RETRACTED mir-423-5p knockdown enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway

Transcription

1 mir-423-5p knockdown enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway Tumor Biology December 2018: 1 Ó The Author(s) 2018 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: / journals.sagepub.com/home/tub At the request of the authors and the Editor-in-Chief, the following article has been retracted. Yi Wan, Xifeng Fei, Zhimin Wang, Dongyi Jiang, Hanchun Chen, Mian Wang and Shijun Zhou. MiR-423-5p knockdown enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway. Tumor Biology 2017;39(4):1-10. doi: / Upon repetition of the experiment, the authors discovered that the analysis of the primary glioblastoma cell cdna identified not only human GAPDH and b-actin genes, but also mouse GAPDH and b-actin genes. The authors have found that the primary glioblastoma cells were contaminated by mouse cells and therefore definitive conclusions cannot be drawn. Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License ( which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (

2 695526TUB / Tumor BiologyWan et al. research-article2017 Original Article - mir-423-5p knockdown enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway Tumor Biology December 2018: 1 10 The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: journals.sagepub.com/home/tub Yi Wan, Xifeng Fei, Zhimin Wang, Dongyi Jiang, Hanchun Chen, Mian Wang and Shijun Zhou Abstract This study aimed to investigate the effect of mir-423-5p on the sensitivity of glioma stem cells to apigenin and to explore the potential mechanism. Previous research indicated that apigenin can effectively inhibit the proliferation of many cancer cells, including glioma cells, though our data unexpectedly showed that apigenin had no effect on glioma stem cell apoptosis. As many studies have reported that malignant transformation and progression of glioma are due to glioma stem cells, an anti-glioma stem cell approach has become an important direction for glioma treatment. In this study, we found mir-423-5p to be overexpressed in glioma tissues and corresponding glioma stem cells. Downregulation of mir-423-5p repressed glioma stem cell growth but did not cause apoptosis. Based on the concept of PharmacomiR, this study further demonstrated that the combination of mir-423-5p knockdown and apigenin had a notable additive effect on inhibiting proliferation and promoting apoptosis in glioma stem cells. Hoechst staining showed higher apoptosis rates and typical apoptotic morphological changes of the cell nucleus, and JC-1 (5,5,6,6 -tetrachloro-1,1,3,3 - tetraethylbenzimi-dazolylcarbocya-nine iodide) staining revealed reduced mitochondrial membrane potential. Further research demonstrated that the mechanism is associated with a shift in the Bax/Bcl-2 ratio, an increased cytochrome c level, Apaf-1 induction, and caspase-3 activation. In conclusion, this study indicates that downregulation of mir-423-5p enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway. Keywords mir-423-5p, apigenin, glioma stem cells, apoptosis, mitochondrial Date received: 21 August 2016; accepted: 24 December 2016 Introduction Glioblastoma multiforme (GBM) is one of the most common primary malignant brain tumors, with highly aggressive, invasive, and neurologically destructive characters. GBM has high mortality and morbidity: Despite a combination of approaches, including surgery, radiation therapy, and chemotherapy, the median 5-year survival rate of patients is less than 5%. 1 To date, studies have reported that the main cause of mortality in GBM patients is recurrence, and glioma stem cells (GSCs) are enriched in GBM compared with other types of gliomas. Thus, it is considered that the presence of GSCs a subpopulation of highly tumorigenic glioma cells characterized by indefinite proliferation, self-renewal, and drug resistance capabilities is an important factor for GBM recurrence. GSCs are proven to be major contributors to glioma progression and play key roles in therapy resistance. Therefore, it is essential to find novel and effective anti-gsc therapeutic approaches. Department of Neurosurgery, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, Suzhou, P.R. China Corresponding author: Zhimin Wang, Department of Neurosurgery, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, Suzhou , P.R. China. wanyiwzm@126.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License ( which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (

