MOLECULAR MEDICINE REPORTS 12: 5443-5448, 2015

MicroRNA-7 inhibits the malignant phenotypes of non‑small cell lung cancer in vitro by targeting Pax6

JIASHUN LUO1,2, HUI LI1 and CHUNFANG ZHANG2

1Institute of Medical Research, University College of Medicine, Jishou, 416000; 2Department of Cardiothoracic Surgery, Xiangya Hospital of , , Hunan 410008, P.R.

Received June 25, 2014; Accepted June 15, 2015

DOI: 10.3892/mmr.2015.4032

Abstract. MicroRNA (miR)-7 has been reported to act as a chemotherapy, the overall survival rate of patients with lung suppressor in several types of cancer, including non-small cancer remains poor, with only 14% of patients surviving cell lung cancer (NSCLC). In addition, paired box 6 (Pax6), a 5 years from the date of diagnosis (1). As non-small cell lung highly conserved transcriptional factor, has been implicated in cancer (NSCLC) is the most common type of lung cancer, NSCLC. However, the exact role of miR-7, and the association the development of effective therapeutic targets for NSCLC is between miR-7 and Pax6 in NSCLC cells remain to be fully urgently required (1). elucidated. The present study demonstrated that miR-7 was The development and progression of NSCLC are associ- downregulated and Pax6 was upregulated in NSCLC cell ated with the dysregulation of oncogenes or tumor suppressors, lines. Subsequently, it was demonstrated that overexpression of including microRNAs (miRNAs) (2). miRNAs are 18-25 nucle- miR-7 notably inhibited the protein expression of Pax6, while otide, non-coding RNAs, which can result in the inhibition of inhibition of miR-7 enhanced the protein expression of Pax6 in gene expression at the post-transcriptional level, through direct NSCLC A549 cells. Further investigation identified Pax6 as a binding to the 3'-untranslational region (UTR) of mRNAs (3). target of miR-7 in A549 NSCLC cells. Ina ddition, the overex- In addition, the dysregulation of miRNAs has been reported to pression of miR-7 significantly inhibited A549 cell proliferation be associated with the development and progression of NSCLC, and invasion, which was reversed by upregulation of Pax6. including miRNA (miR)-7 (4,5). miR-7 has been demonstrated Investigation of the underlying molecular mechanism revealed to act as a tumor suppressor in NSCLC, through targeting that the extracellular signal-regulated kinase (ERK) and p38 B-cell lymphoma (BCL)-2 and PA28γ (6,7). As one miRNA mitogen-activated protein kinase (MAPK) signaling pathways can directly bind to several target mRNAs, whether other target were downregulated in the miR-7-overexpressed A549 cells, but genes of miR-7 exist in NSCLC remains to be fully elucidate. were activated in the Pax6-overexpressed A549 cells. Based on Paired box 6 (Pax6) has been demonstrated as a highly these findings, it was suggested that miR-7 negatively regulates conserved transcription factor during embryogenesis, and is the protein level of Pax6, which can promote the proliferation involved in the development and function of the central nervous and invasion of NSCLC cells via activation of the ERK and system, endocrine glands, eyes and pancreas (8-12). The role of MAPK signaling pathways. Therefore, miR-7/Pax6 may offer Pax6 in the development and progression of cancer been gradu- potential for use as a target for the treatment of NSCLC. ally revealed (13,14). Zhao et al reported that shRNA-induced RETRACTEDPAX6 downregulation notably suppressed proliferation and Introduction cell cycle progression in NCSLC cells. They further demon- strated that the extracellular signal-regulated kinase (ERK) Lung cancer is the leading cause of cancer-associated mortality, and p38 mitogen-activated protein kinase (MAPK) signaling with an increasing incidence worldwide (1). Despite improve- pathways are involved in Pax6-mediated cell cycle progression ments in diagnostic imaging, surgery, radiotherapy and in NCSLC cells (15). The present study aimed to examine the role of miR-7 in the regulation of NSCLC in vitro. In addition, as a previous study demonstrated a target association between miR-7 and Pax6 in colon cancer (16), the present study also investigated whether Correspondence to: Professor Chunfang Zhang, Department of Cardiothoracic Surgery, Xiangya Hospital of Central South Pax6 is involved in the regulatory effect of miR-7 on NSCLC University, 87 Xiangya Road, Changsha, Hunan 410008, P.R. China cells. E-mail: [email protected] Materials and methods Key words: non small cell lung cancer, microRNA-7, paired box 6, proliferation, invasion Agents. TRIzol reagent, fetal bovine serum (FBS) Lipofectamine 2000 and the mirVana™ qRT-PCR miRNA Detection kit were purchased from Invitrogen Life 5444 LUO et al: MicroRNA-7 INHIBITS NSCLC BY TARGETING PAX6 in vitro

