In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal–vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca The AV axis can be traced back to the unfertilized egg Boveri, , and the3). It has been shown that DAG activates protein kinase C (PKC), and that InsP3 promotes an increase in intracellular Ca2+ concentration  is sufficient to vegetalize the embryos. In addition, the presumptive ectoderm blastomeres could be vegetalized by a lithium ion (Li+) that modulates the metabolism of inositol phospholipids, and the vegetalizing effect of lithium was reversed by preinjection of the blastomere with myo-inositol , which regulates the activity of the transcription factors and β-catenin  hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate  constructed from the amino acid sequence of the Hemicentrotus pulcherrimus PKC and a specific antibody against β-catenin of H. pulcherrimus (Hpβ-catenin). First of all, contrary to a previous report  the nuclear β-catenin activates not only the endodermal genes but also ectodermal genes, depending on their timing and location, without interfering with the formation of the animal–vegetal and oral–aboral axes.Sea urchin embryos exhibit polarized CaHemicentrotus pulcherrimus were supplied from the Misaki Marine Biological Station, the University of Tokyo, and Paracentrotus lividus from the Stazione Zoologica Anton Dohrn in Naples, Italy. Gametes were obtained by intracoelomic injection of 0.5 M KCl for H. pulcherrimus. P. lividus eggs were released from the dissected gonad into in natural seawater filtered with a Millipore-filter set. Eggs were washed and inseminated in seawater that contained 5 mM p-aminobenzoic acid (PABA-SW). Fertilization envelopes were removed by pipetting, and the embryos were cultured in filtered seawater at 15°C or 18°C (H. pulcherrimus), or at room temperature (20–23°C) for P. lividus.Individual animals of H. pulcherrimus PKC  and myristoylated at the N-terminus of the peptide  to enable it to permeate cell membranes . The overexpressed protein was extracted and purified by affinity chromatography using a ‘GST-Bind Resin’ column (Novagen). The GST-tag was removed using the Thrombin Cleavage Capture Kit (Novagen), and the remaining polypeptide was used to immunize rabbits to generate polyclonal antibodies against Hpβ-catenin.The HpPKC pseudosubstrate domain (FARRGALRQ) was synthesized according to GenBank information on H. pulcherrimus embryos was prepared by sonication in Laemmli's sample buffer , 10% glycerol, 0.005% bromophenol blue, 5% β-mercaptoethanol) and boiled for 5 min. SDS-PAGE was carried out with 10% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes  using a semi-dry blotting apparatus. Membranes were probed with the rabbit antiserum against Hpβ-catenin  and the horseradish peroxidase (HRP)-conjugated swine anti-rabbit IgG . Immunoreactivity signals were detected with the Millipore Immobilon western chemiluminescent HRP substrate (Millipore).Whole lysate of et al.  containing 2% paraformaldehyde for 10 min at room temperature, and then passed briefly through 100% MeOH on ice to permeabilize the cell membrane. After three cycles of washing in phosphate-buffered saline (PBS) by sedimentation, embryos were treated with 3% bovine serum albumin in PBS containing 0.1% Tween 20 (TPBS) for 30 min, and then incubated with rabbit polyclonal antiserum against Hpβ-catenin  for 1 h with gentle agitation. Following one wash in TPBS and three subsequent washes in PBS, embryos were incubated for 1 h in a solution that contained the secondary antibody: the Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probe) at 1 μg/ml (1:2000 dilution of the stock solution) in 0.1 M Na-phosphate, 0.1 M NaCl, 5 mM NaN3, pH 7.5. In the last10 min of the immuno-reaction, a DNA-binding dye PI (KPL) was added at the 1:600 dilution ratio of the stock solution (1 mg/ml PBS). After four washes in PBS, the specimens were placed in a well on a glass slide surrounded by lens paper soaked in mineral oil and were monitored with an Olympus laser-scanning confocal microscope (LSM-GB200).Following the methodology by Logan et al. , embryosP. lividus embryos were used for the observation of F-actin in accordance with the method described previously  that contained 0.1% Triton X-100, the embryos were incubated for 30 min with 3–10 U/ml of Alexa Fluor 488-conjugated phalloidin in PBT  and then rinsed in PBT for three times. All steps were performed at room temperature. The embryos stained with fluorescent phalloidin were observed under an Olympus Fluoview 