Dewetting of Thin Liquid Films Surrounding Air Bubbles in

Dec 14, 2017 - As an air bubble translates in a microchannel, a thin film of liquid is formed on the bounding walls. In a microchannel with a rectangu...
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Dewetting of thin liquid films surrounding air bubbles in microchannels sepideh khodaparast, Omer Atasi, Antoine Deblais, Benoit Scheid, and Howard A. Stone Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03839 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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RNA binding protein RNPC1 inhibits breast cancer cells metastasis via activating STARD13-correlated ceRNA network Zhiting Zhang a,b , Chenxi Xiang a,b, Xinwei Guo a,b, Shufang Zhang a,b, Qianqian Guo a,b

, Feng Zhang a,b , Lanlan Gao a,b, Haiwei Ni a,b, Tao Xi a,b,* , Lufeng Zheng a,b,*

a, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, People’s Republic of China b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, People’s Republic of China * Correspondence: Doctor Lufeng Zheng, [email protected]. Professor Tao Xi, [email protected]. School of Life Science and Technology, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China. Abstract RNA binding proteins (RBPs) are pivotal post-transcriptional regulators. RNPC1, an RBP, acts as a tumor suppressor through binding and regulating the expression of target genes in cancer cells. This study disclosed that RNPC1 expression was positively correlated with breast cancer patients’ relapse free and overall survival, and RNPC1suppressed breast cancer cells metastasis. Mechanistically, RNPC1 promoting a competing endogenous network (ceRNA) crosstalk between STARD13, CDH5, HOXD10, and HOXD1 (STARD13-correlated ceRNA network) that we previously confirmed in breast cancer cells through stabilizing the transcripts and thus facilitating the expression of these four genes in breast cancer cells. Furthermore, RNPC1 overexpression restrained the promotion of STARD13, CDH5, HOXD10, and HOXD1 knockdown on cell metastasis. Notably, RNPC1 expression was positively correlated with CDH5, HOXD1 and HOXD10 expression in breast cancer tissues, and attenuated adriamycin resistance. Taken together, these results identified that RNPC1 could inhibit breast cancer cells metastasis via promoting STARD13-correlated ceRNA network. Keywords RNPC1 ceRNA STARD13 breast cancer metastasis Introduction ACS Paragon Plus Environment

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Breast cancer is the most commonly malignancy in women, and leading cause of cancer-related deaths in the developed world [1]. Despite the booming development of diagnosis and treatment in breast cancer, it remains a major health problem in women [2]. Thus, it is an urgent need to elucidate the mechanisms and find new targets for breast cancer progression. RNA binding proteins (RBPs) have been realized as potent regulators of gene expression and participate in carcinogenesis processes. RBPs post-transcriptionally regulate the expression of genes involved in the maturation, modification, transport, stability and translation of coding and non-coding RNAs [3]. RNA binding motif protein 38 (RBM38), also known as RNPC1, belongs to the RNA recognition motif (RRM) family of RBPs, which could influence cancer cells proliferation, cell cycle arrest and epithelial-mesenchymal transition (EMT) [4-6]. Recent studies indicate that RNPC1 is capable of binding to the 3ˊ untranslated region (UTR) of mRNAs including p21, p73, macrophage inhibitory cytokine-1 (MIC) and human antigen R (HuR) [5, 7-9]. On the other hand, RNPC1 could bind to the mRNAs of p63, murine double minute-2 (MDM2) and p53, mediate an instability in their mRNA levels and attenuation of their translation, which suggests a negative role of RNPC1 in regulating the transcripts of its targets [10-12]. Recently, RNPC1 has been identified as a tumor suppressor in breast cancer, characterized as inducing cell cycle arrest [6]. Moreover, RNPC1 can bind to the 3′UTR of Estrogen receptors (ERs) and progesterone receptor (PR), both of which are pivotal markers for endocrine therapies in breast cancer [13]. However, that the mechanisms by which RNPC1 regulate breast cancer metastasis and EMT are still confused. The competing endogenous RNAs (ceRNAs) hypothesis posits that RNA transcripts sharing sequences called microRNA recognition elements (MREs) can modulate each other’s expression by competing for the same pool of microRNAs (miRNAs), which have a potential as therapeutic targets for their importance in different aspects of cancer etiology [14]. In our previous study, we revealed a competing endogenous network (ceRNA) crosstalk between STARD13, CDH5, HOXD10, and HOXD1 (STARD13-correlated ceRNA network) which suppress ACS Paragon Plus Environment