Technologies (Carlsbad, CA, USA). Mouse anti-Pax6, -total Cybernetics, Inc., Rockville, MD, USA), and presented as the ERK, -phosphorylated (p)-ERK, -p38 MAPK and -GAPDH density ratio, vs. GAPDH. primary antibodies, and rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK). Transfection. The plasmid of Pax6, scramble miRNA mimics, Bioinformatics analysis was conducted to predicate the puta- miR-7 mimics and miR-7 inhibitor were constructed by tive target genes of miR-7 using the Targetscan online software Nlunbio (Changsha, China). Lipofectamine 2000 was used (http://www.targetscan.org/). The Quick‑Change Site-Directed to perform transfection, according to the manufacture's Mutagenesis kit was purchased from Stratagene (La Jolla, instruction. Briefly, the plasmid or miRNA mimics and CA, USA). The PsiCHECK™2 vector was purchased from Lipofectamine 2000 were diluted with 100 nM serum-free Promega Corporation (Madison, WI, USA). The enhanced medium, respectively. The diluted Lipofectamine 2000 was chemiluminescence (ECL) kit was purchased from Pierce added into the diluted plasmid or miRNA mimics, respectively, Biotechnology, Inc. (Rockford, IL, USA). and incubated for 20 min at room temperature, following which they were added to the cell suspension. The cells (105 cells/ml), Cell lines and cell culture. The A549, H460, SK-MES-1 and which were suspended in DMEM were then incubated at 37˚C,

SPC-A1 human NSCLC cell lines, and normal BEAS-2B 5% CO2 for 6 h. Following incubation, the medium in each human lung epithelial cell line were purchased from the Cell well was replaced with normal serum‑containing medium, and bank of Central South University (Changsha, China). All the cells were cultured for 24 h prior to performing the subse- cells were cultured in Dulbecco's modified Eagle's medium quent assays. The successful transfection was confirmed by (DMEM; Invitrogen Life Technologies) supplemented with detecting the expression levels of PAX6 or miR-7, respectively.