200 laser-scanning microscope using the BP510540 emission filter. Transmitted light and fluorescent images were acquired from the same confocal plane.H. pulcherrimus embryos at the final concentration of 40 mM as described by Ciapa & Maggio -6,7,12,13-tetrahydro-13-methyl-5-oxo-5-idolo pyrrolo-carbazole  was applied at a concentration of 400 nM during the same period as the treatment with HpPKC-I. Gadolinium chloride , a stretch-activated calcium channel blocker  and then lined on a protamine sulphate-coated dish filled with PABA seawater. A mixture of 10 mg/ml of calcium green® BAPTA conjugated with 10 kDa of dextran (Molecular Probe) and 3 mg/ml of rhodamine red was prepared in the injection buffer , and microinjected into eggs within 5 min after fertilization. The eggs were then collected by a mouth pipette and transferred to agar-coated dishes filled with Millipore-filtered natural seawater. About10 min before the 2nd cleavage, zygotes were transferred into the Ca2+-free seawater . Blastomeres were separated at the 4-cell stage by gentle pipetting. These cells give rise to quarter embryos, which are able to configure their cells as 1-cell layer: two mesomeres, one macromere and one micromere to enable photometry on each cell. Ca2+ imaging was performed in the period from 5 to20 min after the 4th cleavage, the time of the micromere-specific Ca2+elevation had established in normal embryos, using a charged coupled device (CCD) camera with a ×63 objective and MetaMorph analyzing software. Calcium levels were expressed as the ratios of fluorescence intensities between calcium green® BAPTA (Ex 485 nm/Em 535 nm for 200 ms) and the internal control rhodamine red (Ex 555 nm/Em 565 nm for 100 ms exposure). When a time course measurement was needed, the time resolution was 5 s.Strongylocentrotus purpuratus embryos at pertinent developmental stages with the RNeasy mini kit columns (Qiagen), and subjected to cDNA synthesis by use of the SuperScript First-Strand Synthesis System (Invitrogen). Polymerase chain reaction (PCR) was carried out with specific primers synthesised from mRNA sequences of target genes in Strongylocentrotus purpuratus. The sequences (5′→3′) of each primer set (forward and reverse) are as follows: Gsc, F_ggattgatggcgatacgact and R_gcagaggaagacaccgagac; Delta, F_tgtgcaataaccagggtgaa and R_tgcaagagtttggtgagtcg; Nodal, F_gacctgctctggacctcttg and R_agtggtcgaagtttggttgg; Lim, F_gtatctttcgggcaaggaca and R_ctcttccatcggatgtggat; Gcm, F_atcgttcaccatcgacaaca and R_aggcatggtaggcagctaag; FoxG, F_gtgaaagtccctcgccatta and R_tcagcttacccgttgtaccc; Brain 1/2/4, F_gacgcattaagctcggctac and R_aagctcaactgcaaggcttc; FoxA, F_caattcgccacagtctctca and R_ctcggacatattcccagcat; NK22, F_actttggttcggttggtcag and R_ttcacggaaaagctcgtctt; Coquilette, F_ctgaccaggcatttcatgtg and R_gtgcaccagggcactatttt; Blimp, F_cagggaatctccgattcaaa and R_tggcacctcctcttctctgt; Deadringer, F_cagagctgcaatcagccata and R_gggaaccagaaggggtatgt; Sm 30, F_caaccagtctggatcggtct and R_ataggggccatcattccttc; Sm 50, F_gccttttcgcaggataatca and R_ccgtttggataagctggtgt; G-cadhedrin, F_cggtggaacttgtgttgatg and R_cgtaaccgttgttgaagctg; Bra, F_aggacacggaattgtggaag and R_gcgatgttagccgagagaac. The automated thermal cycler used was the Chromo4™ Real-Time Detector (Biorad) utilizing Sybr Green in the reaction mixture that is intercalated into double-stranded PCR amplicons and emits fluorescence. For each query transcript, the cycle threshold value (Ct) was normalized to the Ct value obtained on the same cDNA preparations with ubiquitin primers: i.e., Ct = Ct (sample)/Ct (ubiquitin). To evaluate the effect of the PKC modulator on the transcript level of each query gene, the difference (D) between the Ct values of the control and experimental samples were obtained, i.e., D = Ct (control) – Ct . Thus, positive D values indicate an increase in the transcript after treatment. To convert D into the ratio (R) of the transcript concentrations, we applied the formula R = 1.94 × D, where 1.94 is the mass amplification coefficient per PCR cycle for this particular case.Total RNA was extracted from t-test and one-way analysis of variance (ANOVA) with Tukey's multiple comparison test were performed using Prism 6.0 software (Graph Pad Software); P-values less than 0.05 were considered to be statistically significant.Paired Hemicentrotus pulcherrimus embryos, we prepared a specific peptide inhibitor of PKC and antibody against β-catenin, which is a potential downstream target of PKC in the micromeres  was completely identical to Lytechinus pictus PKC . When HpPKC-I (7 μM) was applied to the embryo 20 min before cleavage, the next cell division stopped, although the nuclear division progressed at the normal schedule (C). At a lower dose (5 μM), we found that treatment with HpPKC-I strikingly reorganized the actin cytoskeleton in the blastomeres. Whereas the Alexa 488-phalloidin-stained F-actin filaments in the normal blastomeres were localized predominantly at the plasma membrane, the distribution of F-actin in the blastomeres of the HpPKC-I-treated embryos was shifted more towards the cytoplasm . In contrast, when the myristoylated pseudosubstrate of human PKC differing by only one amino acid was used , cell division of sea urchin embryos was not affected, even at 28 μM (data not shown). This finding suggested that the inhibitory effect on the target enzyme requires highly stringent specificity.To study the roles of the PKC pathway in the early development of schedule C. At a lLytechinus variegatus β-catenin (Lvβ-catenin)  was 95% homologous to that of Lvβ-catenin (821 amino acids). Antibody to Hpβ-catenin was prepared against amino acids 1–250, in consideration of the antigenic region of Lvβ-catenin . In this region, four residues of serine/threonine (asterisked) matched the consensus substrate sequences of the glycogen synthase kinase-3β (GSK-3β) . As expected, when a western blot of the proteins extracted from H. pulcherrimus eggs was probed with the Hpβ-catenin antibody, a single band of 103 kDa was recognized as Hpβ-catenin . To the best of our knowledge, this finding is the first information for the molecular weight of sea urchin β-catenin. Taken together, these results assured that our PKC inhibitor and anti β-catenin antibody were highly specific.As to the specificity of the anti-Hpβ-catenin antibody, we used a part of the Hpβ-catenin as the epitope. To this end, the homologous protein to C). This result is consistent with immunostaining with anti-Lvβ-catenin antibody . Interestingly, Hpβ-catenin appeared to be concentrated at the place where amphiaster is located . We found Hpβ-catenin either surrounding the equatorial plate of metaphase chromosomes  or localized in the space between the separating chromosomes . In the latter case, β-catenin was scarce near the mitotic asters. In Fig. 3C, four micromeres in the centre were evident with metaphase chromosomes, but the eight surrounding cells were already in telophase. When the chromosomes construct the nuclei in the newly divided cells at telophase, the Hpβ-catenin was evidently outside the constructing nuclei . In Fig. 3D, the outer eight macromere derivatives are in interphase, and Hpβ-catenin accumulated in the interphase nuclei . Hpβ-catenin changed its subcellular distribution during mitosis presumably by its interaction with the actin cytoskeletons through cadherin .In the normal sea urchin embryos, we found Hpβ-catenin protein localized in the cytoplasm and in the apical and intercellular regions of plasma membrane by the 8-cell stage C. This r Wilson, . At metan Bienz,  rather tA), although in some batches of embryos it was detectable also in the micromeres, but its fluorescence intensity was always much weaker than in the macromeres (data not shown). In the late 16-cell stage , the macromeres and mesomeres entered the next cleavage cycle (anaphase chromosomes were visible in Mac) and the four micromeres (mic) were still in the interphase. At the early 28-cell stage, the micromeres were in metaphase with more condensed stain with PI, while Mac had already divided and attained the telophase. It is noticeable that β-catenin accumulated around the newly formed metaphase chromosomal plates of mic . When the Mac and meso had completed the 5th division and formed interphase nuclei, the micromeres belatedly entered anaphase of the 5th division, displaying nuclear-β-catenin-like distribution . Nuclear β-catenin was still detected only in the daughter cells of the macromeres. At the 56-cell stage , four small (s-mic) and four large micromeres (l-mic) were formed, and the macromere descendants were located in the veg1 and veg2 cell layers. Nuclear β-catenin was now detected in the interphase nuclei of both s-mic and l-mic, and as well as in the veg2 cell layer. A small weak signal seemed to be present in the nuclei of veg1 cells. However, nuclei of animal cells (an) always exhibited no β-catenin signal.Fig. 4B), and the nuclei were more densely stained in the cells at the vegetal side in comparison with the control, although the nuclei of the cells in the animal side were largely devoid