MEG3 targets miR-184 and Wnt/β-catenin and modulates properties of osteosarcoma
Recent studies indicate that MEG3, a long non-coding RNA (lncRNA) and microRNA-184 (miR-184) are abnormally expressed in osteosarcoma (OS). To this end, we show here that MEG3 negatively regulates the expression of miR-184 and down-stream effectors of WNT/β-catenin pathway including β-catenin, T-cell factor 4 (TCF4) and c-MYC. MEG3 overexpression by adenoviral vectors down-regulate proliferation, migration and apoptosis of OS in vitro and restrict the tumor growth in vivo. We also show that the effects of MEG3 can be effectively reversed by miR-184 mimic. Together these studies show that both MEG3 and miR-184 cooperatively regulate the proliferation, migration and apoptosis of OS.
Osteosarcoma (OS) is one of the most common solid bone cancers in children and adolescents that carries a high morbidity and mortality. This tumor usualy affects proximal humerus or tibia and the metaphysis of distal femur (1). Typical symptoms of OS include limitation of joint movement, trabecular fracture, local swelling and pain (2). Other than surgery, the traditional treatments including post-operative radiotherapy and chemotherapy may lead to life-threatening side effects such as nephrotoxicity, infertility and cardiotoxicity. Therefore, There is a need for better understanding of the pathogenesis of OS with the hope to develop better treatment strategies with fewer side effects.
Maternally Expressed Gene 3 (MEG3), is a lncRNA, with key role in various cancers (3,4). Some earlier work suggested that MEG3 is poorly expressed in OS and might be a potential target for the treatment of this cancer (5). Other studies have shown that the expression of diverse miRNAs including miRNA-184 is deregulated in OS and other cancers and these changes might be used for assessment of prognosis (6-9). MEG3 interacted with miR-184 and subsequently alleviated the proliferation and invasion of leukemia cells by down-regulating related proteins (10). Wnt/β-catenin pathway plays essential roles in the tumorigenesis and EMT and is thought to be involved in the occurrence and tumor progression and metastasis of OS (11,12). It has also been shown that in retinoblastoma, reduced MEG3, resulted led to the tumor progression by regulating the activity of Wnt/β-catenin pathway (13-14). Based on such a background, we hypothesized and tesed whether miR-184/MEG3/β-catenin axis may be involved in the pathogenesis of OS.
3. Materials and methods
3.1. Cell lines, clinical samples and animals
MG63, U2OS and normal osteoblast hFOB1.19 cell lines were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). All cells were cultured in RPMI-1640 (Gibco, Rockville, MD, USA) supplemented with 10% FBS at 37°C with 5% CO2. Samples of OS tumors the adjacent non-involved tissues of 55 patients who underwent surgery were frozen and stored at -80°C from following an approved protocol of the Ethics Committee of Nanchong central hospital. Patients did not receive radiotherapy or chemotherapy before surgery. Nude mice (6-8 weeks old) were obtained from Animal Experimental Center of Nanchong central hospital following a protocol approved by the Committee on the Ethics of Nanchong central hospital. MG63 cells with good growth were made as a single cell suspension of 5 × 106 cells. U2OS cells with good growth were made as a single cell suspension of 5 × 105. 0.4 mL 5×106 MG63 cells were subcutaneously injected into the right armpit and 2×106 MG63 cells were introduced to the skin to form xenograft tumors.
3.2. Cell transfection and MEG3 luciferase reporter assay
TargetScan and miRanda software revealed the existence of a high probability of interaction between miR-184 and MEG3. MEG3 wild type (MEG3 wt), MEG3 mutant (MEG3 mut), negative control mimics (miR-NC), miR-184 mimic, pcDNA-MEG3 and pcDNA vector were obtained from GenePharma Co., Ltd (Shanghai, China). MG63 and U2OS cells were transfected using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA). The experimental protocol was performed according to the manufacture’s instruction. Cells were used 48 h after transduction with pcDNA or after transfection.
3.3. Osteosarcoma model and groups
Nude mice were randomly divided into two groups (n=18). Control group was comprised of nude mice receiving subcutaneous injection of 2×106 MG63 cells transfected with empty vector; pcDNA-MEG3, and those receiving subcutaneous injection of 2×106 MG63 cells transfected with pcDNA-MEG3. Tumor volume was assessed every five days including the day 30 after injection using the formula: volume=length×width2×0.5. Percent survival was recorded every three days up to and including the day 27 after injection.