3 2 Tumor Biology MicroRNAs (mirnas) are small, endogenous noncoding RNAs that have recently been found to function as tumor suppressors or oncogenes involved in tumor pathogenesis, and related mirna pathways are reportedly involved in stem cell proliferation and apoptosis. Morgado et al. 2 showed that mir-145 regulates neural stem cell differentiation through the Sox2-Lin28/let-7 signaling pathway and that mir-145-mediated modulation of the Sox2-Lin28/let-7 network is crucial for neurogenesis progression. Zheng et al. 3 reported that overexpression of mir-186 inhibits the proliferation, migration, and invasion of GSCs and promotes apoptosis, and Esposito et al. 4 found that combining mir-137 and anti-mir-10b could prevent GSC expansion. Recently, the Pharmaco-miR concept has been put forth through mirna pharmacogenomics, which is defined as the study of mirnas and polymorphisms affecting mirna function to predict drug behavior and improve drug efficacy. Prospective studies have demonstrated that mirnas play a central role as a novel regulatory layer affecting drug metabolism and targets. 5 7 Chen et al. 8 reported that mir-125b is upregulated in temozolomide (TMZ)-resistant cells and that inhibition of this mirna resulted in a marked increase in TMZinduced cytotoxicity and apoptosis and a subsequent decrease in TMZ resistance in GSCs. Shi et al. 9 also showed that mir-125b functions in the development of GSC resistance to TMZ and that inhibiting mir-125b enhances GSC sensitivity to TMZ with respect to cell invasion. In addition, Zhang et al. 10 found that neither mir-21 inhibitor nor TMZ induced GSC apoptosis but that mir-21 inhibitor combined with TMZ significantly enhanced apoptosis in GSCs. We recently identified a mirna named mir-423-5p as being overexpressed in GBMs. 11 Our data showed mir p to be increased in World Health Organization (WHO)-III and IV gliomas but not in WHO-II gliomas; it was also overexpressed in the corresponding primary GSCs. Downregulation of mir-423-5p expression resulted in decreased cell proliferation but not in cell apoptosis. These data demonstrate that mir-423-5p functions as an oncogene in GSCs. Apigenin is a bioflavonoid found in citrus, and similar to other flavonoids, it possesses antioxidant, anti-inflammatory, and anti-tumor properties. Recent studies have shown that apigenin can inhibit U87 glioma cell growth and U251 GSC proliferation. 12,13 However, in this study, we found that apigenin has no effect on GSC apoptosis. Based on Pharmaco-miR, we further demonstrated that the combination of mir-423-5p knockdown and apigenin had a marked additive effect on inhibiting proliferation and increasing apoptosis in GSCs. This is the first report of the combined functions and mechanisms of mir-423-5p knockdown and apigenin in GSCs. This study focused on the cell apoptotic inducing effect of mir p knockdown and apigenin on GSCs and its related mechanism. Materials and methods Primary glioblastoma cell cultures Human glioma tissues were collected from adult patients after obtaining informed consent. During surgery, fresh samples of glioma tissues were placed in sterile saline. After removing any blood present, the tissue was washed with phosphate-buffered saline (PBS, ph 7.40). The tissue was then cut and passed through a 200 mesh screen. Serumfree Dulbecco s Modified Eagle s Medium (DMEM) was added and the sample was centrifuged for 5 min at 2000 r/ min. The supernatant was removed and 0.25% trypsin was added for digestion. The sample was incubated for 15 min at room temperature and DMEM containing 10% fetal bovine serum (FBS) was added to terminate the reaction. After centrifugation, the supernatant was discarded and DMEM culture containing 10% fetal calf serum was added. The sample was again centrifuged and the supernatant was discarded. The cell suspension was added to a culture bottle with 2% DMEM/F-12 and incubated for a week, after which the medium was replaced with DMEM/F-12 medium containing 10% FBS. The cells were cultured at 37 C in a 5% CO 2 incubator. After reaching 70% 80% confluence, adherent cells were digested and passaged. Glial origin was confirmed by morphology and staining with an anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (mab) clone 6F2 (Dako, Glostrup, Denmark). 