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breast cancer metastasis via inhibiting EMT in vitro and in vivo [15]. However, how this ceRNA crosstalk is regulated in breast cancer remains unknown. Here, we identified a RNPC1-engaged in molecular mechanism contributing to activation of STARD13-correlated ceRNA network, by which breast cancer metastasis is suppressed. Results 1. RNPC1 suppressed cells metastasis and attenuated adriamycin resistance in MCF-7 cells Firstly, Kaplan-Meier survival analysis (R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl)) revealed that level of RNPC1 was positively correlated with the longer relapse free survival (Fig. 1A) and overall survival (Fig. 1B) of breast cancer patients. Next, we measured the effects of RNPC1 on breast cancer cells metastasis by wound healing and transwell migration assays. RNPC1 was overexpressed in MDA-MB-231 and knocked down in MCF-7 cells due to its relative lower expression in MDA-MB-231 and higher expression in MCF-7 cells [6]. Knockdown and overexpression efficiency of RNPC1 was confirmed in MCF-7 and MDA-MB-231 cells (Supplementary Fig. 1 and Fig. 2). RNPC1 siRNA #3 was chosen for the subsequent experiments. Knockdown of RNPC1 remarkably increased the rate of wound closure, migratory and invasive ability compared with negative control (Fig. 1C, D and E, F), while RNPC1 overexpression inhibited cells migratory and invasive ability in MDA-MB-231 cells (Supplementary Fig. 3A, B and C, D). As EMT is involved in the major steps of cancer cells metastatic developments [16], we sought to determine the role of RNPC1 in EMT. As shown in Fig. 1G and 1H, RNPC1 level was positively correlated with epithelial marker E-cadherin, and negatively correlated with mesenchymal marker vimentin level in breast cancer tissues, respectively by a biologist web R2 analysis. Moreover, western blot showed that RNPC1 knockdown resulted in down-regulation of E-cadherin and up-regulation of mesenchymal markers including N-cadherin and MMP-9 (Fig. 1I). Opposite effects were observed in MDA-MB-231 cells with RNPC1 overexpression (Supplementary Fig. 3E). Furthermore, we investigated the function of RNPC1 in chemoresistance. ACS Paragon Plus Environment

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The IC50 for adriamycin in the adriamycin-resistance MCF-7 cells was 5.931

1.194

µM, while there is an increased lethality of adriamycin-resistant MCF-7 overexpressing with RNPC1, as its IC50 was 1.046

0.262 µM (Fig. 1J). Taken

together, these data demonstrated that RNPC1 could suppress breast cancer cells metastasis and sensitize MCF-7 cells to adriamycin treatment. 2. RNPC1 facilitated STARD13-correlated ceRNA network development in breast cancer cells Next, we found that RNPC1 level was positively correlated with CDH5, HOXD10 and HOXD1 levels in breast cancer tissues, respectively by a biologist web R2 analysis (Fig. 2A-C), thus we speculated that RNPC1 could positively regulate STARD13, CDH5, HOXD10, and HOXD1 expressions in breast cancer cells. As shown in Fig. 2D-G, RNPC1 overexpression increased these four genes expression in MDA-MB-231 cells, while knockdown of RNPC1 decreased their levels in MCF-7 cells. These results suggested that RNPC1 could facilitate STARD13-correlated ceRNA network development in breast cancer cells. 3. RNPC1 stabilized CDH5 and HOXD10 via directly binding to their mRNAs We further sought to investigate whether RNPC1 could bind with STARD13, CDH5, HOXD10, and HOXD1 transcripts, stabilize them and thus promote STARD13-correlated ceRNA network progression. To test for this speculation, whole-cell lysates of MCF-7 cells with RNPC1 knockdown were subjected to RIP assay. The binding of RNPC1 to CDH5 and HOXD10 transcripts was confirmed where the CDH5 and HOXD10 transcripts were decreased in RNA extracted from RNPC1 knockdown cell as compared to control (Fig. 3A and 3B). However, no effect was observed on STARD13 and HOXD1 transcripts (Fig. 3C and 3D). P21 mRNA, a well-known target of RNPC1 protein, was used as a positive control (Fig. 3E). Next, When RNPC1 was knocked-down and then de novo synthesis was blocked with actinomycin D, the half-life of CDH5 was shortened from 5.4 h to 2.5 h and HOXD10 from 5.8 h to 1.1 h (Fig. 3F and 3G), but the decay rate of STARD13 and HOXD1 mRNAs was unaffected (Fig. 3H and 3I), this effect was consistent with RIP results. 4. RNPC1 enhanced CDH5 and HOXD10 mRNA stability via binding to their ACS Paragon Plus Environment

