MiR-21-5p inhibition attenuates Warburg effect and stemness maintenance in osteosarcoma cells via inactivation of Wnt/β- catenin signaling

MicroRNA (miR)-21 has been found to be overexpressed in osteosarcoma (OS). The aim of the present study was to investigate the effect of miR-21-5p on the Warburg effect and stemness maintenance in OS cells and its potential molecular mechanism. Herein, miR-21-5p was overexpressed or inhibited in MG-63 cells via transfection with mimics or inhibitors. The effect of miR-21-5p on cell viability, apoptosis, Warburg effect and stemness maintenance were explored in OS cells. The results demonstrated that miR-21-5p inhibition suppressed MG-63 cell viability and enhanced their apoptosis. Additionally, miR-21-5p inhibition attenuated the stemness maintenance of MG-63 cells, as demonstrated by the reduced proportion of CD133-positive MG-63 cells, the decrease in tumorsphere formation capacity, and the downregulation of Sox2, Oct4, and Nanog proteins. Moreover, miR-21-5p inhibition suppressed the Warburg effect in MG-63 cells, as indicated by the decrease in glucose uptake, lactic acid production, and ATP level and the downregulation of proteins involved in the Warburg effect (GLUT1, LDHA, HK2, and PKM2). Furthermore, the results suggested that the effect of miR-21-5p suppression on stemness and the Warburg effect may be associated with the decreased activity of the Wnt/β-catenin pathway in OS cells. Our findings suggest a novel potential biomarker for OS therapy.


INTRODUCTION
Osteosarcoma (OS) is the most prevalent primary bone tumor occurring in young adults and teenagers, exhibiting high aggressiveness and metastatic potential Otoukesh et al., 2018). Epidemiological investigation indicated that three out of every million young adults and children suffer from OS, and the incidence reaches a peak at 11-15 years of age with six in every million children . Although much effort has been dedicated to OS therapy, the five-year survival and prognosis of OS patients remain unimproved, particularly for patients with recurrence, multidrug resistance, or lung metastasis, due to insufficient diagnosis at the early stage and ineffective therapeutic approaches . Therefore, it is necessary to identify novel clinical prognostic biomarkers and investigate the potential molecular mechanisms of OS to develop more effective therapeutic strategies (Tu et al., 2020).
MicroRNAs (miRNAs) are endogenous, non-coding, and single-stranded small molecular RNA containing about 22 nucleotides . They have demonstrated important functions in biological processes including cell proliferation, differentiation, and apoptosis (Najm et al., 2019). MiRNAs are involved in regulating gene expression by complete or incomplete complementary pairing with the 3'-untranslated regions of the target mRNA, resulting in target mRNA degradation to silence specific genes or the inhibition of protein translation (Bartel, 2018). Numerous investigations have revealed that miRNAs are abnormally expressed in a variety of tumors, suggesting that they play an essential role in tumorigenesis and tumor progression (Iacona & Lutz, 2019;Zhang et al., 2019).  is located at 17q23.2 in the human chromosome and has exhibited proto-oncogenic activity (Li et al., 2018b;Ribas et al., 2012). Previous studies have shown that miR-21 was highly expressed in hepatocellular carcinoma , hepatoblastoma , and colorectal cancer (Xie et al., 2019). MiR-21 is also associated with the regulation of tumors cell proliferation and apoptosis through its interaction with target genes (Bautista-Sanchez et al., 2020). In addition, miR-21 was involved in the modulation of glycolysis and stemness maintenance in tumor cells by regulating relevant signaling pathways, thereby affecting tumor development. Zhu and others  found that miR-21 inhibition suppressed glycolysis in breast cancer cells by regulating the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt pathway, in turn inhibiting breast cancer tumorigenesis. Fu and others (Fu et al., 2013) reported that NPV-LDE-225 inhibited the self-renewal of glioblastoma cells by downregulating the expression of Nanog, Oct4, and Sox2 through suppressing miR-21. MiR-21 has been identified as a biomarker of poor prognosis in OS patients, as it was shown to be overexpressed in OS and associated with poor clinicopathological characteristics (Ren et al., 2016). However, the effect of miR-21 on glycolysis and stemness maintenance in OS cells remains to be elucidated. Moreover, evidence suggests that miR-21 was involved in the regulation of Wnt/β-catenin signaling , which is associated with glycolysis and stemness maintenance in tumor cells (Manna et al., 2014;Udoh et al., 2019). Whether miR-21 has an effect on the activity of the Wnt/βcatenin pathway in OS cells remains unknown. On this basis, the current study was performed to explore the effect of miR-21-5p on glycolysis and stemness maintenance in OS cells and clarify the underlying molecular mechanism involved therein.