14 Magnetic cell separation of CD133-positive cells Cells were collected and added to 1% FBS-RPMI, and the cell suspension was pre-cooled at 4 C and mixed with 50 µl CD133 antibody beads (Miltenyi Biotec, Bergisch Gladbach, Germany). After cooling again at 4 C, the cell suspension was placed in a magnetic particle separator for 3 min. The CD133-positive cell fraction was collected and centrifuged at 2000 r/min for 5 min, and the supernatant was removed. The CD133-positive cells were collected and washed twice with PBS. After centrifugation at 2000 r/ min for 5 min, the CD133-positive cells were cultured in neural stem cell culture medium including DMEM/F-12 supplemented with 20 ng/ml each of human recombinant epidermal growth factor, human recombinant basic fibroblast growth factor (both from R&D Systems, Minneapolis, MN, USA), and human leukemia inhibitory factor (Chemicon, Millipore(R), USA), in addition to 2% B27 (Life Technologies, Darmstadt, Germany). Cell growth assay To assess the effects of mir-423-5p and/or apigenin on the growth of CD133-positive GSCs, these cells were plated at cells per well in 96-well plates, with six replicate wells at the indicated concentrations of mir-423-5p inhibitor and/or apigenin. To analyze cell proliferation at

4 Wan et al. 3 certain time points after transfection, the MTT assay (3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide) was performed as described previously. 15 Hoechst staining for morphological analysis of apoptosis Cells were cultured in six-well plates. After the indicated treatment, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 1 h. The PBS was removed and 50 µm Hoechst staining solution was added for 10 min. Apoptotic morphological changes were detected under a fluorescence microscope. Mitochondrial membrane potential analysis Mitochondria play an important role in inducing apoptosis, and decreases in mitochondrial membrane potential (ΔΨm) are an early hallmark of apoptosis. JC-1 (5,5,6,6 -tetrachloro- 1,1,3,3 -tetraethylbenzimi-dazolylcarbocya-nine iodide) is a probe that is used for detecting mitochondrial membrane potential. It exists as a monomer when the mitochondrial membrane potential is low, and cells exhibit green fluorescence. However, a dimer forms when the mitochondrial membrane potential increases, and the cells exhibit red fluorescence. To measure the mitochondrial membrane potential for apoptosis analysis, JC-1 staining was performed using Accuri s JC-1 Mitochondrial Potential Assay Kit (Invitrogen, Carlsbad, CA, USA) according to the instructions. Annexin V/PI apoptosis assays Cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA) and centrifuged at 300g for 5 min at 4 C. The cells were washed twice with pre-cooled PBS, with centrifugation at 300g at 4 C for 5 min each. Binding buffer (100 µl) was added to the suspension cells; 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) staining solution were then added, followed by gentle mixing. The samples were incubated at room temperature for 10 min in dark. Then, the binding buffer (400 µl) was added and mixed well. The samples were evaluated for apoptosis percentages using flow cytometry. Western blot analysis After treatment with mir-423-5p inhibitor and/or apigenin, cells were collected, and proteins were isolated using lysis buffer (Bio-Rad Laboratories, Richmond, CA, USA). After electrophoresis and transfer to a membrane, the proteins were incubated with primary antibodies against Bax, Bcl-2, cytochrome c, and Apaf-1 (used at a 1/200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 1.5 h at 37 C in a water bath. The membranes were then incubated with secondary antibodies for 1.5 h at 37 C in a water bath. Bands were detected using enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA). An anti-glyceraldehyde-3-phosphate dehydrogenase (anti-gapdh) antibody (1/2000, Santa Cruz Biotechnology) was used for equal loading of the gel. Caspase-3 activity assay A caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China) was used to detect caspase-3 activity. Adherent cells were digested with trypsin, and collected and centrifuged at 600g at 4 C for 5 min. The supernatant was carefully removed and the cells were washed once with PBS. After centrifugation, the supernatant was removed. Each of the 2 million cells were added to 100 µl of lysate added lysate, suspend the precipitation and cracking on the ice bath for 15 min. The sample was centrifuged for min at 4 C at 16,000g 20,000g and the supernatant was transferred to pre-cooled centrifuge tubes. After adding Ac-DEVD-pNA (2 mm) and mixing, the sample was incubated at 37 C for min. Samples were measured using an enzyme-linked immunosorbent assay (ELISA) reader at an absorbance of 405 nm. Statistical analysis All