10% FBS at 37˚C with 5% CO2. Dual luciferase reporter assays. A Quick-Change Site‑Directed RNA extraction and reverse transcription-quantitative poly- Mutagenesis kit was used to generate a mutant 3'-UTR of Pax6, merase chain reaction (RT-qPCR). Total RNA was extracted according to the manufacturer's instructions. The wild-type or from the cells using TRIzol reagent, according to the manu- mutant 3'-UTR of Pax6 were inserted into the psiCHECK™2 facturer's instructions. The relative expression level of miR-7 vector, respectively. A549 cells (105 cells/well), which were was determined by RT-qPCR using a mirVana™ qRT-PCR cultured to ~70% confluence, were the transfected with either miRNA Detection kit, according to the manufacturer's the psiCHECK™2-Pax6-3'-UTR or psiCHECK™2-mutant instruction. Specific primer sets for miR-7 and U6 (internal Pax6 -3'-UTR vector, with or without 100 nM miR-7 mimic, reference) were obtained from GeneCopoeia, Inc. (Maryland respectively. Following transfection for 48 h, the luciferase Rockville, MD, USA). The PCR cycling conditions were as activities were determined using an LD400 luminometer follows: 95˚C for 10 min, denaturation at 95˚C for 15 sec and an (Beckman Coulter, Fullerton, CA, USA). The activity of annealing/elongation step at 60˚C for 1 min for 40 cycles. The Renilla luciferase was normalized to that of firefly luciferase. data were analyzed with SDS relative quantification software version 2.2.2 (Applied Biosystems, Foster City, CA, USA). The Cell proliferation assay. An MTT assay was performed to 2-ΔΔCt method was used for the relative quantification of the measure cell proliferation. Cells in the exponential growth differences in expression levels of each target. were plated, at a final concentration of 2,000 cells per well, into 96-well plates. The viability of the cells were evaluated using Western blotting. The cells (107) were solubilized in cold radio- an MTT assay (20 µl MTT) 24, 48, 72 and 96 h after seeding. immunoprecipitation assay lysis buffer (Beyotime Institute of The optical density (OD) at 570 nm of each well was measured Biotechnology, Shanghai, China). The proteins (60 µg) were using an ELISA reader (ELX-800; BioTek Instruments, Inc., separated with 12% SDS-PAGE (Beijing Biolab Science and Winooski, VT, USA). Technology Co., Ltd., Beijing, China), and transferred onto a polyvinylidene difluorideRETRACTED (PVDF) membrane (Invitrogen Life Cell invasion assay. The invasive abilities of the A549 Technologies), which was then incubated with Tris-buffered cells were determined using 24-well Transwell chambers saline with Tween 20 (TBST; Fanke Biotech Co., Ltd., (Chemicon, Temecula, CA, USA), which contained a layer of Shanghai, China) containing 5% milk at room temperature for Matrigel. For each group, the cell suspension was added to the 3 h. The PVDF membrane was then incubated with primary upper chamber, and DMEM, containing 10% FBS, was added antibodies at room temperature for 3 h. The primary antibodies to the lower chamber. Following incubation for 24 h at 37˚C, were as follows: Monoclonal mouse anti-human PAX6 (1:100, the non-invading cells and the matrix gel on the interior of the cat. no. ab78545), monoclonal mouse anti-human ERK (1:100, inserts were removed using a cotton-tipped swab. The inva- cat. no. ab119933), polyclonal rabbit anti-human p-ERK (1:200, sive cells on the lower surface of the membrane were stained cat. no. ab131438), monoclonal mouse anti-human p38 MAPK, with gentian violet, followed by rinsing with water and air (1:100, cat. no. ab31828) and monoclonal rabbit anti-human drying. Subsequently, the number of cells were counted in five p-p38 MAPK, (1:100, cat. no. EPR16587). This was followed randomly-selected fields under an inverted microscope (IX71; by incubation with rabbit anti-mouse IgG, (cat. no. ab46540) Olympus, Tokyo, Japan). and mouse anti-rabbit IgG (cat. no. ab99700) secondary antibodies at room temperature for 40 min. All antibodies Statistical analysis. The results are expressed as the were purchased from Abcam. Chemiluminescent detection mean ± standard deviation of at least three independent was performed using an ECL kit. The relative protein expres- experiments. Statistical analysis was performed using SPSS 17 sion was analyzed using Image-Pro plus software 6.0 (Media software (SPSS, Inc., Chicago, IL, USA). Statistical analysis of MOLECULAR MEDICINE REPORTS 12: 5443-5448, 2015 5445 differences was performed using one-way analysis of variance. Overexpression of miR-7 inhibits NSCLC A549 cell invasion P<0.01 was considered to indicate a statistically significant through inhibition of Pax6. The present study subsequently difference. further investigated the roles of miR-7 and Pax6 in the regula- tion of A549 NSCLC cell invasion. The findings demonstrated Results that upregulation of miR-7 inhibited A549 NSCLC cell inva- sion, whereas in the Pax6-overexpressed A549 NSCLC cells, miR-7 is downregulated, while Pax6 is upregulated in NSCLC cell invasion was upregulated. Furthermore, restoration of the cell lines. To determine the role of miR-7 in NSCLC, the expression of Pax6 reversed the suppressive effect of miR-7 present study first examined the expression level of miR-7 in overexpression on NSCLC A549 cell proliferation. These data the A549, H460, SK-MES-1 and SPC-A1 human NSCLC cell suggested that overexpression of miR-7 inhibited NSCLC