3.4. RT-PCR and quantitative real-time PCR (qPCR)
Total RNA and miRNA were isolated from OS, and its adjacent non-involved tissues and MG63, U2OS and hFOB1.19 cell lines according to the manufacturer’s instructions. The PCR primers for MEG3, miR-184 and GAPDH were purchased from RiBoBio (Guangzhou, China) (the primer sequences in Table 1). 10 ng total RNA of each sample was used to analysis miRNA expression.Total RNA transcribed into cDNA using TaqMan® MicroRNA Reverse Transcription kit (Huiying, Shanghai, China). TaqMan® 2× Universal PCR Master Mix no UNG (Applied Biosystems) was used to performe qRT-PCR. PCR parameters were as follows 55°C1 min, followed by 35 cycles(denature:92°C for 30 sec then 58°C for 45 sec; elongation at 72°C for 35 sec). CT values of all samples and standard reference were calculated by fluorescent quantitative PCR cycler (PE, US). All experiments were carried out in triplicates and results of qPCR were assessed using ABI 7500 Fast Realtime PCR system (Applied Biosystems, Life Technologies, USA). The relative expression of miR-184 and MEG3 was determined using the 2-ΔΔCt method.
|Gene||Forward primer 5’-3’||Reverse primer 5’-3’|
|miR-184||TACGACTATGTGACCTGCCTG||TGGTTCAACT CTCCT TTCCA|
3.5. CCK-8 assay
The viability of MG63 and U2OS cell lines was evaluated using CCK-8 assay that allows sensitive colorimetric determination of cytotoxicity, cell viability and cell proliferation as described previously (15). The kit was obtained from Shanghai Xiya Biotechnology Co., Ltd (Shanghai, China). and analysis was carried out 48 h post-transfection. The OD values were assessed using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA).
3.6. Analysis of apoptosis
Using fluorescein isothiocyanate (FITC) -labeled Annexin V (KeyGEN Biotech, Nanjing, China), apoptosis in MG63 and U2OS cells was assessed by flow cytomerty 48 h after transfection as described previously (16). Apoptotic cells were also identified in 3 µm thick sections of OS tumors from mice by TUNEL assay by a kite (Name of company, City and state) and as described previously (17). Images (×400) were captured using a light microscope (Olympus, Tokyo, Japan).
3.7. Transwell mobility assay
The transfected cells were lysed with trypsin and adjusted at 1 × 105 cells/mL density. Then cells added into the upper chambers coated with 150 µg of Matrigel (BD, New Jersey, USA) and the lower chamber was filled with RPMI-1640 medium containing 10% FBS. Upon 24h incubation, non-invasive cells were washed away and the invasive cells were fixed in methanol and stained with crystal violet (Sigma, MO, USA) for 15 min. Finally, the invading cells were photographed under an optical microscope (Olympus, Tokyo, Japan).
3.8. Western bloting
The abundance of Ki67, cleaved caspase-3, vascular endothelial growth factor (VEGF), β-catenin, T-cell factor 4 (TCF4) and c-MYC (Abcam, Cambridge, UK) were assessed by Western bloting refered to previous literature reports (18,19). GAPDH was used as the house-keeping control. Immunoreactive bands were visualized by Chemiluminescent ECL Reagent Kit (Millipore, Bedford, MA, USA). Quantitation of signal intensities was performed by densitometry on a Xerox scanner using NIH ImageJ software.
3.9. TOP/FOP-FLASH luciferase reporter assay
TOP/FOP-FLASH is a method for measuring intracellular β-catenin-mediated transcriptional activity (classic wnt signaling pathway). Interactions between Tcf4 and β-catenin are responsible for activation of transcription of many tumor-related proteins (20). β-catenin and TCF4 can show co-localization relationship.TOP/FOP-FLASH luciferase reporter assay was performed as reported previously (21). Briefly, transfected MG63 cells in each group were incubated with mixture containing serum-free DMEM (20 µL), β-catenin expression vector (1.5 µg), TCF4 expression vector (0.15 µg) and Renilla luciferase vector phRG-TK (0.8 ng). 24 h later, luciferase activity was measured by a luminometer (Zhongmei, Xi'an, China).
