separated were then transferred to PVDF membranes. Skimmed milk (5%) was used for blocking the membranes for 1 h at room temperature. The PVDF membranes were treated with primary antibodies against CDK6 (1:1000, Cell Signaling Technology, Danvers, MA, USA). This was followed by the incubation of PVDF membranes with secondary antibodies conjugated with horseradish peroxidase. The specific protein bands were visualized with an PierceTM ECL kit (Thermo Fisher Scientific). β-actin was used as a reference protein.

MiR target prediction and dual luciferase reporter assay

Potential targets of miR-15 were predicted with TargetScan7.0 (http://www.targetscan.org) online software. The synthetic luciferase reporter plasmids of 3′-UTR segment of CDK6 gene with wild type (WT, pGL3-CDK6-WT) or mutant miR-15 binding site (MUT, pGL3-CDK6-MUT) were obtained from GenePharma (Shanghai, China). Briefly, MG-63 cells were seeded into 24-well plates with a density of 1.5×105/well and co-transfected with pGL3-CDK6-WT/MUT (5 ng) luciferase and miR-15 mimics or miR-NC (10 nmol final concentration) into MG-63 osteosarcoma cells using Lipofectamine 2000 transfection reagent and cells were harvested in logarithmic growth phase after 48 h of transfection. Finally, the dual-luciferase reporter system (Promega, Madison, WI, USA) was used for the analysis of luciferase activity of the transfected cells. The host-cell luciferase activities were normalized to Renilla luciferase activity.

Statistical analysis

The analysis of statistical data was performed using the GraphPad Prism 7.0 offline software (San Diego Inc, CA. USA). Results were presented as mean ± standard deviation (SD). Student’s t-test (two-tailed) was carried out to make comparisons between two treatment groups. P<0.05 was taken to represent a statistically significant difference.

RESULTS

MiR-15 is downregulated in osteosarcoma regulates its proliferation

The results of the qRT-PCR showed that osteosarcoma tissues express significantly (P=0.03) lower miR-15 transcript levels than the normal adjacent tissues (Fig. 1A). Further, relative to hFOB 1.19, the normal human foetal osteoblastic cell line, the osteosarcoma cells (HOS, 143B, MG-63 and U2OS) were found to exhibit significantly (P<0.05) lower miR-15 transcript levels (Fig. 1B). Among osteosarcoma cell lines, MG-63 cells exhibit the lowest transcript levels of miR-15 and was thus used in further experiments. To proceed with the characterization of miR-15 role in osteosarcoma, MG-63 cells were transfected with miR-15 mimics to induce miR-15 overexpression. The RT-PCR showed more than 7-fold upregulation of miR-15 in miR-15 mimics transfected MG-63 cells (Fig. 1C). The overexpression of miR-15 in MG-63 cancer cells was shown to significantly inhibit (P=0.02) their viability relative to the negative control cells at different time intervals (Fig. 1D). The inhibition of viability was found to be due to the induction of apoptosis. The percentage of early and late apoptosis increased from 0.71% and 0.21% in miR-NC transfected to 20.3% and 16.5% in miR-15 mimics transfected MG-63 cells (Fig. 1E). In addition, the miR-15 overexpression decreased the colony formation of the MG-63 cancer cells by more than 60% (Fig. 1F). These findings suggest the tumor-suppressive role of miR-15 in osteosarcoma.

MiR-15 inhibits the migration, invasion of MG-63 cells

The effects of miR-15 on the migration and invasion were assessed by transwell assays. The results showed that overexpression of miR-15 lead to a significant (P=0.03) decrease in the migration of the MG-63 cells. The migration was inhibited by 60% (Fig. 2A). Similarly, the invasion of the MG-63 cells was inhibited by 65% (Fig. 2B). The qRT-PCR was used to study the effect of miR-15 on the expression of epithelial and mesenchymal markers in MG-63 cancer cells. The results showed that E-cadherin (epithelial marker) levels significantly increased (P=0.02) while the expression levels of N-cadherin, Snail and Vimentin (Mesenchymal markers) were significantly decreased (P=0.02) by miR-15 overexpression (Fig. 2C). This indicates that overexpression of miR-15 inhibited the migration and invasion of MG-63 osteosarcoma cells, in vitro.

MiR-15 exerts its tumor-suppressive effects via CDK6

The in-silico analysis showed that CDK6 acts as the potential regulatory target of miR-15 and predicted that miR-15 interacts with a specific binding site in 3′-UTR of CDK6 gene (Fig. 3A). To confirm this, the luciferase reporter plasmid of CDK6 3′-UTR with wild type (WT) or mutant (MUT) binding site was co-transfected with miR-15 mimics or miR-NC into MG-63 cells. Dual luciferase reporter assay showed that luciferase activity of host cells was significantly declined (P=0.02) only when UTR segment with wild type miR-15 binding site was used, confirming the specific interaction of miR-15 with 3′-UTR of CDK6 (Fig. 3B). Also, the osteosarcoma tissues and cell lines expectedly showed significantly higher (P<0.05) CDK6 gene expression relative to the normal tissues and cells (Fig. 3C and 3D). Overexpression of miR-15 suppressed the expression of CDK6 (Fig. 4E). Whether miR-15 exercises its regulatory role in osteosarcoma via suppression of CDK6 protein levels, CDK6 was silenced in MG-63 cancer cells by transfecting them with si-CDK6 while si-NC transfected cells served as a negative control. Downregulation of CDK6 significantly (P=0.03) inhibited the proliferation and colony formation of MG-63 cells (Fig. 3F and 3G). Additionally, the effects of CDK6 silencing on the migration and invasion as well as the expression of epithelial to mesenchymal transition markers in MG-63 cells were similar to that of miR-15 overexpression (Fig. 4A–C).

