MicroRNA-200b reduced ZEB1 to inhibit cell proliferation and migration in human gastric cancer

BACKGROUND : Gastric cancer (GC) is one of the most common malignant cancers, with high morbidity and mortality rates worldwide. The present study was to explore whether miR-200b is a tumor suppressor in GC and to unveil the potential mechanisms. METHODS: Levels of c-Myc, Cyclin D1, MMP-3 and MMP-9 expression were detected respectively by qRT-PCR and Western blot assay. BrdU proliferation assay, Cell cycle analysis, Wound-healing and Transwell assays were used to study the role of miR-200b with inhibitor, mimics or ZEB1-RNAi in TGF-β1-treated SGC-7901/DDP cells. The xenograft model with nude mice was established to unveil the role of miR-200b in vivo . RESULTS : Compared with the paracancerous tissues, miR-200b was decreased in GC patients and SGC-7901/DDP cells. Lower level of miR-200b induced by its inhibitor promoted TGF-β1-treated SGC-7901/DDP cells proliferation and migration, and increased the levels of c-Myc, Cyclin D1, MMP-3, MMP-9, β-catenin and APC. Interestingly, miR-200b mimics and ZEB1-RNAi were able to reduce the proliferation and migration of TGF-β1-induced SGC-7901/DDP cells as well as their levels of c-Myc, Cyclin D1, MMP-3 MMP-9, β-catenin and APC. In addition, ZEB1 was indeed the potential target of miR-200b identi�ed by dual luciferase reporter gene assay. Xenograft model also suggested that over-expression of miR-200b suppressed the growth of tumor in vivo. CONCLUSION


Background
Gastric cancer (GC) is a malignant tumor originating from the gastric mucosal epithelium with highest morbidity and mortality [1].Most GC patients with early have no obvious symptoms and be ignored, however, the 5-year overall survival rate less than 30% with prognosis of advanced GC due to the local and systemic metastasis [2].Transforming growth factor-beta1 (TGF-β1), includes polypeptides play important roles in regulating tumor cells, tumor-associated broblasts and immunorelated cells, has been shown to play an important role in GC.The TGF-β was increased in the cancer microenvironment after radiotherapy [3] and promoted activation of epithelial-mesenchymal transition in GC [4].Therefore, it is very important to study the molecular mechanism and effective molecular biomarkers of GC.The microRNAs (miRNAs) are a class of 18-24 nucleotides small noncoding RNAs, which regulate biological processes and pathogenesis of cancer by targeting genes [5,6], such as miR-124a in non-small cell lung cancer [7] and miR-7 for prostate cancer [8].On the other hand, accumulating evidence strongly suggests that miR-200c could reverse drug resistance of GC. [9].The miR-200 family members (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) [10] could inhibite epithelial-mesenchymal transition (EMT) by targeting E-cadherin, ZEB1 or ZEB2 [11].Interestingly, It was found ZEB1could bind to the promoter regions ofmiR-200b/200a/429 or miR-200c/141, suggesting that miR-200b could target inhibition of ZEB1 and control cell migration, invasion, and EMT [12].
In this study, we investigated the expressions of miR-200b in GC, paracancerous tissues and SGC-7901/DDP cells, and further explored the functions of miR-200b in GC.

Methods
Human GC tissue collection All GC tissues (n=6) were obtained from GC from First A liated Hospital, Anhui Medical University, Hefei, China.After patients who take part in the study were informed and signed written informed consents to their participation, tissues were performed in accordance with the institutional guidelines.The study protocol was approved by the ethics boards of Anhui Medical University (Ethics LLSC20150030), and specimen acquisition.
Animal experiments 20g nude mice were subcutaneously injected with 5×10 6 SGC-7901/DDP cells with miR-200b mimics or NC lentivirus.After 2 weeks, the tumor size wad observed and measured.At the 3rd week, the mice were sacri ced by neck dragging.Tumors were photographed and weighed.All experimental protocols used on the animals were approved by the institutions' subcommittees on animal care of Anhui Medical University.

Immunohistochemistry (IHC) staining
Tissues were xed wtih 4% paraformaldehyde and embedded with para n.IHC was performed according to standard procedures and assessed and photographed under CaseViewer (3DHISTECH Ltd., Hungary).
Cell culture SGC-7901 cells and SGC-7901/DDP cells were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10 % (v/v) FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin (both from Beyotime, China) at 37 °C and 5 % CO 2 .

Cell cycle analysis
After xed in ethanol overnight, SGC-7901/DDP cells were stained with 0.5 ml of PI (Beyotime, China) and RNase A for 30 min in dark.Flow cytometric analysis was performed on Beckman Coulter.