3.10. Immunohistochemical staining
Tumor were fixed in 10% formalin, embedded in paraffin, sectioned at 3 µm and immunostained using antibodies to Ki67 (Abcam, Cambridge, UK), anti-caspase-3 (Abcam, Cambridge, UK), anti-VEGF (Abcam, Cambridge, UK), anti-β-catenin (Abcam, Cambridge, UK). using antibodies. Sections were photographed (×400) using a optical microscope.
3.11. Statistical analysis
All statistical analysis were conducted using the SPSS statistical software program (version 17.0; SPSS, Inc., Chicago, IL). The data presented in this study were expressed as means± SD. Statistical differences were analyzed by one-way ANOVA. P values < 0.05 were considered statistically significant.
4.1. Expression of MEG3 and miR-184 in OS
The expression of MEG3 and miR-184 was assed by RT-PCR followed by qPCR. The expression of MEG3 was reduced while the expression of miR-184 was increased in OS as compared to adjacent non-involved tissues showing an inverse relationship between the expression of MEG3 and miR-184 (Figure 1A-B). Carrying out the same analysis in normal osteoblast cell line, hFOB1.19, and human OS cell lines (MG63 and U2OS) showed the same trend and of existence of an inverse relation between the expression of MEG3 and miR-184 (Figure 1C).
To decipher of the cause of such an inverse correlation, using the online software Diana Tools (http://diana.imis.athena-innovation.gr), we analyzed the MEG3 elements that were complementary to and were potetinal binding site for miR-184. This analysis revealed of existence of many elements that were complementary to miR-184 in MEG3 (Figure 2A). To formally test such an interaction, we transduced MEG63 and U2OS cells with empty control, pcDNA vector and pcDNA-MEG3(20 μg/mL). Following transduction, while the expression of MEG3 was increased significantly in MG63 and U2OS cells, the expression of miR-184 was significantly reduced (Figure 2B). In addition, we transfected MEG63 and U2OS cells with empty control, NC mimics and miR-184 mimic. Upon transfection, the expression of miR-184 was increased significantly in MG63 and U2OS cells compared with transfected by empty control and NC mimics (Figure 2C).
To further validate direct evidence for the interaction between MEG3 and miR-184, we subcloned wild-type (MEG3 -wt) and mutated (MEG3 -mut) miR-184 binding site into dual-luciferase reporters. Figure 2D shows that the relative luciferase activity of MEG3 wt in MG63 and U2OS cells were obviously reduced after co-transfection of miR-106a-3p mimic, but did not change the activity of MEG3 mut, which suggest that miR-184 is a direct target of MEG3.
4.2. MEG3 regulates the OS tumor cell properties by targeting miR-184
Based on existence of a reverse relation between expression of MEG3 and miR-184, we formally tested the biologic impact of this relation as it relates to the proliferation, and apoptosis of OS cells. As shown in Figure 3A, cells over-expression of MEG3 inhibited cell proliferation. However, while transfection with miR-184 mimic did not by itself changed proliferation as compared to the control group, its transfection in conjunction with pc-MEG3 erased the effect of MEG3 on proliferation. Consistent with these results, flow cyomtetric analysis of apoptosis showed miR-184 mimic did not alter apoptosis while transduction with pcDNA-MEG3 increased apoptosis in OS cells (Figure 3B). However, joint transduction of cells with pcDNA-MEG3 and transfection with miR-184 decreased the apoptosis which was inducible by pcDNA-MEG3 (Figure 3B). These findings were further validated by examining the relative abundance of Ki67 as a marker of proliferation as well as cleaved caspase 3 as a measure of apoptosis (Figure 3C).
We, then, examined the effect of MEG3 and miR-184 mimic on in vitro mobility and invasiveness of OS. Figure 3D shows that while cells that transduction with pcDNA-MEG3 decreased the number of cells that migrated from the upper to the lower chamber, this mobility was enhanced by transfection of cells with miR-184 mimic (Figure 3D). Moreover, the number of invasive cells was reduced in pcDNA-MEG3 group as compared to the control or miR-184 mimic group and this enhanced mobility was recovered after transfection with miR-184 (Figure 3D).
4.3. MEG3 represses the Wnt/β-catenin pathway via targeting miR-184
Given that Wnt/β-catenin is involved in tumor progression, we considered the possibility that MEG3/miR-184 might down-regulate Wnt/β-catenin in OS. To do this, we carried out Western blotting of Wnt/β-catenin including its members and effectors such as GSK3 β Axin1, APC, β -catenin, TCF4, c-Myc, using GAPDH as a houskeeping control. Transduction with pcDNA-MEG3 decreased β-catenin, TCF4 and c-MYC while transfection with miR-184 enhanced such effects (Figure 4A).