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3′UTR Since numerous studies have indicated that RNPC1 is capable of specifically binding to several transcripts in 3ˊUTR, which is implicated in translational regulation, we speculated that RNPC1 could regulate CDH5 and HOXD10 mRNA stability via binding to their 3ˊUTRs. We constructed luciferase reporter plasmids carrying CDH5or HOXD10-3′UTR (LUC-CDH5, LUC-HOXD10, Fig. 4A), and MCF-7 cells were transfected with LUC-CDH5 or LUC-HOXD10 or not, followed by RIP analysis. The precipitated mRNAs were subjected to qRT-PCR analysis and results showed that the chimeric LUC-CDH5 and LUC-HOXD10 mRNAs were pulled down by RNPC1 antibody rather than IgG (Fig. 4B). Besides, the chimeric RNA containing CDH5-or HOXD10-3ˊUTR was found to be enriched compared to control vector (Fig. 4C). These results demonstrated that RNPC1 could directly bind to the CDH5-and HOXD10-3ˊUTR. To further confirm that CDH5 and HOXD10 mRNA were stabilized by associating with RNPC1, MCF-7 cells were co-transfected with siRNPC1 and LUC-CDH5 or LUC-HOXD10. The degradation rates of LUC-CDH5 and LUC-HOXD10 mRNA were strikingly increased by RNPC1 knockdown, and the half-life was shortened from 8.1 h to 2.7 h and from 6.0 h to 1.2 h, respectively (Fig. 4D and 4E). Additionally, we confirmed that the level of LUC containing CDH5- or HOXD10-3ˊUTR was greatly decreased by siRNPC1 in MCF-7 cells, and was increased upon overexpression of RNPC1 in MDA-MB-231 cells compared with control plasmid, respectively (Fig. 4F and 4G). Together, these findings indicated that RNPC1 stabilized CDH5 and HOXD10 mRNAs through directly interacting with their 3ˊUTRs. 5. RNPC1 regulated STARD13 and HOXD1 expression by the ceRNA interaction in STARD13-correlated ceRNA network Since RNPC1 could regulate the expressions of STARD13 and HOXD1 even when RNPC1 could not directly bind to their mRNA transcripts (Fig. 2D-G), we assumed that this regulation might be due to the crosstalk of STARD13-, CDH5-, HOXD10-, and HOXD1-3ˊUTRs [15]. A same experiment was performed to procure a luciferase reporter vector carrying STARD13 or HOXD1 3ˊUTR (LUC-STARD13, ACS Paragon Plus Environment