of β-catenin . Thus, the addition of LiCl and PMA appeared to intensify the AV gradient of nuclear β-catenin, which is found in the control. By contrast, 5 μM HpPKC-I markedly increased β-catenin predominantly near the plasma membrane with the concomitant decrease in the cytoplasm. In addition, it was noted that β-catenin was present in the nuclei of all blastomeres . Embryos treated with another PKC inhibitor Gö6976 produced virtually the same results as in the embryos treated with HpPKC-I . Hence, whereas a pharmacological activator of PKC mimics some of the effects displayed by Li+ in terms of β-catenin distribution, the peptide inhibitor of the PKC interfered with the normal subcellular distribution of β-catenin, negating its cell-specific nuclear localization.In the 28-cell embryo, nuclear localization of the β-catenin was evident preferentially in the cells at the vegetal side, although the micromeres had not yet arrived at interphase and therefore exhibited no clear sign of nuclear β-catenin . When thFig. 52+influxes. L-type Ca2+ channels derived from the egg plasma membrane function from the first cleavage and show AV axis polarity , we have monitored changes in the intracellular Ca2+ levels 5–10 min after the 4th cleavage. The Ca2+ levels of each blastomere were monitored by single photometry at the 16-cell stage  were slightly, but significantly, higher than those of the macromeres  and the mesomeres . In embryos treated with the PKC activator PMA (8 nM), the intracellular Ca2+ levels in all blastomeres were significantly lowered in comparison with the control  to the levels in the macromeres and mesomeres, which were virtually unchanged in comparison with the controls  and gastrulation (A). We found that gastrulation was delayed by GdCl3 when it was applied at the 4th and 5th cleavages, but not before or after the period. When GdCl3 was added from the 5th cleavage, which included micromere cleavage to form small and large micromeres during treatment, gastrulation was delayed to a lesser extent than when treatment was carried out at the 4th cleavage (A). By contrast, the ingression of PMCs into the blastocoel was not altered in all treatment schemes.The earliest function of the micromeres appears to be endoderm induction, as the signal is emitted from the micromeres during the 16–60-cell stages . To quantify the progression of gastrulation, we rated the embryos into four grades based on the length of the invaginated archenteron: 0G, uninvaginated embryo; 1/4G, embryo with an indent at the vegetal pole; 2/4G, embryo with the archenteron elongated to the midpoint; 4/4G, embryo with the archenteron attained to the animal pole. The control embryos underwent gastrulation by 23 h (B). About 30% of embryos nearly completed gastrulation (3/4G + 4/4G), and the rest elongated their archenteron and arrived at the 2/4G stage. In contrast, the embryos exposed to HpPKC-I barely started gastrulation by 23 h. Two hours later , all the control embryos had finished gastrulation, but the embryos treated with HpPKC-I were still gastrulating (most embryos were estimated to be at 2/4G). The extent of the delay was not changed by the duration of the treatment, from 70 min (8–16-cell stage) to 150 min (8–60-cell stage). Thus, the delay of gastrulation by HpPKC-I appeared to have a critical period of about 70 min that was between the 8-cell and 16-cell stages. Indeed, inhibition of PKC from the 7th cleavage (124-cell stage) caused no delay in gastrulation , whereas the largest delay in gastrulation was provoked by the treatment during the 8–60-cell stages . In contrast, in all these conditions, PMC ingression took place on a normal schedule (data not shown), analogous to the results obtained with GdCl3 . Hence, inhibition of the stretch-activated Ca2+ channel (GdCl3) and PKC (HpPKC-I) both led to a delay in gastrulation but not to alteration of the micromere specifications.Similar to finding with GdCl HpPKC-I B, C. To  by 23 h B. About H. pulcherrimus and P. lividus were treated with 5 μM HpPKC-I (C), the total length of the rod structures in the embryos treated with HpPKC-I was about 10% reduced compared with the control embryos, while the skeleton supporting oral ectoderm, alr, remained the same . To discriminate the shape clearly, the embryos marked with asterisks in the left panels were delineated on the right to indicate the oral (arrowheads) and the aboral ectoderms (arrow) in the normal embryos (B). Similar morphological changes were observed in the embryos treated with HpPKC-I after the 4th