Cell culture and transfection
MG-63 human osteosarcoma cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Science and maintained in minimum essential medium supplemented with 10% fetal bovine serum (Gibco, MD, USA) at 37°C with 5% CO 2 . After the cell confluence reached 70%, the cells were transfected with commercially generated miR-21-5p mimic or inhibitor, or their corresponding negative control (NC) (40 pmol/ml, Ribo Biotechnology Co. LTD, Guangzhou, China) for 24 h using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture's instruction to overexpress or silence miR-21-5p expression. The transfection efficiency was then evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Doxorubicin (200 nM; Kamata et al., 2017, Aladdin, Shanghai, China), a prevalent antineoplastic drug (Qin et al., 2018), served as positive control (PC).

Flow cytometry
Flow cytometry was performed to detect the apoptosis and proportion of CD133-positive cells. For apoptosis: After treatment, 1×10 6 cells were collected and centrifuged at 4°C at 400 × g for 5 min, followed by resuspension in 1 ml of phosphate-buffered saline (PBS) and centrifugation at 4°C at 400 × g for 5 min. Then, the cells were resuspended in 200 µl of PBS and incubated with 10 µl of Annexin V-fluorescein isothiocyanate (FITC) (BD, Shanghai, China) and 10 µl of propidium iodide (PI) in the dark at 4°C for 30 min. After 300 µl of PBS was added, the cells were evaluated by flow cytometry (ACEA Biosciences, San Diego, California, USA) For the detection of CD133-positive cells: Cells (1×10 6 ) were resuspended in 100 µl of PBS, followed by the addition of 2 µl of phycoerythrin-conjugated CD133 antibodies (eBioscience, CA, USA). The cells were cultured in the dark at 4°C for 30 min. Then, the cells were centrifuged at 4°C at 400 × g for 5 min, resuspended in the dark in 400 µl of PBS, and evaluated by flow cytometry (ACEA Biosciences).

Biochemical detection
Adenosine triphosphate (ATP) content (A095-1-1) and lactic acid levels (A019-2-1) in MG-63 cells were detected using corresponding commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer's instruction. Glucose uptake (361510) was evaluated using a corresponding commercial kit purchased from Shanghai Rongsheng Biotechnology Co., LTD following the manufacturer's instruction.

Statistical analysis
Data are shown as the mean ± standard deviation (S.D.). Differences among groups were analyzed using one-way analysis of variance followed by Tukey test. P<0.05 was considered to be statistically significant.

miR-21-5p inhibition attenuates proliferation while promoting apoptosis of MG-63 cells
As shown in Fig. 1A, the expression of miR-21-5p in MG-63 cells was increased and decreased by miR-21-5p mimic and inhibitor transfection, respectively. The corresponding NC showed no effect. Compared to the control or NC, the viability of MG-63 cells (Fig. 1B,  P<0.05) was decreased by miR-21-5p inhibition, while apoptosis (Fig. 1C, P<0.05) was enhanced. The effect of miR-21-5p inhibition on cell viability and apoptosis were similar to that of doxorubicin. These results demonstrated that miR-21-5p inhibition attenuates the proliferation while promoting the apoptosis of MG-63 cells.

miR-21-5p inhibition attenuates stemness maintenance in MG-63 cells
Tumorspheres are spherical and solid structures originating from cancer stem cells. Figure 2A indicates that miR-21-5p inhibition suppressed tumorsphere formation of MG-63 cells. Flow cytometry demonstrated that miR-21-5p inhibition reduced the proportion of MG-63 cells with positive expression of CD133 (Fig.  2B), a specific marker of cancer stem cells (Bi et al., 2016). In addition, the expression of stem cell-related proteins was evaluated. miR-21-5p inhibition attenuated the protein expression of Sox2, Oct4, and Nanog in MG-63 cells, while miR-21-5p overexpression showed an opposite effect (Fig. 2C). The effect of miR-21-5p inhibition on the stemness of MG-63 cells was similar to that of doxorubicin. These results indicated that miR-21-5p inhibition attenuates stemness maintenance in MG-63 cells

miR-21-5p inhibition attenuates the activity of Wnt/βcatenin signaling
As shown in Fig. 4, the expression of Wnt and β-catenin was decreased by miR-21-5p inhibitors compared to that of the control or inhibitor NC groups (P<0.05), while it was increased by miR-21-5p mimics compared to that of the control or mimic NC groups (P<0.05). These results demonstrated that miR-21-5p inhibition attenuates the activity of Wnt/β-catenin signaling.