Wound-healing analysis
Ater cultured 2 days; SGC-7901/DDP cells (5 × 10 5 /ml cells/well) were scratched with serum deprived in 24-well plate.24 h later, cells were xed with methanol, stained with crystal violet, and viewed under an Olympus BX-51 microscope.Transwell assay After coating with matrigel, SGC-7901/DDP cells were placed into the upper chamber (8 μm pore size; Millipore, USA) in 1% FBS media.48h later, cells were stained with methanol and 0.1% crystal violet, and viewed under an Olympus BX-51 microscope.

Statistical analysis
Data were presented as means ± SD and analyzed using SPSS16.0software.Statistical signi cances were determined by one-way ANOVA with the post-hoc Dunnett's test.In all assays, values of P < 0.05 were considered to be statistically signi cant.
To explore the role of miR-200b, GC and paracancerous tissues were collected.As shown in Fig. 1A, the miR-200b was decreased in GC compared with the paracancerous tissues by qRT-PCR.Likewise, compared with SGC-7901, the miR-200b was also reduced in SGC-7901/DDP cells.Moreover, the miR-200b was down-regulated by various concentrations of TGF-β1 in SGC-7901/DDP cells (Fig. 1 B).These results demonstrated that the miR-200b may be closely associated with the incidence of GC.

Down-regulation of ZEB1 weakened proliferation and migration of TGF-β1-induced SGC-7901/DDP cells
After ZEB1 siRNA was obtained to knockdown the ZEB1 expression in SGC-7901/DDP cells (Fig. 4A, C and D), c-Myc and CyclinD1 were remarkably reduced in TGF-β1-induced SGC-7901/DDP cells (Fig. 4C  and 4D).Similar to the miR-200b mimics, the ZEB1-RNAi signi cantly reduced the proportions of G2/M phases as shown in Fig. 4E.In addition, BrdU cell proliferation ELISA showed that the cell proliferation was inhibited substantially by the ZEB1-RNAi (Fig. 4B).Meanwhile, MMP-3 and MMP-9 (Fig. 4F and 4G) were also decreased by the ZEB1-RNAi.Moreover, wound-healing and transwell assay exhibited that the cell migration ability was inhibited signi cantly with the ZEB1-RNAi (Fig. 4H and 4I).All the ndings suggest that ZEB1-RNAi be likely to weaken the ability of TGF-β1-induced SGC-7901/DDP cells proliferation and migration.