To further validate these results, cells were transfected with Renilla luciferase vector phRG-TK (0.8 ng), β-catenin expression vector (1.5 µg), or TCF4 expression vector (0.15 µg). Compared with the control group, the expression of β-catenin was decreased in cells transfected with pcDNA-MEG3, showing lower reporter gene activity, while the cells transfected with miR-184 mimics significantly enhanced β-catenin expression, showing higher TOP-FLASH transactivation (Figure 4B).
4.4. MEG3 inhibits the OS by targeting miR-184 in vivo
Together, these findings show that MEG3 regulates different attributes of OS. To further validate these findings, in vivo, MG63 tumor cells were transduced without and with pcDNA-MEG3 and then the tumor volume was assessed every five days up to 30 days. In these while the expression of MEG3 was significantly increased the expression of miR-184 was highly suppressed (Figure 5C). Moreover, tumor volume was significantly decreased and the survival of mice increased in mice with tumors that were transduced with pcDNA-MEG3 (Figure 5A-B).
Tumors that were tranduced with pcDNA-MEG3 showed a decreased β-catenin, marked reduced Ki67 and a higher level of apoptosis as evidenced by TUNEL and caspase 3 immunostaining as compared with the control group (Figure 5D ).
Available evidence suggests that lncRNAs are implicated in tumor development and that their expression is subjected to silencing X chromosome, transcriptional activation, genomic imprinting and chromatin modification (22). Here, we carried out TargetScan and miRanda and, as orginally suggested by Li J et al., the results predicted the probability of an interaction between miR-184 and MEG3 (10). Moreover, we find that the expression of miRNA-184 is signficantly increased in OS, and OS cell lines (MG63 and U2OS) while in the same tumors MEG3 was represssed. We further valided of the inverse relation between miR-184 and MEG3 using the online software Diana Tools (http://diana.imis.athena-innovation.gr), and find that the there are elements in MEG3 that are complementary to and are potetinal binding site for miR-184. Following transduction of cells with MEG3, while the expression of MEG3 was increased significantly in MG63 and U2OS cells, the expression of miR-184 was significantly reduced. We also showed that while the luciferase activity of miR-184 is reduced in MG63 and U2OS cells that overexpressed MEG3. Thus, the results of this study conform of existence of an inverse correlation between the expression of miR-184 and MEG3. Moreover, we showed that the interplay between MEG3 and miR-184 jointly regulate tumor cell attributes including proliferation, apoptosis and mobility of cancer cells lines in vitro. MEG3 overexpression led to a decreased Ki67 positive cells, while increasing caspase-3.
As previoulsy suggested, our findings are consistent with the notion that MEG3 lncRNA appears to be a tumor suppressor in OS (23). Sahin et al. showed that miR-664a and MEG3 interact and that inhibition of miR-664a interferes with the migration of osteosarcoma cells via modulation of MEG3 (5). Sun et al. demonstrated that growth and metastasis of OS cells were promoted by lncRNA EWSAT1 by repressing the expression of MEG3 (24). Moreover, the expression of miRNAs, which is regulated at a post-transcriptional level, has been shown to be deregulated in diverse forms of cancer (25). Among these, miR-184 seems to control proliferation, and apoptosis of diverse forms of cancers including ovarian cancer (7). In line with such role, the upregulation of miR-184 enhanced the malignant biological behavior of human glioma cell line A172 by targeting FIH-1 (26).
These findings are consistent with other reports including regulation of growth and migration and apoptosis in diverse tumors and cell types. For example, the proliferation of human hepatoma cells was strongly suppressed and apoptosis was induced by ectopic expression of MEG3 in vivo (27). The migration and proliferation of artery smooth muscle cells were triggered by down-regulation of MEG3 through p53 signaling pathway (28). As a prognostic factor for colorectal cancer, MEG3 promoted chemosensitivity by elevating oxapliplatin-induced cell apoptosis (29). It has also been reported that miR-184 plays an important role in proliferation of hepatocellular carcinoma by loss of INPPL1 and serves as an anti-apoptotic factor by suppressing the expression of caspase-3 and caspase-7 (30). In a nude mouse breast cancer xenograft model, overexpression of MEG3 suppressed angiogenesis and tumorigenesis (31). Similarly, in our study, the growth rate of tumor was significantly repressed while survival rate and apoptosis remarkably elevated by MEG3 overexpression in vivo.
