European Review for Medical and Pharmacological Sciences 2019; 23: 2838-2846 LncRNA MAFG-AS1 promotes the aggressiveness of breast carcinoma through regulating miR-339-5p/MMP15 H. LI1, G.-Y. ZHANG2, C.-H. PAN3, X.-Y. ZHANG1, X.-Y. SU4 1Department of Obstetrics and Gynecology, Shandong Jiyang Public Hospital, Ji’nan, Shandong, China 2Department of Anesthesiology, Shandong Jiyang Public Hospital, Ji’nan, Shandong, China 3Department of Obstetrics and Gynecology, LanCun central hospital, Jimo, Shandong, China 4Department of Critical Care Medicine, Tai’an Central Hospital, Tai’an, Shandong, China Abstract. – OBJECTIVE: The main purposes of and is the leading cause of cancer-related deaths this study are to investigate the possible effects worldwide1. Recently, treatment strategies, such of long noncoding RNAs (lncRNAs) MAFG-AS1 on as chemotherapy, radiotherapy and molecular tar- the growth and metastasis of breast carcinoma. PATIENTS AND METHODS: geting treatment significantly improve the thera- The quantitative 2 Real Time-Polymerase Chain Reaction (qRT- peutic outcome of patients . However, the clinical PCR) assay was used to assess the MAFG-AS1 outcome of patients with breast cancer needs to level in breast cancer tissues and cells. The improve. The metastasis of cancer cells is one ma- wound healing and transwell invasion analy- jor difficulty of overcoming the poor prognosis of sis were applied to explore the invasion and mi- breast cancer patients. The epithelial-mesenchy- gration of breast cancer cell in vitro. The ex- mal transition (EMT) process of cancer cells is a pressions of epithelial-mesenchymal transition 3,4 (EMT) related markers were determined by West- crucial step during metastasis . Therefore, inhi- ern blotting. Xenograft model and lung metasta- bition of EMT may be helpful for inhibiting breast sis model were used to assess the progression cancer cell metastasis and for improving the poor of breast carcinoma cell in vivo. clinical outcome of patients. RESULTS: The level of lncRNA MAFG-AS1 is Earlier investigations have proved that ln- higher in breast carcinoma, and the aggressive cRNAs epigenetically regulate their target genes phenotypes of breast carcinoma cell are en- and play crucial roles in the development of hanced by MAFG-AS1 transfection. Moreover, we 5-7 identify that MAFG-AS1 overexpression reduces cancers . For example, lncRNA maternally ex- the expression of miR-339-5p and miR-339-5p is pressed gene 3 (MEG3) suppresses the growth the target of MAFG-AS1 in breast carcinoma. In and metastasis of gastric cancer cell via regulat- addition, matrix metalloproteinase 15 (MMP15) is ing the p53 signaling pathway8. In colorectal can- the functional regulated gene of miR-339-5p in cer, lncRNA MAFG-AS1 promotes the develop- breast carcinoma. The aggressiveness of breast ment of tumor through regulating miR-147b and carcinoma induced by lncRNA MAFG-AS1 is activating NADH dehydrogenase (Ubiquinone) 1 weakened by the miR-339-5p. Finally, we demon- 9 strated that the development of breast carcinoma Alpha Subcomplex, 4 (NDUFA4) . MicroRNAs cell is enhanced by MAFG-AS1 in vivo. (miRNAs), which are small endogenous nucle- CONCLUSIONS: MAFG-AS1 appears to play otides, regulate the expression of target genes. an oncogene role in breast carcinoma by regu- Currently, more than 2000 miRNAs have been lating the miR-339-5p/MMP15. identified and have been proved to be related to diseases, including cancer10,11. Key Words: Breast cancer, Metastasis, MAFG-AS1, MMP15, MiR- Here, we demonstrated that MAFA-AS1 was 339-5p. over-regulated in breast cancer tissue compared to that in adjacent normal tissue. Moreover, we identified that miR-339-5p was modulated by Introduction MAFA-AS1 in breast cancer. Additionally, we verified that the aggressiveness of breast cancer Breast cancer has been recognized as one of cell was enhanced by MAFG-AS1. Functional the most common types of malignant cancers experiments demonstrated that lncRNA MAFG- 2838 Corresponding Authors: Xiao Yan Su, MD; email: [email protected] LncRNA MAFG-AS1 and breast cancer aggressiveness by regulating miR-339-5p/MMP15 AS1 facilitated the aggressiveness of breast cancer scribed by PrimeScript RT Reagent Kit and oligo through the modulation of miR-339-5p-MMP15. dT primer (TaKaRa, Dalian, Liaoning, China). The SYBR PrimeScript miRNA RT PCR kit (Ta- KaRa, Dalian, Liaoning, China) was used to de- Materials and Methods tect the level of miRNA-363. The 2(-∆∆Ct) method was adapted to the analysis of MMP15, lncRNA Breast Carcinoma Tissues and Cells MAFG-AS1, and miR-339-5p. The primer se- Breast carcinoma cells (MDA-MB-231, quences are the as following: MMP15 forward, HCC1937, MCF-7, and MDA-MB-468) and the 5’-ATGACGACCCCCAATAAAGGA-3’, and normal epithelial breast cell line, MCF-10A, were reverse, 5’-CACCGACATGGTTACCAGC-3’; obtained from Nanjing Cobioer Biotechnolo- GAPDH forward, 5′-AGGTCGGTGTGAACG- gy Co., Ltd (Nanjing, Jiangsu, China). The cells GATTTG-3′, and reverse: 5′-TGTAGACCAT- were was cultured using Dulbecco’s Modified Ea- GTAGTTGAGGTCA-3′; miR-339-5p, forward gle’s Medium (DMEM; Thermo Fisher Scientif- 5’-GGGTCCCTGTCCTCCA-3’ and reverse: ic, Waltham, MA, USA) containing 10% of FBS 5’-TGCGTGTCGTGGAGTC-3’; U6 forward, (Thermo Fisher Scientific, Waltham, MA, USA). 