Discussion
As all know that miRNAs are involved in biological processes, such as cell proliferation, differentiation and apoptosis, through inhibiting target genes expression [6].Many miRNAs have been proved to play in cancers, such as breast cancer [14], gastric cancer [15] and hepatocellular carcinoma [16].It has been proved that miRNAs play important role in GC including miR-15b, miR-16 [17], miR-146a [18] and miR-200 family [19].And miR-200b is proved widely expressed in many tumors, including breast cancer, pancreatic cancer and GC [20].Interestingly, miRNA-200b could inhibit the proliferation, invasion, and migration of glioma cells through targeting CD133 in glioma [21].Moreover, Chen [22] has proved miRNA-200 could inhibit the growth of melanoma cells.The previous study showed that the miRNA-200b was decreased in GC tissues.And Wu et al. [23] also found miRNA-200 family (miRNA-200a, miRNA-200b and miRNA-200c) was down-regulated in GC.Moreover, the miR-200b was also down-regulated in SGC-7901/DDP compared with SGC-7901 cells.Hence, we supposed that miR-200b would play a pivotal role in GC.We also proved that the expression of miR-200b was decreased by various concentrations of TGF-β1 in SGC-7901/DDP cells.Indeed, it is necessary to consider that miR-200b is likely to be closely related to the tumor genesis of GC.
To identify the effects of miR-200b in GC, miR-200b inhibitor was used in TGF-β1-induced SGC-7901/DDP cells.It is generally known that c-Myc and Cyclin D1 are important downstream molecules in proliferation pathway.C-Myc regulates G-phase of cell cycle as proto-oncogene protein, and Cyclin D promotes G1/S phase transition and accelerates the process of cell cycle [24].The results illustrated that miR-200b inhibitor up-regulated c-Myc and Cyclin D1 in TGF-β1-induced SGC-7901/DDP cells.Moreover, BrdU cell proliferation assay and cell cycle analysis also showed that miR-200b inhibitor promoted proliferation of TGF-β1-induced SGC-7901/DDP cells.Migration is another important biological process in malignant tumors, and it was related by matrix metalloproteinases (MMPs) [25,26].More signi cantly, MMP-3 and MMP-9 were up-regulated signi cantly by miR-200b inhibitor.Remarkably, wound-healing and transwell assay further proved the cell migration was dramatically promoted with miR-200b inhibitor.These data suggest that miR-200b inhibitor might enhance both proliferation and migration abilities of TGF-β1induced SGC-7901/DDP cells.The uncontrollable proliferation of cells is always corresponding with maladjusted cell cycle and shorter passage time [27].In this study, the construction and transfection of ZEB1 3'UTR-WT and miR-200b mimics into SGC-7901/DDP cells changed the uorescence, and ZEB1 was identi ed as the target of miR-200b.Title et al. [12] reported that regulation of ZEB1 by miR-200 was su cient to drive EMT in tumor progression and invasion in vivo.ZEB1 is a critical member of the ZEB family of transcription factors [28], and involved in regulation of key factors at the invasive front of carcinomas by triggering EMT.EMT could control protein stabilization mechanisms, transcription and translation, alternative splicing and expression of non-coding RNAs [29].In the present study, after treating the SGC-7901/DDP cells with the miR-200 inhibitor, the ZEB1 were up-regulated in GC.Similarly, miR-200 mimics signi cantly inhibited the ZEB1 levels.
Coincidentally, we also found the ZEB1 mRNA and protein levels were over-expressed in GC.Moreover, it was also increased signi cantly by TGF-β1 in SGC-7901/DDP cells.Additionally, inhibition of ZEB1 induced by ZEB1-RNAi signi cantly suppressed the mRNA and protein expressions of c-Myc, CyclinD1, MMP-3 and MMP-9 in TGF-β1-induced SGC-7901/DDP cells.Interestingly, ZEB1-RNAi reduced the proportion of G2/M-phase cells remarkably.Notably, BrdU cell proliferation assay proved ZEB1-RNAi signi cantly the proliferation.Wound-healing and transwell assay further proved that the cell migration was hindered dramatically with the ZEB1-RNAi.These results indicate that the proliferation and migration abilities were compromised by ZEB1-RNAi.Zhou et al. [30] also found that miR-200c/141 decreased ZEB1/2 and increased E-cadherin expressions to repress the migration and invasion of gastric cancer cells.Therefore, we speculate that miR-200b is likely to inhibit cell proliferation and migration of TGF-β1induced SGC-7901/DDP cells through targeting ZEB1.

Conclusions
In conclusion, as our study demonstrated, consistent with experiments in vitro, miR-200b plays an important role in activating TGF-β1-induced SGC-7901/DDP cells and may perform as a tumor suppressor by targeting ZEB1 in GC.Therefore, it is likely to provide new insight for the diagnosis and new targeted therapy of GC.    Figure 3

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MiR-200b suppressed tumor growth in vivo.We established xenograft model with overexpression of mir-200b SGC-7901/DDP cells.The tumor growth was remarkably slower in mir-200b mimics group (Fig. 5A).As shown in Fig 5B, the tumor volume was observably decreased at 2 weeks.Interestingly, the average tumor weight was also down-regulated in the mir-200b mimics (Fig. 5C).It has proved that mir-200b could suppress tumor growth in vivo.7. miR-200b may modulate proliferation and migration of by SGC-7901/DDP Wnt/β-catenin signaling pathwayWe also found Wnt/β-catenin signaling was closely associated with the effect of miR-200b in SGC-7901/DDP cells.Western blot have performed that the expression of β-catenin and APC were up-regulated obviously by miR-200b inhibitor, and down-regulated obviously by miR-200b mimics in TGF-β1-induced SGC-7901/DDP cells, as show as Fig.5D and E. In particular, Fig.5Fshowed that ZEB1-RNAi also reduced the β-catenin and APC.Taken together, all the above results indicated that miR-200b could modulate proliferation and migration of TGF-β1-induced SGC-7901/DDP cells by activation of Wnt/βcatenin signaling pathway.
Next, miR-200b mimics were transfected into TGF-β1-induced SGC-7901/DDP cells to simulate endogenous overexpression of miR-200b.The results showed that u miR-200b mimics decreased c-Myc, Cyclin D1, MMP-3 and MMP-9 expressions in TGF-β1-induced SGC-7901/DDP cells.Similarly, the percentages of cells at G2/M stages were decreased.Moreover, BrdU cell proliferation assay revealed that the cell proliferation was inhibited considerably by miR-200b mimics.Wound-healing and transwell assay proved that the cell migration ability was signi cantly weakened with miR-200b mimics.Xenograft model was established by SGC-7901/DDP cells with miR-200b mimics.The miR-200b mimics also suppress tumor growth and weight in vivo.These ndings indicated that miR-200b mimics could signi cantly inhibit the proliferation and migration of GC cells.

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