cleavage but to a lesser extent (C). Again, added after the 7th cleavage, HpPKC-I had no effect on the morphology of the embryos (data not shown).As mentioned for Fig. 6iderably B. To dis embryos . When Hp embryos B. Similar extent C. Again,Nodal, Deadringer and Lim, markedly increased their expression after the embryos were exposed to HpPKC-I, while the transcripts of the aboral ectoderm genes, NK2.2 and Coquilette, decreased. In accordance with the morphology data  transcripts. Together with Lim, Nodal and Deadringer (Dri) are involved in the development of oral ectoderm, but the expression times of these genes are known to be slightly different. In the beginning of gastrulation, Nodal declines by half  are expressed from the blastula stage  shared the same myristoylated pseudosubstrate region that we have employed as a specific PKC inhibitor (HpPKC-I). HpPKC-I was highly effective and completely blocked cell division at 7 μM concentration. At 5 μM, phalloidin-stained F-actin filaments were apparently released from the plasma membrane region, disrupting the intercellular adhesion system . In addition, we have found that the initial nuclear localization of β-catenin takes place in the macromeres (A and 5A). While the significance of this finding is not entirely clear, our result is at variance with a previous study using another sea urchin species L. variegatus in which the initial nuclear entry of β-catenin was reported to take place in the micromeres at the 16-cell stage  that was in general agreement with the molecular weights of the β-catenins in Xenopus (92 kDa)  axis and oral–aboral (OA) axis. Both axes are initially inherent in the cytoarchitecture of the unfertilized egg .The results of our experiment using HpPKC-I raised intriguing questions as to how PKC regulates subcellular distribution of β-catenin, and as to the roles of nuclear β-catenin. In sea urchin embryos, it has been proposed that the specific nuclear entrance of β-catenin in the cells on the vegetal side is mediated by glycogen synthase kinase 3β (GSK-3β), which is a serine/threonine kinase comprising the Wnt pathway. In line with this idea, Lhomond et al.,  reportedet al., et al., et al., et al., et al., et al., et al., In view of the fact that β-catenin entering the nucleus functions as a transcriptional activator of target genes , an activator of PKC, promotes the development of endoderm and mesoderm . In support of the idea that GSK-3β-dependent modulation of β-catenin level is largely responsible for regional specification, overexpression of GSK-3β produced animalized embryos, while the kinase-dead GSK-3β markedly vegetalized embryos , leading to vegetalization of the sea urchin embryo  and in the macromeres (2.3 ± 2.7 μA/cm2) at the 16-cell stage, but not in the micromeres  or even at the 16- to 60-cell stages (H. pulcherrimus) . The observation that PKC activity may modulate actin dynamics in a subcellular-specific manner offers two intriguing implications. Firstly, the actin network subjacent the plasma membrane may modulate the functions of adherence junctions. Yazaki  gene (SpDri) is first expressed in the precursor cells of PMCs but disappears altogether with PMCs ingression. Just before gastrulation, SpDri restarts its expression in the presumptive oral ectoderm  formation. A chimera that consisted of normal micromeres and micromere-deleted embryo using SpDri morpholino developed into an alr-deficient pluteus. In the present study, we have observed that, as a result of PKC inhibition during the 16–60-cell stages, alr was larger than the other skeletons, br and por  and Lim. Our result suggesting that Nodal gene is modulated by PKC activity is of keen interest, as Nodal is probably the earliest zygotic marker that determines OA polarity following the mitochondrial distribution gradient and the local redox state .In this study, we have also found that inhibition of PKC interfered with gene expression in later embryonic stages . HpPKC-I control . As for  control . Likewisri) gene , which ie et al.  found th and por . Increasmal side . We have pluteus . Overexpll stage , althougll stage B, C.2+influx conducted and amplified through Wnt/Ca2+  and Wnt/β-catenin signalling pathways  may add to activate PKC, whose enzyme activity is further enhanced by DAG that is produced by Ca2+-dependent phospholipase C. From the first cleavage, PKC in the sea urchin embryo may function in association with the actin cytoskeletons to direct various downstream events: (i) regulation of β-catenin localization; (ii) guiding intercellular communications by constructing cell adhesive structures by the 60-cell stage; and (iii) establishment of the embryonic axes, animal–vegetal and oral–aboral axes.In summary, we propose a model in which Ca