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LUC-HOXD1, Fig. 4A). To our surprise, similar effect was observed in the other two chimeric RNA, LUC-STARD13 and LUC-HOXD1 (Fig. 5A and 5B), which implied that RNPC1 might regulate the expressions of STARD13 and HOXD1 through their 3ˊUTR indirectly without binding to them. Notably, Protein and mRNA levels of STARD13 and HOXD1 was profoundly attenuated and even reversed after knockdown of CDH5 or HOXD10 in RNPC1-overexpressed MDA-MB-231 cells (Fig. 5C-E). Similarly, after overexpressing of CDH5- and HOXD10-3ˊUTR, STARD13 and HOXD1 levels inhibited by siRNPC1 was reversed in MCF-7 cells (Fig. 5F-H). Additionally, knockdown efficiency of RNPC1, CDH5 and HOXD10 and overexpressing efficiency of RNPC1, CDH5- and HOXD10-3ˊUTR were confirmed by qRT-PCR (Supplementary Fig. 2A-F). Altogether, these observations indicated that RNPC1 modulated STARD13 and HOXD1 expressions through the ceRNA crosstalk between STARD13-, CDH5-, HOXD10-, and HOXD1-3ˊUTRs. 6. RNPC1 suppressed MCF-7 cells metastasis by regulating the expressions of STARD13, CDH5, HOXD10, and HOXD1 in vitro As our previous study have found that STARD13-correlated ceRNA network suppress breast cancer metastasis and EMT [17], and RNPC1 could activate this ceRNA network, we further explored whether the inhibition of RNPC1 on cell metastasis was via regulating STARD13-correlated ceRNA network. MCF-7 cells that were stably transfected with shSTARD13, shCDH5, shHOXD10 and shHOXD1 by lentiviral infection were used to detect the capabilities of RNPC1 on cells migration and invasion. Wound-healing and transwell assays showed that compared with MCF-7 cells transfected with scramble shRNA (shScramble), there was a more significant wound healing, accompanied with an increased number of cells migrating or invading from the upper transwell chambers in MCF-7 cells infected with shSTARD13, shCDH5, shHOXD10 and shHOXD1. However, these effects were attenuated with RNPC1 overexpression (Fig. 6A-C, Fig. 7A and 7B). Notably, the protein levels of these four genes were resumed with RNPC1 overexpression (Fig. 6D). The overexpression efficiency of RNPC1 was shown in Supplementary Fig. 4A and 4B. As STARD13-correlated ceRNA network could inhibit EMT process in breast ACS Paragon Plus Environment

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cancer cells, we further detected whether RNPC1 would restrain EMT by regulating this ceRNA network. As expected, the protein level of E-cadherin was reduced, while N-cadherin and MMP-9 protein levels were increased in MCF-7 cells that were stably knocked down of STARD13, CDH5, HOXD10 and HOXD1 compared with shScramble (Fig. 7C-F), these effects were attenuated by RNPC1 overexpression (Fig. 7C-F). The data above suggested that RNPC1 suppressed the metastasis of MCF-7 cells by regulating the STARD13-correlated ceRNA network. Discussion In the present study, we elucidated that knockdown of RNPC1 decreased, whereas overexpression of RNPC1 increased, the expression levels of STARD13, CDH5, HOXD10, and HOXD1 in MCF-7 and MDA-MB-231 cells. RNA immunoprecipitation (RIP) showed that RNPC1 could only bind and stabilize the transcripts of CDH5 and HOXD10. In addition, qRT-PCR and western blot indicated that the expression of STARD13 and HOXD1 modulated by RNPC1 is dependent on CDH5 and HOXD10. Moreover, functional assays proved that RNPC1 suppressed the metastasis of MCF-7 cells by up-regulating the expressions of STARD13, CDH5, HOXD10, and HOXD1, thus activating STARD13-correlated ceRNA network. EMT has a large impact on cancer cell dissemination and metastasis, characterized as loss of markers like E-cadherin for the epithelial trait and upregulation of markers such as vimentin and N-cadherin for the mesenchymal [18]. In our previous study, we have discovered that STARD13 and its ceRNAs CDH5, HOXD10, and HOXD1, targets of miR-9, miR-10b and miR-125b in vitro and in vivo, suppress breast cancer metastasis and EMT [15]. To further hunt for the molecular mechanisms by which this ceRNAs network is regulated, we focused on the RNPC1, a RBP which holds critical roles in tumor progression. Previous study has shown that RNPC1 targeted by transcription factor Snail is significantly reduced after triggering EMT by TGF-β, and insufficient RNPC1 precluded its function on stabilizing the mRNA of ZO-1, an epithelial marker, following a promotion of cell invasion and migration [19]. Furthermore, we found that RNPC1 level was positively correlated with CDH5, HOXD10 and HOXD1 levels in breast cancer tissues respectively by a ACS Paragon Plus Environment