tests were performed using SPSS Graduate Pack 19.0 statistical software (SPSS, Chicago, IL, USA). Descriptive statistics, including the mean and standard error (SE), and one-way analysis of variance (ANOVA) were used to determine significant differences. A value of p < 0.05 was considered statistically significant. Results Evaluation of mir-423-5p expression in human glioma tissues and corresponding GSCs Li et al. 11 showed that mir-423-5p is highly expressed in human glioma tissues and that its expression is positively correlated with the grade of glioma. However, we found that mir-423-5p exhibited different expression patterns in all glioma grades. In this study, we collected normal and glioma tissue samples, including 7 normal brain tissues, 10 cases of grade II glioma, 11 cases of grade III glioma, and 12 cases of grade IV glioma to investigate mir-423-5p expression in different grades of glioma. As shown in Figure 1(a), mir-423-5p was expressed in a low level in normal brain tissues and was not obviously overexpressed in grade II gliomas. In contrast, mir-423-5p expression was much higher in grade II and III gliomas compared with normal and grade II glioma tissues (p < 0.05; Figure 1(b)). Regardless, statistical analysis showed no significant differences for levels of mir-423-5p between grade III and IV glioma tissues (p > 0.05; Figure 1(b)). The level of mir-423-5p expression in GSCs is unknown to date. To confirm that GSCs are more tumorigenic in vivo than non-gscs, /ml of GSCs or

5 4 Tumor Biology Figure 1. (a) mir-423-5p expression was detected in human glioma tissues and normal brain tissues by stem-loop quantitative realtime (qrt)-pcr (n = 3 independent experiments). (b) Significant differences in mir-423-5p in different grades of gliomas and normal brain tissues were analyzed using SPSS Graduate Pack (c) The tumor volume of GSCs or non-gscs injected subcutaneously into nude mice for 30 days was determined using caliper measurements (n = 3 independent experiments). (d) mir-423-5p expression was detected in GSCs and corresponding WHO-IV glioma tissues by stem-loop qrt-pcr (n = 3 independent experiments). **p < non-gscs were directly injected subcutaneously into the flanks of nude mice; after 30 days, the tumor volume resulting from GSC injection was greater than that for non- GSCs (Figure 1(c)). We cultured primary GSCs from WHO-IV glioma tissues identified by CD133 and Nestin expression. Our data showed higher expression of mir p in GSCs than in corresponding WHO-IV glioma tissues (Figure 1(d)). Evaluation of mir-423-5p inhibitor or apigenin regarding GSC viability and apoptosis To measure the effects of mir-423-5p knockdown on the proliferation of primary GSCs, these cells were transfected with mir-423-5p inhibitor. As shown in Figure 2(a), mir p expression in GSCs was decreased by, approximately, 96.3% at 48 h after transfection in the mir-423-5p inhibitor model compared with the control (p < 0.05). The MTT assay was then used to detect the in vitro growth ability of GSCs. As shown in Figure 2(b), GSC growth rates with mir-423-5p inhibitor transfection were, approximately, ± 1.73%, ± 2.37%, and ± 3.16% at 24, 48, and, 72 h, respectively. Furthermore, Hoechst staining was used to observe the morphological features of apoptosis after mir-423-5p knockdown, and we found no significant differences with regard to the amount of apoptotic death in the mir-423-5p inhibitor group compared with the control group (Figure 2(c)). Figure 2(d) shows that the proliferation ability of GSCs was inhibited by different concentrations of apigenin at 24, 48, or 72 h of treatment, and the inhibition effect was time and dose dependent. Moreover, no obvious morphological features of apoptosis were observed by Hoechst staining after 20 or 40 µm apigenin treatment for 48 h (Figure 2(e)). mir-423-5p inhibitor and apigenin had additive effects on inhibiting GSC proliferation and promoting apoptosis As shown in Figure 3(a), pre-treatment with mir-423-5p inhibitor and 10, 20, or 40 µm apigenin for 48 h increased the growth inhibition rate in GSCs, and the increased inhibition was positively correlated with the concentration of apigenin. After combined treatment for 48 h, morphological features of apoptosis were also observed in the mir p inhibitor + apigenin group by Hoechst staining (Figure 3(b)), and increases in apoptosis were observed with increasing apigenin concentration. Therefore, we speculate that knockdown of mir-423-5p actually increased the sensitivity of GSCs to apigenin.