DISCUSSION
The phenomenon wherein cancer cells preferentially utilize glucose for energy via glycolysis, even under aerobic conditions, was identified as the "Warburg effect" (Spencer & Stanton, 2019). It is associated with oncogenic expression, abnormal expression of glycometabolic enzymes, the tumor microenvironment, and tumor development. Thus, the Warburg effect has been widely characterized as a hallmark of tumor, and anticancer therapeutic strategies targeting the Warburg ef-fect are being development (Li et al., 2018a). Compared to non-tumorigenic cells, tumorigenic cells exhibit dramatically increased glucose uptake, followed by the rapid conversion of pyruvate into lactic acid even under aerobic conditions, during which the glycolysis rate is upregulated and ATP is produced (Lebelo et al., 2019;Lu et al., 2015). Evidence has suggested that the glycolytic process is associated with GLUTs, LDHA and rate-limiting enzymes including HK2, and PKM2 (Ruzzo et al., 2020;Wiese & Hitosugi, 2018). GLUTs are involved in the transport of glucose across the plasma membrane, thereby mediating glucose uptake (Barron et al., 2016). GLUT1 is upregulated in numerous types of cancer and can act as a target for anti-cancer treatment because of its effect on glucose uptake (Lebelo et al., 2019). HK2 facilitates glucose phosphorylation to generate glucose-6-phosphate, which is the first irreversible step in the glucose metabolism pathway, thereby enhancing aerobic glycolysis . Thus, it is identified as a vital player in the Warburg effect, and like GLUT1, it has been regarded as a therapeutic target for tumors (Patra et al., 2013). Previous studies have found that HK2 deficiency exhibited remarkable reduced LDHA expression, a crucial functional enzyme in glycolysis that promotes pyruvate conversion into lactic acid (Pathria et al., 2018;Xu et al., 2019). PKM2 is another important component of the Warburg effect that promotes rapid energy generation during glycolysis (Palsson-McDermott & O'Neill, 2013). Overexpression of HK2, PKM2, LDHA, and GLUT1 contributed to the Warburg effect in cancer cells Yu et al., 2019). The present work showed that miR-21-5p inhibition suppressed the expression of HK2, PKM2, LDHA, and GLUT1, thereby inhibiting glucose uptake and the levels of lactic acid and ATP in OS cells, indicating that miR-21-5p inhibition attenuates the Warburg effect in OS cells. As for HK2, PKM2, LDHA, and GLUT1 proteins detection, GAPDH, another glycolytic enzyme , was used as the loading control. The present study showed no effect of miR-21-5p on GAPDH expression, as indicated by no statistical difference of gray value in different group, which was in consistence with previous studies that miR-21-5p inhibition or overexpression did not affect GAPDH expression (Huang et al., 2021;Zhang et al., 2021) As an important characteristic of metabolic reprogramming in tumor cells, the Warburg effect contrib- utes to growth, survival, and proliferation of cancer stem cells (CSCs) as well as enables the maintenance of the CSCs state (Vaupel et al., 2019). Tumorigenic cells with stem cell-like characteristics are regarded as CSCs, which have strong multi-directional differentiation potential and self-renewal ability (Ayob & Ramasamy, 2018;Hajizadeh et al., 2019). Emerging evidence has demonstrated that the capacity of tumor reproduction and development depends on the stem-like characteristics of cancer cells, which allow cells to remain in a dormant state and retain the ability to evade therapeutic drugs and metastasis (Adorno-Cruz et al., 2015). Therefore, inhibiting the maintenance of the CSC state or the proportion of CSCs in cancer cells is an effective strategy for cancer treatment (Yoshida & Saya, 2016). The current study revealed that miR-21-5p inhibition suppressed tumorsphere formation (initiated from CSCs) of OS cells and reduced the proportion of MG-63 cells with positive expression of CD133, a specific marker of CSCs (Bi et al., 2016). In addition, miR-21-5p inhibition attenuated the protein expression of Sox2, Oct4, and Nanog, which are key transcription factors that keep the self-renewal of CSC, in turn regulating cancer recurrence, progression, and drug resistance (Hattermann et al., 2016). Taken together, these results indicate that miR-21-5p inhibition suppressed the stemness maintenance in OS cells, as demonstrated by the decrease in the proportion of CD133-positive OS cells and the downregulation of Sox2, Oct4, and Nanog. Whether this is associated with the suppression of the Warburg effect induced by miR-21-5p inhibition needs to be further elucidated.
The current results also demonstrated that miR-21-5p inhibition attenuates the activity of the Wnt/βcatenin signal pathway, which has been shown to be associated with glycolysis and stemness maintenance in tumor cells (Manna et al., 2014;Udoh et al., 2019). The activation of Wnt/β-catenin signaling promoted the Warburg effect in tumor cells by increasing the level of acetyl-coenzyme A and ATP level produced by the oxidation of fatty acids (Manna et al., 2014). Additionally, it was previously reported that Wnt/βcatenin pathway inactivation may assist in eradicating OS stem cells by regulating CD133, Sox2, Oct4, and Nanog expression (Yi et al., 2015). In agreement with previous research, the current study indicates that miR-21-5p inhibition suppresses the Warburg effect and stemness maintenance in OS cells, possibly by Wnt/β-catenin signal pathway.
In conclusion, the current study provides insight suggesting that miR-21-5p inhibition suppresses the Warburg effect in OS cells by regulating the expression of proteins involved in the Warburg effect (HK2, PKM2, LDHA, and GLUT1), thereby inhibiting glucose uptake and lactic acid and ATP level in OS cells. In addition, miR-21-5p inhibition suppresses stemness maintenance in OS cells by regulating the expression of transcription factors that promote the self-renewal of CSCs (Sox2, Oct4, and Nanog). The underlying mechanism might be associated with the Wnt/βcatenin signaling pathway.