cell lines, and BEAS-2B normal human lung epithelial cell line. As invasion through the inhibition of Pax6 (Fig. 4). shown in Fig. 1A, the expression level of miR-133a was signifi- cantly reduced in the NSCLC cell lines, compared with the Activities of the ERK and p38 MAPK pathways are mediated BEAS-2B normal human lung epithelial cells. Subsequently, by miR-7 and Pax6 in A549 NSCLC cells. The activities of the the expression level of Pax6 was determined by performing ERK and p38 MAPK signaling pathways were determined RT-qPCR. As shown in Fig. 1B, the mRNA level of Pax6 following the overexpression of Pax6 or miR-7 in the A549 was upregulated in the NSCLC cell lines, compared with the human NSCLC cells. As shown in Fig. 5, the phosphorylated BEAS-2B normal human lung epithelial cells. Accordingly, protein levels of ERK and p38 MAPK were suppressed in the these data demonstrated that miR-7 was downregulated, while miR-7-overexpressed A549 cells, but were increased in the Pax6 was upregulated in the NSCLC cell lines. Pax6-overexpressed A549 cells. These data indicated that As the A549 cells exhibited the most marked changes in the activities of the ERK and p38 MAPK signalingpathways expression levels of miR-7 and Pax6 among the four NSCLC were downregulated by the overexpression of miR-7, but cell lines, when compared with the BEAS-2B normal human upregulated by the overexpression of Pax6 in A549 NSCLC lung epithelial cells (Fig. 1B), the A549 cell line was selected cells. for examination in the subsequent experiments. Discussion miR-7 negatively regulates the protein expression of its target, Pax6, in A549 NSCLC cells. Based on the above data, In the present study, the roles of miR-7 and Pax6, as well as the regulatory association between miR-7 and Pax6 in A549 their associations, were investigated in NSCLC cells. The NSCLC cells were further investigated. Following transfection results demonstrated that miR-7 was downregulated, while with miR-7 mimics or inhibitor, the level of miR-7 in the A549 Pax6 upregulated in the NSCLC cell lines. Further investiga- NSCLC cells was examined, and the data revealed that the tion identified Pax6 as a target of miR-7 in the A549 NSCLC transfection had been successful (Fig. 2A). The protein level cells, and the protein expression of Pax6 was negatively medi- of Pax6 in each group was then determined by performing ated by miR-7. Overexpression of miR-7 significantly inhibited western blotting, which demonstrated that, in the A549 NSCLC A549 cell proliferation and invasion, which was reversed by cells transfected with miR-7 mimics, the protein level of Pax6 the upregulation of Pax6. In addition, the ERK and p38 MAPK was reduced (Fig. 2B). By contrast, inhibition of miR-7 led signaling pathways were downregulated in miR-7-overex- to an increase in the protein expression of Pax6 in the A549 pressed A549 cells, but activated in Pax6-overexpressed A549 NSCLC cells (Fig. 2B). Based on the bioinformatical predic- cells. tion that the putative seed sequences for miR-7 at the 3'UTR It has been demonstrated that miR-7 acts as a tumor of Pax6 is conserved (Fig. 2C), the present study performed suppressor in various types of cancer. Reduced levels of a luciferase reporter assay to determine whether Pax6 was a miR-7 have been linked to the development of cancer and target of miR-7. The resultingRETRACTED data indicated that the luciferase metastasis (17). Zhou et al reported that miR-7 inhibits tumor activity was reduced only in the A549 NSCLC cells that were metastasis and reverses epithelial-mesenchymal transition co-transfected with the miR-7 mimics and wild‑type Pax6 through inhibition of AKT and ERK signaling in epithelial 3'UTR. In the remaining groups, the luciferase activity was ovarian cancer (18). Xie et al demonstrated that miR-7 inhibits unchanged (Fig. 2D). These findings indicated that Pax6 was a gastric cancer cell invasion and metastasis by suppressing direct target of miR-7 in the A549 NSCLC cells. the expression of epidermal growth factor receptor (19). In adddition, miR-7 has been reported to inhibit colorectal Overexpression of miR-7 inhibits NSCLC A549 cell prolif- cancer cell proliferation and induce apoptosis (20). The role of eration by targeting Pax6. To further investigate the effect of miR-7 in NSCLC has also been reported. Xiong et al revealed Pax6 and miR-7 on NSCLC cell proliferation, an MTT assay that miR-7 has a suppressive effect in NSCLC by targeting was performed. As shown in Fig. 3, in the miR-7-overex- PA28γ (6). In addition miR-7 has been observed to inhibit the pressed A549 cells, the cell proliferation was downregulated, growth of A549 NSCLC cells through targeting BCL-2 (7). whereas in the Pax6-overexpressed A549 cells, the cell As a single miRNA has multiple targets, and one gene can proliferation rate was upregulated. In addition, upregulation be regulated by various miRNAs (21), the presents study of Pax6 attenuated the inhibitory effect of miR-7 overexpres- aimed to identify other targets of miR-7 in NSCLC cells, and sion on A549 cell proliferation. These data suggested that demonstrated that the inhibition of Pax6 was involved in miR- overexpression of miR-7 inhibited A549 cell proliferation by 7‑overexpression‑induced downregulation of A549 NSCLC targeting Pax6. cell proliferation and invasion. 5446 LUO et al: MicroRNA-7 INHIBITS NSCLC BY TARGETING PAX6 in vitro