6 Wan et al. 5 Figure 2. (a) mir-423-5p expression was detected in GSCs by stem-loop qrt-pcr after mir-423-5p inhibitor transfection for 48 h (n = 6 duplicate wells). (b) The growth inhibition rate of GSCs after mir-423-5p inhibitor transfection for 24, 48, and 72 h was assessed by the MTT assay (n = 6 duplicate wells). (c) The morphology of the nucleus in GSCs treated with mir-423-5p inhibitor was observed by Hoechst staining. (d) The growth inhibition rate of GSCs treated with apigenin for 24, 48, and 72 h was assessed using the MTT assay (n = 6 duplicate wells). (e) The morphology of the cell nucleus in GSCs treated with apigenin was observed after Hoechst staining. **p < Flow cytometry analysis revealed apoptosis rates of 7.3%, 13.6%, and 17.6%, respectively (Figure 3(c)). Mitochondrial membrane potential was downregulated by apigenin treatment JC-1 is an ideal fluorescent probe which is widely used for detecting mitochondrial membrane potential (ΔΨm) in cells, tissues, or purified fractions, and a decrease in mitochondrial membrane potential is a hallmark of the early stage of apoptosis. JC-1 aggregates in the mitochondrial matrix when the mitochondrial membrane potential is high, forming polymers (J-aggregates) that produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and exists as a monomer that produces green fluorescence. Therefore, this probe is very convenient for detecting changes in mitochondrial membrane potential based on fluorescent color changes, which can be expressed as a detection index of early apoptotic cells. As shown in Figure 4, the mitochondria membrane potentials of GSCs treated with mir-423-5p inhibitor combined with 20 or 40 µg/ml apigenin were significantly reduced compared with mir-423-5p inhibitor or 20 or 40 µg/ml apigenin alone. Therefore, we hypothesized that this could reflect apoptosis induced by the combined treatment of mir p inhibitor and apigenin in association with the mitochondrial pathway. mir-423-5p inhibitor + apigenin induced cell apoptosis by activating Bax/Bcl-2-caspase-3 signaling Liu et al. 16 showed that expression of Bcl-2 family proteins has an important role in GSC apoptosis; they also showed that mir-21 inhibitor sensitized U251 stem cells to TMZ by regulating the Bax/Bcl-2 ratio. Bax functions as an apoptotic activator in GSCs, and this protein is

7 6 Tumor Biology Figure 3. (a) The growth inhibition rate of GSCs treated with mir-423-5p inhibitor and apigenin for 48 h was assessed by the MTT assay (n = 6 duplicate wells). (b) The morphology of the nucleus in GSCs treated with mir-423-5p inhibitor and apigenin was observed by Hoechst staining. (c) The apoptosis rates of GSCs treated with mir-423-5p inhibitor and apigenin were analyzed by flow cytometry (n = 6 duplicate wells). **p < reported to interact with and increase the opening of the mitochondrial voltage-dependent anion channel (VDAC), which leads to loss in membrane potential and the release of cytochrome c. 17 Bcl-2 acts as an anti-apoptotic regulator in GSCs, and Qiu et al. 18 showed that levels of Bcl-2 expression were higher in GSCs and that its expression was related to glioma malignancy. As an anti-apoptotic gene, expression of Bcl-2 coincides with the pivotal biological features of GSCs. High Bcl-2 expression allows GSCs to evade apoptosis, and therapies targeting Bcl-2 induce apoptosis in GSCs. Zhang et al. 19 also showed that epigallocatechin gallate (EGCG) induced apoptosis in U87 GSCs by downregulating Bcl-2 and cleaving poly (ADP-ribose) polymerase (PARP). To confirm that the synergistic anti-cancer mechanisms of mir-423-5p inhibitor + apigenin in GSCs relies on Bax and Bcl-2 alterations, the levels of Bax and Bcl-2 were measured after mir-423-5p inhibitor + apigenin treatment by western blot analysis. Our data showed that combined treatment of mir-423-5p inhibitor + 20 µg/ml apigenin significantly increased Bax expression and decreased Bcl-2 expression compared with mir-423-5p inhibitor or apigenin alone