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biologist web R2 analysis (Fig. 2A-C). Thus, we speculated that aberrant RNPC1 expression had impact on expression of STARD13, CDH5, HOXD10, and HOXD1, RIP and luciferase reporter assays confirmed our speculation. In this study, due to the deficiency of vimentin in MCF-7 cells [20], a substitute marker was detected known as MMP-9, a kind of matrix metalloproteinase related to the induction of EMT [21]. It is widely testified that RNPC1 bind to AU/U-rich elements (AREs) in the 3ˊUTR of many mRNAs and mediates an increased stability of the transcripts [5, 7-9]. Consistent with this, our result showed that RNPC1 directly bound to CDH5 and HOXD10 3ˊUTRs and prolonged the half-life of their transcripts. It seems that RNPC1 influenced the expression levels of STARD13 and HOXD1 in a more complex way rather than by binding to their 3ˊUTR. It has been reported that RNPC1 could bind to 5ˊUTR of hypoxia-inducible factor 1 (HIF1) and p53, but it mediates the process of inhibiting the transcripts [22]. Thus, RNPC1 might also bind to the other regions of STARD13 and HOXD1 mRNA, which should be further explored. Furthermore, the detailed AREs on CDH5- and HOXD10- 3ˊUTR bound by RNPC1 remains to be concreted. Notably, overexpression of RNPC1 exerted almost no influence on the expression of STARD13, CDH5, HOXD10, and HOXD1 in shScramble MCF-7 cells (Supplementary Fig. 4C), as well as the EMT and cell migration and invasion (Supplementary Fig. 4D, Fig. 6B and 6C, Fig. 7A and 7B). However, we confirmed that overexpression of RNPC1 could markedly inhibit the metastasis in MDA-MB-231 cells (Supplementary Fig. 3A-E). This phenomenon may due to the higher expression of STARD13, CDH5, HOXD10, and HOXD1 in MCF-7 cells that hold relative low metastatic ability compared with highly metastatic breast cancer cells [15], which may weaken the effects of RNPC1 on them. Additionally, empirical evidence have demonstrated the complex relationships between miRNAs and RBPs, especially in the aspect of modulating shared target mRNAs [23]. For example, RBM38 effectively counteracted the function of miR-17/106b on the p21-3ˊUTR, miR-125b on the RBM38-3ˊUTR, miR-153 on the DDIT4-3ˊUTR, and miR-372/373 on the LATS2-3ˊUTR [24]. Therefore, it needs ACS Paragon Plus Environment

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further exploration whether miRNAs are engaged in RNPC1-involved ceRNA network.

Materials and Methods 1. Cell culture HEK-293T, MCF-7 and MDA-MB-231 were obtained from ATCC (Manassas, VA, USA). In a humidified atmosphere with 5% CO2, HEK-293T and MCF-7 were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) and MDA-MB-231 cells were cultured in L-15 medium (KeyGENBioTECH, Nanjing) with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) at 37 °C, respectively. To obtain the adriamycin-resistance cells, MCF-7 cells were treated with 0.2 µg/ml adriamycin as the starting concentration. After 24 h, adriamycin was washed out and fresh medium was added. When cells status returned to normal, adriamycin drug concentration gradually reached 2.0 µg/ml. Long-term culture was performed in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C with 5% CO2 and saturated humidity. The adriamycin-resistance MCF-7 cells were continuously cultured in the above medium containing 2.0 µg/ml ADM to maintain the drug-resistance. Two weeks prior to experiments, cells were placed in adriamycin-free medium for culture. For stable knockdown of STARD13, CDH5, HOXD10 and HOXD1, we used their knock down (plko.1) lentivirus to infect MCF-7 cells. DMEM medium with puromycin (Sigma, 2 µg/ml) was changed every other day for 2 weeks. After two rounds infection, western blot analyses were used for verification. 2. Plasmid, siRNA and transfection RNPC1 were amplified using polymerase chain reaction (PCR) from human cDNA templates and PCR products were cloned into pcDNA 3.1 (+) vector (Ambion, Austin, USA). Luciferase reporter vector carrying 3ˊUTRs of STARD13, CDH5, HOXD10 and HOXD1 were obtained from our lab [15]. All the primer sequences were listed in Supplementary table S1. Sequences of siRNA against specific target in this study were synthesized in Genepharma (Shanghai, China) and listed in ACS Paragon Plus Environment