5’-CTCGCTTCGGCAGCACATATACT-3’, and 42 cases of breast cancer and normal tissues were reverse, 5’-ACGCTTCACGAATTTGCGT- obtained from the Tai’an Central Hospital (Tai’an, GTC-3’; MAFG-AS1 forward, 5’-ATGACGAC- Shandong, China). The experiment protocol was CCCCAATAAAGGA-3’, and reverse, 5’-CAC- approved by the Ethics Committee of the Tai’an CGACATGGTTACCAGC-3’. Central Hospital. Luciferase Reporter Gene Assay Transfections The pmirGLO Dual-Luciferase miRNA Tar- MiRNA-control inhibitor (miR-NC inhibitor), get Expression Vector was obtained from Prome- miR-339-5p inhibitor, miRNA control (miR-NC) ga (Madison, WI, USA). The wild type 3’-UTR and miR-339-5p were obtained from GeneCopoe- of MMP15 or MAFG-AS1 (wt-MMP15 or wt- ia (Guangzhou, Guangdong, China). Scramble or MAFG-AS1) was inserted into the vector. The MAFG-AS1, which was provided by GeneCopoe- mutated type 3’-UTR of MAFG-AS1 or MMP15 ia and was cloned into the pcDNA3.1 (GeneCo- (mut-MAFG-AS1 or mut-MMP15) was produced poeia). MDA-MB-231 cell was transfected with using the quikChange XL site-directed muta- miRNAs using Lipofectamine 3000 (Thermo genesis kit (Agilent Technologies, Santa Clara, Fisher Scientific, Waltham, MA, USA). CA, USA). MAFG-AS1 and miR-339-5p or miR- 339-5p and MMP15 were co-transfected into Migration Assay MDA-MB-231 cell using the Lipofectamine 3000 5 x 105 MDA-MB-231 or MCF-7 cell was seed- (Thermo Fisher Scientific, Waltham, MA, USA). ed into 6 well plates. After 24 h, a wound was The luciferase reporter assay kit (Promega, Mad- made by a 100 μl pipette tip. The picture of the ison, WI, USA) was applied to the analysis of the wound width was taken at 0 h and 48 h, respec- luciferase activity. tively. Transplanted Tumor Model Invasion Assay 100 μl MDA-MB-231 cells (2x107) were subcu- 200 μl MCF-7 or MDA-MB-231 cells (1 x 105) taneous injected subcutaneously into nude mice. were seeded in the upper chamber of the transwell The tumor size was weekly recorded and the (Corning, Shanghai, China). 600 μl medium con- tumor volumes were calculated. Volume = 0.5× tained 20% FBS was used as a chemo-attractant length × width2. After 35 days, all mice were sac- and plated into the lower chamber. After 24 h, rificed and the tumor was extracted for the sub- 4% of paraformaldehyde and 1% of crystal vio- sequent immunohistochemical staining analysis. let were used to fix and stain the invaded cells, respectively. Experimental Metastasis Model MAFG-AS1 transfected MDA-MB-231 cell Quantitative Real-time (qRT-PCR) Assay (5×10 5) was injected into BALB/c mice through RNAs were extracted with TRIzol (Thermo the tail vein. After two weeks, the mice were sac- Fisher Scientific, Waltham, MA, USA). For the rificed and lung tissues were applied for H&E analysis the level of MMP15, RNA was tran- staining. All animal experiments were approved 2839 H. Li, G.-Y. Zhang, C.-H. Pan, X.-Y. Zhang, X.-Y. Su by the Ethics Committee of Tai’an Central Hospi- higher in breast carcinoma in contrast to normal tal (Shandong, China). (Figure 1B). Meanwhile, MAFG-AS1 was dis- tinctly over-expressed in breast carcinoma cells, Statistical Analysis including MDA-MB-231, MCF-7, MDA-MB-468, Results were expressed as the Mean ± SD. The and HCC1937) than that in the normal epitheli- difference was analyzed using either two-tailed al breast cell, MCF-10A (Figure 1C). These data Student’s t-test or one-way ANOVA followed by suggested that MAFG-AS1 was up-regulated in post-hoc Dunnett’s test. p<0.05 was statistically breast carcinoma. significant. The Aggressive Phenotypes of Breast Carcinoma was Enhanced by MAFG-AS1 Results The pcDNA3.1 containing MAFG-AS1 was transfected into the MDA-MB-231 cell to raise MAFG-AS1 Is Up-regulated in Breast MAFG-AS1 level (Figure 2A). Transwell inva- Carcinoma sion and wound closure analysis were applied to The statistical analysis of dysregulation of analyze the function of MAFG-AS1 on the MDA- lncRNAs was conducted using the dataset MB-231 cell invasion and migration. We observed (GSE113851) which contained both cancer and that the migration and invasion of MDA-MB-231 normal tissues12. We found that MAFG-AS1 was cell were enhanced by the transfection of MAFG- overexpressed in breast carcinoma tissue in con- AS1 (Figure 2B-2C). At the same time, the aggres- trast to that in the normal tissue (Figure 1A). siveness of the MCF-7 cell was also promoted by Meanwhile, MAFG-AS1 in breast cancer, as MAFG-AS1 (Figure 2D-2E).