A B

Figure 1. (A) Expression levels of miR-7 were determined using RT-qPCR in the A549, H460, SK-MES-1 and SPC-A1 human NSCLC cell lines, and BEAS‑2B normal human lung epithelial cells. **P<0.01, vs. BEAS-2B. (B) mRNA expression levels of Pax6 were examined using RT-qPCR in the A549, H460, SK-MES-1 and SPC-A1 human NSCLC cell lines, and BEAS‑2B normal human lung epithelial cells. *P<0.05, vs. BEAS-2B; **P<0.01 vs. BEAS-2B. GAPDH was used as an internal control. Data are expressed as the mean ± standard deviation. NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. miR, microRNA.

A B

C D

Figure 2. (A) Expression levels of miR-7 were examined using RT-qPCR in A549 NSCLC cells transfected with miR-7 mimics or inhibitor, respectively. Untreated A549 cells were used as a control (**P<0.01, vs. control). (B) Protein levels of Pax6 were determined using western blotting in A549 NSCLC cells transfected with miR-7 mimics or inhibitor, respectively. Untreated A549 cells were used as a control (**P<0.01, vs. control). (C) Putative seed sequences of miR-7 in the 3'-UTR of Pax6 mRNA were indicated from online bioinformatics analysis software (Targetscan; http://www.targetscan.org). (D) Data of the luciferase reporter assay demonstrated that the luciferase activity was reduced only in the A549 NSCLC cells co-transfected with the miR-7 mimics and wild‑type Pax6 3'UTR, and were unchanged in the remaining groups. A549 cells transfected only with wild-type or mutant type Pax6 3'-UTR were used as controls (**P<0.01, vs. control). Data are expressed as the mean ± standard deviation. NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription- quantitative polymerase chain reaction. miR, microRNA; UTR, untranslated region.

As a transcription factor,RETRACTED Pax6 mediates the expression of study, that overexpression of Pax6 induced upregulation in the various genes. In addition, the expression of Pax6 has been activity of ERK and p38 MAPK signaling. It has been well reported to be upregulated in pancreatic cancer and retino- established that the ERK and p38 MAPK signaling pathways blastoma, but downregulated in glioma and prostate cancer, are involved in the development and progression of various suggesting that Pax6 may have different roles in different types types of cancer, including NSCLC, and targeting components of cancer (13,22-24). In the present study, the results suggested of the ERK and MAPK signaling pathways offers an attractive that, as a target of miR-7, Pax6 acts as an oncogene in NSCLC strategy in the development of novel therapeutic approaches to through promoting NSCLC cell proliferation and invasion. treat NSCLC (25,26). Previously, Zhao et al investigated the role of Pax6 in NSCLC In addition, the present study demonstrated for the first cell proliferation and reported that inhibition of the expression time, to the best of our knowledge, that miR-7/Pax6 is involved of Pax6 suppresses cell growth and colony formation in A549 in the regulation of NSCLC cell invasion. The role of Pax6 and H1299 NSCLC cells (15). In addition, the percentage of in cancer cell invasion has also been demonstrated in several NSCLC cells in the G1-phase is increased following a reduc- other types of cancers, including glioma and colon cancer (27). tion in the expression of Pax6, indicating that the inhibition Cheng et al reported that Pax6 inhibits cell proliferation and of Pax6 induces cell cycle arrest at the G1 phase (15). It was invasion in glioma cells (27), and, Liu et al suggested that also demonstrated that the activitites of the ERK and p38 brain fatty acid-binding protein 7 is a target gene of Pax6, and MAPK signaling pathways were suppressed in Pax6 knock- is involved in the regulation of glioma cell invasion (28). In down cells (15), consistent with the data obtained the present glioblastoma cells, Pax6 has been reported to suppress cell MOLECULAR MEDICINE REPORTS 12: 5443-5448, 2015 5447

Figure 3. An MTT assay was used to measure cell proliferation in the A549 NSCLC cells transfected with the miR-7 mimic, Pax6 plasmid, or co-transfected with the miR-7 mimics and Pax6 plasmid, respectively. Untransfected A549 NSCLC cells were used as a control. NSCLC, non-small cell lung cancer; miR, microRNA; OD, optical density.