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Supplementary table S2. Cells at the density of 70% -90 % per well were seeded in 6-well plates. Four micrograms of DNA or 50 nM of RNA were transfected or co-transfected into the cells by using the Lipofectamine® 2000 Reagent (Invitrogen, Carlsbad, CA) on MCF-7

cells,

transIT®-BrCa

Transfection

Reagent

(Mirusbio,

USA)

on

MDA-MB-231 cells and Lentifection (abm, Vancouver, Canada) on HEK-293T cells according to the manufacturer’s protocol. 3. RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (qRT-PCR) Total RNA was isolated with the Trizol reagent (Invitrogen, USA), and cDNA was synthesized by using M-MLV (Vazyme Biotech Co. Ltd, China) following standard protocols. qRT-PCR was performed by using the ChamQ SYBR qPCR Master Mix (High ROX Premixed, Vazamy, China) on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Gene expression was normalized to GAPDH. Primers used for qPCR are listed in Supplementary table S3. The expression level of mRNA was performed by 2-ΔΔct method. 4. Western blot analysis Total proteins were extracted from whole-cell using RIPA lysis buffer, 20 µg of proteins

were

separated

by

12%

SDS-PAGE

and

transferred

onto

polyvinylidinedifluoride membranes (Millipore, Bedford, Massachusetts). Proteins were detected with antibodies against RNPC1, HOXD1 (1:1000, Santa Cruz Biotechnology, USA), STARD13, CDH5 (1:1000, Abgent, China), HOXD10 (1:5000, Abcam, USA), E-cadherin (Proteintech, USA), N-cadherin, MMP-9 (1:1000, Wanlei, China). Anti-human β-actin monoclonal antibody (1:5000, Yifeixue Biotech Ltd., Nanjing, China) was considered as an internal reference. Finally, the corresponding secondary antibodies were used according to protocols and detection of specific signals was performed using Chemistar High-sig ECL Western Blotting Substrate (Thermo Fisher, USA). Protein levels were quantified by density analysis using Quantity One software (BioRad). 5. RNA Immunoprecipitation (RIP) assay ACS Paragon Plus Environment

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The breast cancer cells were lysed in NP-40 lysis buffer (Beyotime, China). After the extract was centrifuged, 100 µl supernate was incubated with rabbit polyclonal anti-RBM38 (1:50) or non-immunized rabbit IgG (1:50) at 4°C overnight. Then, Protein A/G Agarose Resin (YEASEN, China) that was used to pull down the RNA-protein immunocomplexes was incubated with protease K (TEASEN, China). After that, RNA was extracted, converted into cDNA and performed qRT-PCR. 6. Wound Healing Assay MCF-7 cells were plated in 6-well plates, and allowed to grow to 90% confluence in complete medium after transfection for 24 h. A vertical wound was made to cells with a sterile micropipette tip (1 mm) and wounded monolayers were then washed several times with PBS to remove cell debris. Cells were further incubated for 36 h in serum-free medium and the phase-contrast images of scratch were captured. The average distance of migrating cells was determined under an inverted microscopy at designated time points. Migration rate =(D0-Dt)/D0, of which, D0 stands for the distance measured at 0 h and Dt refers to the distance measured at 24 h. 7. Migration and invasion assays Migration and invasion assays were performed using transwell insert chamber (a pore size of 8 µm) coated with (for invasion assay) or without (for migration assay) BD BioCoatMatrigel (BD Biosciences). A total of 1 × 105 cells suspended in 200 µl serum-free culture medium were added to each insert, and 500 µl culture medium containing 20% FBS was added to the bottom chamber. After incubation for 24h in migration assay and 48h in invasion assay, the cells on the upper filter were removed, and those that migrated or invaded the lower surface of the membrane were fixed in methanol and stained with 0.1% crystal violet. Five random fields (10×10 magnification) for each filter were photographed, and the dye was eluted with glacial acetic acid to perform a quantification by measuring with Microplate Reader (OD570 nm). 8. Statistical analyses The data analysis using Student’s t test were performed by GraphPad Prism 5 software. P-values of less than 0.05 were considered as statistically significant. ACS Paragon Plus Environment

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(*p