Interestingly, the overexpression of CDK6 mitigated the inhibitory effects miR-15 overexpression on proliferation of MG-63 cells which was also evident from the western blots showing the expression of CDK6 cells (Fig. 5A and 5B). Moreover, CDK6 also rescued the inhibitory effects of miR-15 overexpression on the migration and invasion of MG-63 cells (Fig. 5C–D) suggesting that CDK6 acts as the mediator of the miR-15 functional role in osteosarcoma.

DISCUSSION

Molecular irregularities including aberrant expression of miRs have been shown to profoundly affect the tumorigenesis of human osteosarcoma (Czarnecka et al., 2020; Evola et al., 2017; Zhao et al., 2019; Liu et al., 2020). There are growing research evidence that miRs might emerge as prognostic markers and key therapeutic targets against osteosarcoma (Xiao et al., 2020). Different miRs have been shown to act as oncogenes or tumor-suppressors in osteosarcoma to regulate its growth and progression (Jin et al., 2020; Bazavar et al., 2020). MiR-15 known for its tumor-suppressive regulatory function in number of human cancers has been elucidated to negatively regulate diverse hall marks of human osteosarcoma (Cai et al., 2012; Tian et al., 2015). The present study aimed at the further exploration of functional aspects and mechanism of action of miR-15 in osteosarcoma. Osteosarcoma tissues and cell lines were shown to express significantly lower transcript levels of miR-15 suggesting its possible regulatory involvement in osteosarcoma tumorigenesis. To confirm the same, miR-15 was overexpressed in osteosarcoma cells. Interestingly, the overexpression of miR-15 in osteosarcoma cells limited their growth and proliferation, in vitro, which is consistent with the previous reports (Leng et al., 2018; Shi et al., 2018). Moreover, the miR-15 overexpressing osteosarcoma cells exhibited strikingly lower migration and invasion rates which were suggestive of the anti-metastatic molecular function of miR-15 in osteosarcoma. Similar observations have been made by the contemporary researchers regarding the role of miR-15 in cancer (Tian et al., 2015; Shi et al., 2018). Guo and others (Guo et al., 2014) in 2014 revealed that miR-15 inhibits the epithelial to mesenchymal transition of pancreatic cancer cells. Increase in E-cadherin (epithelial marker) expression levels and decrease in the expression of N-cadherin, Snail and Vimentin proteins (mesenchymal markers) by miR-15 overexpression in osteosarcoma indicates that miR-15 negatively regulates EMT in osteosarcoma, which is further indicative of its anti-metastatic regulatory potential. In order to investigate the mechanism of action of miR-15 in osteosarcoma in silico analysis was used to specifically predict the potential regulatory target of miR-15. Although miR-15 has been reported to target CCND1 in osteosarcoma to induce apoptosis and cell cycle arrest (Cai et al., 2012), in the present study we, for the first time, studied cyclin-dependent kinase (CDK6) as a regulatory target of miR-15 in osteosarcoma. The results showed that miR-15 inhibits the expression of CDK6 post-transcriptionally to exert its tumor-suppressive role in osteosarcoma. These findings are in confirmation with previous studies wherein CDK6 has been shown to promote the growth of esophageal squamous cell carcinoma (Baba et al., 2014) and lymphoblastic leukemia (Rodriguez-Otero et al., 2011). The repression of miR-15 transcript levels might be one of the crucial molecular factors responsible for elevation of CDK6 protein levels in osteosarcoma. The resulting overexpression of CDK6 protein might be involved in positively regulating the growth and metastasis of osteosarcoma. Taken together, the results of the present study clarified the regulatory importance of miR-15/CDK6 molecular axis in osteosarcoma and highlighted its therapeutic utility. However, future studies directed at the evaluation of miR-15 in in vivo system and evaluating the effects of different drugs on the expression of miR-15 in osteosarcoma are urgently needed.

CONCLUSION

Taken together, the results of the present study are conclusive that miR-15 transcript levels are significantly repressed in osteosarcoma. Overexpression of miR-15 inhibits the osteosarcoma cell proliferation, migration and invasion. CDK6 acts as the functional target of miR-15 in osteosarcoma at post-transcriptional level. Therefore miR-15/CDK6 might emerge as a possible therapeutic lead against osteosarcoma in the future.

Declarations

Conflict of interest. The authors declare that there are no conflicts of interest.

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