8 Wan et al. 7 Figure 4. The mitochondrial membrane potential of GSCs treated with mir-423-5p inhibitor and apigenin for 48 h was examined using JC-1 staining (n = 6 duplicate wells). (p < 0.05; Figure 5(a)). Furthermore, we examined the downstream signaling pathway of the Bcl-2 family, including cytochrome c, Apaf-1, and caspase-3. As shown in Figure 5(b), expression of cytochrome c and Apaf-1 in GSCs was significantly increased after mir-423-5p inhibitor + apigenin treatment compared with mir p inhibitor or apigenin alone, whereas treatment with mir-423-5p inhibitor or apigenin alone did not result in a significant increase in cytochrome c and Apaf-1 compared with the control group. Caspase-3 activity was measured using a caspase-3 activity kit, and our data showed an obvious increase in caspase-3 activity in the mir-423-5p inhibitor + apigenin group compared with groups treated with mir-423-5p inhibitor or apigenin alone (Figure 5(c)). These results suggest that the combined treatment of GSCs with mir-423-5p inhibitor and apigenin activates apoptosis-related proteins in the mitochondrial pathway. Discussion At present, cancer drug resistance is considered an important factor contributing to the failure of glioma treatment. The presence of GSCs is thought to be responsible for chemotherapy resistance. Similar to normal stem cells, GSCs are capable of self-renewal, differentiation, unlimited proliferation, and resistance to conventional therapy, including radiotherapy and chemotherapy, due to higher expression of multidrug transporters. 20 Recent studies have shown that targeting certain mirnas can successfully interfere with the above-mentioned properties of GSCs and enhance the sensitivity of GSCs to chemotherapeutic drugs. Using human glioblastoma stem cells, Wu et al. 21 showed that mir-218-5p could inhibit the stem cell properties of glioma. Wang et al. 22 found that mir-608 expression was significantly downregulated in GSCs and that mir-608 overexpression could significantly attenuate GSC proliferation, migration, and invasion, and induce apoptosis. Liu et al. 23 reported that mir-143 plays a critical role in the anti-tumor activity of shikonin in GSCs, with mir-143 upregulation significantly enhancing the inhibitory effect of shikonin on GSC growth. In this study, we focused on another mirna, mir p, the dysfunction of which has recently been found in some cancers. Fang et al. 24 found that mir-423-5p is downregulated in the plasma of colorectal cancer (CRC) cells and that it could serve as a potential biomarker for colorectal carcinoma. Stiuso et al. 25 showed that mir p promotes autophagy in hepatocarcinoma cells, and mir-423-5p was also found to regulate cell proliferation and invasion in gastric cancer cells. 26 In gliomas, mir p is reportedly overexpressed in Chinese primary glioblastomas 27 and contributes to the malignant phenotype as well as TMZ chemoresistance. 11 However, the expression and function of mir-423-5p in GSCs have remained unknown. In this study, we confirmed that mir p is overexpressed in primary GSCs compared with corresponding primary glioma tissues. Knockdown of mir-423-5p in primary GSCs could inhibit proliferation but not apoptosis and could enhance the sensitivity of cancer cells to chemotherapeutic drugs, similar to other mir- NAs. Our data also demonstrated that knockdown of mir-423-5p could effectively enhance the anti-gsc effects of apigenin, including increased growth inhibition and apoptosis. Detection of the mitochondrial membrane potential (ΔΨm) in this study showed that combined treatment of mir-423-5p inhibitor and apigenin significantly decreased