Figure 4. A Transwell assay was used to measure the invasion capacity of A549 NSCLC cells transfected with the miR-7 mimics, the Pax6 plasmid, or co- transfected with the miR-7 mimics and Pax6 plasmid (magnification, x100). Untransfected A549 NSCLC cells were used as a control *( P<0.05, vs. control; **P<0.01, vs. control). Data are expressed as the mean ± standard deviation. NSCLC, non-small cell lung cancer; miR, microRNA. RETRACTED

Figure 5. Western blot analysis was performed to determine the protein levels of p-ERK and p-p38 MAPK, as well as the total protein levels of ERK and p38 MAPK in the A549 NSCLC cells transfected with miR-7 mimics or the Pax6 plasmid. GAPDH was used as an internal reference. Untransfected A549 NSCLC cells were used as a control (**P<0.01, vs. control). Data are expressed as the mean ± standard deviation. NSCLC, non-small cell lung cancer; miR, microRNA; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; p-phosphorylated. 5448 LUO et al: MicroRNA-7 INHIBITS NSCLC BY TARGETING PAX6 in vitro invasion, at least in part, through inhibiting the protein expres- 15. Zhao X, Yue W, Zhang L, Ma L, Jia W, Qian Z, Zhang C and Wang Y: Downregulation of PAX6 by shRNA inhibits prolif- sion of matrix metalloproteinase (MMP) 2 (29). Huang et al eration and cell cycle progression of human non-small cell lung also demonstrated that MMP2 and MMP9 are downstream cancer cell lines. PLoS One 9: e85738, 2014. effectors of Pax7 in the regulation of glioblastoma cell inva- 16. Li Y, Li Y, Liu Y, Xie P, Li F and Li G: PAX6, a novel target of microRNA-7, promotes cellular proliferation and invasion in sion (13). human colorectal cancer cells. Dig Dis Sci 59: 598-606, 2014. In conclusion, the present study demonstrated that miR-7 17. Kalinowski FC, Brown RA, Ganda C, Giles KM, Epis MR, inhibited NSCLC cell proliferation and invasion through the Horsham J and Leedman PJ: microRNA-7: A tumor suppressor miRNA with therapeutic potential. Int J Biochem Cell Biol 54: direct targeting of Pax6, suggesting that Pax6 and miR-7 312-317, 2014. may serve as potential therapeutic targets in the treatment of 18. Zhou X, Hu Y, Dai L, Wang Y, Zhou J, Wang W, Di W and Qiu L: NSCLC. MicroRNA-7 inhibits tumor metastasis and reverses epithelial- mesenchymal transition through AKT/ERK1/2 inactivation by targeting EGFR in epithelial ovarian cancer. PLoS One 9: References e96718, 2014. 19. Xie J, Chen M, Zhou J, Mo MS, Zhu LH, Liu YP, Gui QJ, Zhang L 1. Greenlee RT, Murray T, Bolden S and Wingo PA: Cancer and Li GQ: miR-7 inhibits the invasion and metastasis of gastric statistics, 2000. CA Cancer J Clin 50: 7-33, 2000. cancer cells by suppressing epidermal growth factor receptor 2. Kang SM and Lee HJ: MicroRNAs in human lung cancer. Exp expression. Oncol Rep 31: 1715-1722, 2014. Biol Med (Maywood), 239, 1505-1513, 2014. 20. Xu K, Chen Z, Qin C and Song X: miR-7 inhibits colorectal 3. Huang X, Liang M, Dittmar R and Wang L: Extracellular cancer cell proliferation and induces apoptosis by targeting microRNAs in urologic malignancies: Chances and challenges. XRCC2. Onco Targets Ther 7: 325-332, 2014. Int J Mol Sci 14: 14785-14799, 2013. 21. Ambros V: The functions of animal microRNAs. Nature 431: 4. Zagryazhskaya A and Zhivotovsky B: miRNAs in lung cancer: A 350-355, 2004. link to aging. Ageing Res Rev 17: 54-67, 2014. 