Under normal conditions, osteoblast differentiation and bone formation appear to be under the regultion of Wnt/β-catenin pathway (32). Wnt/β-catenin pathway also seems to play a significant role in diverse cancers in regulating their proliferation, motility and differentiation. For example, Gao Y et al. showed that MEG3 negatively regulated the activity of Wnt/β-catenin pathway in the development of retinoblastoma (13). Cisplatin resistance of lung cancer cells was increased by down-regulated MEG3 via the activation of the Wnt/β-catenin (33). In keeping with these studies, we showed that in OS cell lines, MEG3 suppressed the expression of β-catenin, TCF4 and c-MYC that was inducible by miR-184 in MG63 cells in vitro and also suppressed the β-catenin only in tumor cells that were transduced with pcDNA-MEG3 in vivo. Together, the results of this study demonstrated that MEG3 is involved in regulation of OS tumor cell behavior by regulation of miR-184 and impacting the Wnt/β-catenin (Figure 6). This result can provide a basis for target research in the treatment of osteosarcoma.
4. Y. Zhou, H. Yang, W. Xia, L. Cui, R. Xu, H. Lu, X. Dong, Z. Tian, D. Tao, Y. Cao, Q. Shi, X. He: LncRNA MEG3 inhibits the progression of prostate cancer by facilitating H3K27 trimethylation of EN2 through binding to EZH2. Journal of biochemistry, (2019)
5. Y. Sahin, Z. Altan, K. Arman, E. Bozgeyik, M. Koruk Ozer, A. Arslan: Inhibition of miR-664a interferes with the migration of osteosarcoma cells via modulation of MEG3. Biochemical and biophysical research communications, 490(3), 1100-1105 (2017)
6. M.A. Smolle, A. Leithner, F. Posch, J. Szkandera, B. Liegl-Atzwanger, M. Pichler: MicroRNAs in Different Histologies of Soft Tissue Sarcoma: A Comprehensive Review. International journal of molecular sciences, 18(9) (2017)
7. C.Z. Qin, X.Y. Lou, Q.L. Lv, L. Cheng, N.Y. Wu, L. Hu, H.H. Zhou: MicroRNA-184 acts as a potential diagnostic and prognostic marker in epithelial ovarian cancer and regulates cell proliferation, apoptosis and inflammation. Die Pharmazie, 70(10), 668-73 (2015).
8. R. Feng and L. Dong: Inhibitory effect of miR-184 on the potential of proliferation and invasion in human glioma and breast cancer cells
11. J. Luo, Y. Yao, S. Ji, Q. Sun, Y. Xu, K. Liu, Q. Diao, Y. Qiang, Y. Shen: PITX2 enhances progression of lung adenocarcinoma by transcriptionally regulating WNT3A and activating Wnt/beta-catenin signaling pathway. Cancer cell international, 19, 96 (2019)
12. F. Qu, C.B. Li, B.T. Yuan, W. Qi, H.L. Li, X.Z. Shen, G. Zhao, J.T. Wang, Y.J. Liu: MicroRNA-26a induces osteosarcoma cell growth and metastasis via the Wnt/beta-catenin pathway. Oncology letters, 11(2), 1592-1596 (2016)
13. Y. Gao and X. Lu: Decreased expression of MEG3 contributes to retinoblastoma progression and affects retinoblastoma cell growth by regulating the activity of Wnt/beta-catenin pathway. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 37(2), 1461-9 (2016)
14. T.G. He, Z.Y. Xiao, Y.Q. Xing, H.J. Yang, H. Qiu, J.B. Chen: Tumor Suppressor miR-184 Enhances Chemosensitivity by Directly Inhibiting SLC7A5 in Retinoblastoma. Frontiers in oncology, 9, 1163 (2019)
15. Q. Lin, H.L. Wong, F.R. Tian, Y.D. Huang, J. Xu, J.J. Yang, P.P. Chen, Z.L. Fan, C.T. Lu, Y.Z. Zhao: Enhanced neuroprotection with decellularized brain extracellular matrix containing bFGF after intracerebral transplantation in Parkinson's disease rat model. International journal of pharmaceutics, 517(1-2), 383-394 (2017)