9 8 Tumor Biology Figure 5. (a) Expression of Bax and Bcl-2 in GSCs treated with mir-423-5p inhibitor and apigenin for 48 h was examined by western blot analysis (n = 3 independent experiments). (b) Expression of cytochrome c and Apaf-1 in GSCs treated with mir-423-5p inhibitor and apigenin for 48 h was examined by western blot analysis (n = 3 independent experiments). (c) Caspase-3 activity in GSCs treated with mir-423-5p inhibitor and apigenin for 48 h was examined using a caspase-3 activity kit (n = 6 duplicate wells). **p < the ΔΨm in GSCs, suggesting that the synergistic anti-cancer mechanisms of mir-423-5p inhibitor + apigenin involve a mitochondria-related signaling pathway. The mitochondrial apoptosis pathway is considered to be the major target by which anti-cancer drugs induce apoptosis in many cancer cells, causing DNA damage, influencing the proteolysis or dephosphorylation of apoptosis-promoting proteins, activating caspases, and leading to the characteristic changes of apoptosis. 28 In this study, Hoechst staining revealed the characteristic changes of apoptosis in the nucleus of GSCs after mir-423-5p inhibitor + apigenin treatment. The anti-apoptotic protein of Bcl-2 was detected and found to be decreased, whereas the pro-apoptotic protein Bax was increased with the combined treatment. The

10 Wan et al. 9 Bcl-2 family regulates the release of cytochrome c from mitochondria into the cytosol, interacts with Apaf-1, and leads to caspase activation in the apoptosome, which is crucial for the induction of apoptosis. To further verify that the higher apoptosis rate in GSCs induced by mir-423-5p inhibitor + apigenin treatment was achieved through the mitochondrial pathway, we detected the expression of cytochrome c, Apaf-1, and caspase-3. Western blot data confirmed that the expression levels of apoptosis-related proteins, cytochrome c and Apaf-1, were increased after the combined treatment, and caspase-3 activity was also confirmed to be upregulated. In conclusion, these results indicate that either mir p inhibitor or apigenin can inhibit growth but not apoptosis in GSCs. However, combined treatment of mir p inhibitor + apigenin exerts an additive effect, inducing GSC apoptosis primarily through a mitochondria-dependent pathway. Acknowledgements Y.W. and X.F. contributed equally to this work. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This work was supported by Science and Education in Suzhou City Youth Science and Technology Project (kjxw ) and the Suzhou Social Development Foundation (SYS201063). References 1. Kegelman TP, Hu B, Emdad L, et al. In vivo modeling of malignant glioma: the road to effective therapy. Adv Cancer Res 2014; 121: Morgado AL, Rodrigues CM and Solá S. MicroRNA-145 regulates neural stem cell differentiation through the Sox2- Lin28/let-7 signaling pathway. Stem Cells 2016; 34(5): Zheng J, Li XD, Wang P, et al. CRNDE affects the malignant biological characteristics of human glioma stem cells by negatively regulating mir-186. Oncotarget 2015; 6(28): Esposito CL, Nuzzo S, Kumar SA, et al. A combined microrna-based targeted therapeutic approach to eradicate glioblastoma stem-like cells. J Control Release 2016; 238: Mishra PJ and Bertino