22. Mascarenhas JB, Young KP, Littlejohn EL, Yoo BK, Salgia R 5. Giles KM, Barker A, Zhang PM, Epis MR and Leedman PJ: and Lang D: PAX6 is expressed in pancreatic cancer and actively MicroRNA regulation of growth factor receptor signaling in participates in cancer progression through activation of the MET human cancer cells. Methods Mol Biol 676: 147-163, 2011. tyrosine kinase receptor gene. J Biol Chem 284: 27524-27532, 6. Xiong S, Zheng Y, Jiang P, Liu R, Liu X, Qian J, Gu J, Chang L, 2009. Ge D and Chu Y: PA28gamma emerges as a novel functional 23. Wang J, Wang X, Wu G, Hou D and Hu Q: MiR-365b-3p, target of tumour suppressor microRNA-7 in non-small-cell lung down‑regulated in retinoblastoma, regulates cell cycle cancer. Br J Cancer 110: 353-362, 2014. progression and apoptosis of human retinoblastoma cells by 7. Xiong S, Zheng Y, Jiang P, Liu R, Liu X and Chu Y: MicroRNA-7 targeting PAX6. FEBS Lett 587: 1779-1786, 2013. inhibits the growth of human non-small cell lung cancer A549 24. Shyr CR, Tsai MY, Yeh S, Kang HY, Chang YC, Wong PL, cells through targeting BCL-2. Int J Biol Sci 7: 805-814, Huang CC, Huang KE and Chang C: Tumor suppressor PAX6 2011. functions as androgen receptor co-repressor to inhibit prostate 8. Shaham O, Menuchin Y, Farhy C and Ashery-Padan R: Pax6: cancer growth. Prostate 70: 190-199, 2010. A multi-level regulator of ocular development. Prog Retin Eye 25. Ciuffreda L, Incani UC, Steelman LS, Abrams SL, Res 31: 351-376, 2012. Falcone I, Curatolo AD, Chappell WH, Franklin RA, Vari S, 9. Georgala PA, Carr CB and Price DJ: The role of Pax6 in forebrain Cognetti F, et al: Signaling Intermediates (MAPK and PI3K) as development. Dev Neurobiol 71: 690-709, 2011. Therapeutic Targets in NSCLC. Curr Pharm Des 20: 3944-3957, 10. Matsumoto Y and Osumi N: Role of Pax6 in the developing central 2014. nervous system. Brain Nerve 60: 365-374, 2008 (In Japanese). 26. Chen Y, Nowak I, Huang J, Keng PC, Sun H, Xu H, Wei G and 11. Simpson TI and Price DJ: Pax6; a pleiotropic player in devel- Lee SO: Erk/MAP kinase signaling pathway and neuroendocrine opment. BioEssays 24: 1041-1051, 2002. differentiation of non-small-cell lung cancer. J Thorac Oncol 9: 12. Gosmain Y, Cheyssac C, Heddad Masson M, Dibner C and 50-58, 2014. Philippe J: Glucagon gene expression in the endocrine pancreas: 27. Cheng Q, Cao H, Chen Z, Ma Z, Wan X, Peng R and Jiang B: The role of the transcription factor Pax6 in α-cell differentiation, PAX6, a novel target of miR-335, inhibits cell proliferation glucagon biosynthesis and secretion. Diabetes Obes Metab 13 and invasion in glioma cells. Mol Med Rep 10: 399-404, (Suppl 1): 31-38, 2011. 2014. 13. Huang BS, Luo QZ, Han Y, Li XB, Cao LJ and Wu LX: 28. Liu RZ, Monckton EA and Godbout R: Regulation of the FABP7 microRNA-223 promotes the growth and invasion of glio- gene by PAX6 in malignant glioma cells. Biochem Biophys Res blastoma cells by targeting tumor suppressor PAX6. Oncol Commun 422: 482-487, 2012. Rep 30: 2263-2269, 2013. 29. Mayes DA, Hu Y, Teng Y, Siegel E, Wu X, Panda K, Tan F, 14. Bai SW, Li B, Zhang H, Jonas JB, Zhao BW, Shen L and Wang YC: Yung WK and Zhou YH: PAX6 suppresses the invasiveness of Pax6 regulates proliferationRETRACTED and apoptosis of human retino- glioblastoma cells and the expression of the matrix metallopro- blastoma cells. Invest Ophthalmol Vis Sci 52: 4560-4570, 2011. teinase-2 gene. Cancer Res 66: 9809-9817, 2006.