16. K. Yamagata, Y. Izawa, D. Onodera, M. Tagami: Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells. Molecular and cellular biochemistry, 441(1-2), 9-19 (2018)
17. X. Lv, J. Yan, J. Jiang, X. Zhou, Y. Lu, H. Jiang: MicroRNA-27a-3p suppression of peroxisome proliferator-activated receptor-gamma contributes to cognitive impairments resulting from sevoflurane treatment. Journal of neurochemistry, 143(3), 306-319 (2017)
18. G. Wang, S. Wang, C. Li: MiR-183 overexpression inhibits tumorigenesis and enhances DDP-induced cytotoxicity by targeting MTA1 in nasopharyngeal carcinoma. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 39(6), (2017)
20. M. Yan, G. Li, J. An: Discovery of small molecule inhibitors of the Wnt/beta-catenin signaling pathway by targeting beta-catenin/Tcf4 interactions. Experimental biology and medicine, 242(11), 1185-1197 (2017)
22. D. Terracciano, S. Terreri, F. de Nigris, V. Costa, G.A. Calin, A. Cimmino: The role of a new class of long noncoding RNAs transcribed from ultraconserved regions in cancer. Biochimica et biophysica acta. Reviews on cancer, 1868(2), 449-455 (2017)
23. Y. Wang and D. Kong: Knockdown of lncRNA MEG3 inhibits viability, migration, and invasion and promotes apoptosis by sponging miR-127 in osteosarcoma cell. Journal of cellular biochemistry, 119(1), 669-679 (2018)
24. L. Sun, C. Yang, J. Xu, Y. Feng, L. Wang, T. Cui: Long Noncoding RNA EWSAT1 Promotes Osteosarcoma Cell Growth and Metastasis Through Suppression of MEG3 Expression. DNA and cell biology, 35(12), 812-818 (2016)
26. Q. Yuan, W. Gao, B. Liu, W. Ye: Upregulation of miR-184 enhances the malignant biological behavior of human glioma cell line A172 by targeting FIH-1. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 34(4), 1125-36 (2014)
27. L.X. Liu, W. Deng, X.T. Zhou, R.P. Chen, M.Q. Xiang, Y.T. Guo, Z.J. Pu, R. Li, G.F. Wang, L.F. Wu: The mechanism of adenosine-mediated activation of lncRNA MEG3 and its antitumor effects in human hepatoma cells. International journal of oncology, 48(1), 421-9 (2016)
28. Z. Sun, X. Nie, S. Sun, S. Dong, C. Yuan, Y. Li, B. Xiao, D. Jie, Y. Liu: Long Non-Coding RNA MEG3 Downregulation Triggers Human Pulmonary Artery Smooth Muscle Cell Proliferation and Migration via the p53 Signaling Pathway. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 42(6), 2569-2581 (2017)
29. L. Li, J. Shang, Y. Zhang, S. Liu, Y. Peng, Z. Zhou, H. Pan, X. Wang, L. Chen, Q. Zhao: MEG3 is a prognostic factor for CRC and promotes chemosensitivity by enhancing oxaliplatin-induced cell apoptosis. Oncology reports, 38(3), 1383-1392 (2017)
30. B. Gao, K. Gao, L. Li, Z. Huang, L. Lin: miR-184 functions as an oncogenic regulator in hepatocellular carcinoma (HCC). Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 68(2), 143-8 (2014)
31. C.Y. Zhang, M.S. Yu, X. Li, Z. Zhang, C.R. Han, B. Yan: Overexpression of long non-coding RNA MEG3 suppresses breast cancer cell proliferation, invasion, and angiogenesis through AKT pathway. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 39(6), (2017)
32. Y. Wu, S.Y. Xu, S.Y. Liu, L. Xu, S.Y. Deng, Y.B. He, S.C. Xian, Y.H. Liu, G.X. Ni: Upregulated serum sclerostin level in the T2DM patients with femur fracture inhibits the expression of bone formation/remodeling-associated biomarkers via antagonizing Wnt signaling. European review for medical and pharmacological sciences, 21(3), 470-478 (2017).
33. Y. Xia, Z. He, B. Liu, P. Wang, Y. Chen: Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/beta-catenin signaling pathway. Molecular medicine reports, 12(3), 4530-4537 (2015)