JR. MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine. Pharmacogenomics 2009; 10: Rukov JL, Vinther J and Shomron N. Pharmacogenomics genes show varying perceptibility to microrna regulation. Pharmacogenet Genomics 2011; 21: Shomron N. MicroRNAs and pharmacogenomics. Pharmacogenomics 2010; 11: Chen J, Fu X, Wan Y, et al. mir-125b inhibitor enhance the chemosensitivity of glioblastoma stem cells to temozolomide by targeting Bak1. Tumour Biol 2014; 35(7): Shi L, Wan Y, Sun G, et al. mir-125b inhibitor may enhance the invasion-prevention activity of temozolomide in glioblastoma stem cells by targeting PIAS3. BioDrugs 2014; 28(1): Zhang S, Wan Y, Pan T, et al. MicroRNA-21 inhibitor sensitizes human glioblastoma U251 stem cells to chemotherapeutic drug temozolomide. J Mol Neurosci 2012; 47(2): Li S, Zeng A, Hu Q, et al. mir-423-5p contributes to a malignant phenotype and temozolomide chemoresistance in glioblastomas. Neuro Oncol 2017; 19(1): Chen XJ, Wu MY, Li DH, et al. Apigenin inhibits glioma cell growth through promoting microrna-16 and suppression of BCL-2 and nuclear factor-κb/mmp-9. Mol Med Rep 2016; 14(3): Feng X, Zhou Q, Liu C, et al. Drug screening study using glioma stem-like cells. Mol Med Rep 2012; 6(5): Darling JL. The in vitro biology of human brain tumors. In: DGT Thomas (ed.) Neuro-oncology: primary malignant brain tumors. Baltimore, MD: Johns Hopkins University Press, 1990, pp Shi L, Zhang J, Pan T, et al. MiR-125b is critical for the suppression of human U251 glioma stem cell proliferation. Brain Res 2010; 1312: Liu J, Albrecht AM, Ni X, et al. Glioblastoma tumor initiating cells: therapeutic strategies targeting apoptosis and microrna pathways. Curr Mol Med 2013; 13(3): Große L, Wurm CA, Brüser C, et al. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J 2016; 35(4): Qiu B, Wang Y, Tao J, et al. Expression and correlation of Bcl-2 with pathological grades in human glioma stem cells. Oncol Rep 2012; 28(1): Zhang Y, Wang SX, Ma JW, et al. EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition. J Neurooncol 2015; 121(1): Kleinová R, Slabý O and Šána J. The relevance of micro- RNAs in glioblastoma stem cells. Klin Onkol 2015; 28(5): Wu Z, Han Y, Li Y, et al. MiR-218-5p inhibits the stem cell properties and invasive ability of the A2B5+CD133- subgroup of human glioma stem cells. Oncol Rep 2016; 35(2): Wang Z, Xue Y, Wang P, et al. mir-608 inhibits the migration and invasion of glioma stem cells by targeting macrophage migration inhibitory factor. Oncol Rep 2016; 35(5): Liu J, Qu CB, Xue YX, et al. MiR-143 enhances the antitumor activity of shikonin by targeting BAG3 expression in human glioblastoma stem cells. Biochem Biophys Res Commun 2015; 468(1 2): Fang Z, Tang J, Bai Y, et al. Plasma levels of microrna-24, microrna-320a, and microrna-423-5p are potential biomarkers for colorectal carcinoma. J Exp Clin Cancer Res 2015; 34: 86.

11 10 Tumor Biology 25. Stiuso P, Potenza N, Lombardi A, et al. MicroRNA-423-5p promotes autophagy in cancer cells and is increased in serum from hepatocarcinoma patients treated with sorafenib. Mol Ther Nucleic Acids 2015; 4: e Liu J, Wang X, Yang X, et al. mirna423-5p regulates cell proliferation and invasion by targeting trefoil factor 1 in gastric cancer cells. Cancer Lett 2014; 347(1): Yan W, Liu Y, Yang P, et al. MicroRNA profiling of Chinese primary glioblastoma reveals a temozolomide-chemoresistant subtype. Oncotarget 2015; 6(13): Wong IL, Chan KF, Zhao Y, et al. Quinacrine and a novel apigenin dimer can synergistically increase the pentamidine susceptibility of the protozoan parasite Leishmania. J Antimicrob Chemother 2009; 63(6):