WNT-5A plays critical roles in a myriad of processes from embryonic morphogenesis to the maintenance of post-natal homeostasis. WNT-5A knock-out mice fail to survive and present extensive structural malformations. WNT-5A predominantly activates β-catenin-independent WNT signaling cascade but can also activate β-catenin signaling to relay its diverse cellular effects such as cell polarity, migration, proliferation, cell survival, and immunomodulation. Moreover, aberrant WNT-5A signaling is associated with several human pathologies such as cancer, fibrosis, and inflammation. Thus, owing to its diverse functions, WNT-5A is a crucial signaling molecule currently under intense investigation with efforts to not only delineate its signaling mechanisms and functions in physiological and pathological conditions, but also to develop strategies for its therapeutic targeting. The WNT/Ca2+ signaling pathway involves activation of Ca2+-dependent signaling molecules, including protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and nuclear factor of activated T cell (NFAT), whereas the WNT/PCP pathway is mediated by RhoA signaling or activation of c-Jun N-terminal Kinases (JNKs) via small Rho-GTPases . , 5. 4, 5Drosophila, Xenopus, and mouse; in vitro cell-based systems and patient-based reports.In view of the plethora of evidence associating WNT-5A with health and disease, there is considerable interest in understanding its biology. In this review, we discuss our current understanding of various aspects of WNT-5A signaling and its functions derived from studies in wide variety of in vivo models including WNT-5A cDNA was first isolated from mouse fetal tissue  followedWNT-5A is a transcriptional target of an array of cytokines and growth factors. CUTL1 , STAT3 , TBX1 2, and NFκInterestingly, WNT-5A is also regulated at translational level via the numerous AU-rich motifs which are present in the evolutionary conserved 3′-untranslated region of mRNA . AU-rich5 and signaling activity but not required for its secretion [WNT-5A-L and WNT-5A-S, composed of 380 and 365 amino acids, respectively, are heavily glycosylated and lipid-modified proteins. Each isoform consists of an N-terminal hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence and about 22 highly conserved cysteine residues  and β-arrestin to FZD2 leading to the clathrin-mediated internalization of FZD2 [2 is essential for WNT-5A-induced Rac activation [4 [5 also leads to its internalization [WNT-5A has been shown to bind to FZD Xenopus  and zebr Xenopus  and WNT-n injury . WNT-5A  of FZD2 . Internativation . WNT-5A ation [4  in a PKCation [4 . Similarlization . Internalization .8 and RYK receptors leading to the activation of Ca2+-NFATc1 and JNK signaling which mediates TGF-β-induced ECM expression in airway smooth muscle cells [7 activates prosurvival PI3K/AKT cascade in human melanoma cells which can account for the resistance of these cells to BRAF inhibitors [3 in human dermal fibroblasts and promotes integrin-mediated adhesion of these cells [We have recently demonstrated that WNT-5A signals through FZDle cells . WNT-5A hibitors . Similarse cells . In contse cells . Similarse cells . Indeed,6 in hair follicles [5 regulating immune responses by antigen presenting cells and activated T cells in response to mycobacterium infection [WNT-5A is proposed to regulate cell fate via FZDollicles , whereasnfection .αi/o proteins leading to Ca2+-dependent ERK1/2 activation in murine primary microglia [αi in response to WNT-5A stimulation [The FZDs belong to the class of seven transmembrane-spanning G protein-coupled receptors. Recent evidence shows a role for heterotrimeric G proteins in WNT-5A downstream signaling. For instance, G proteins are required for WNT-5A-induced JNK and NFκB activation in human neutrophils . Similaricroglia  and HEK2icroglia . A recenmulation . This inmulation .7 in the presence of ROR2 to form a complex required for DVL polymerization and activation of Rac-dependent WNT signaling [WNT-5A also binds to non-class FZD receptors including ROR2 and RYK receptor tyrosine kinases. ROR2 is a key receptor for WNT-5A-induced effects during development as demonstrated by remarkable phenotypic resemblance between the ROR2 and WNT-5A knock-out mice . Multiplignaling . WNT-5A ignaling , whereasignaling . WNT-5A ignaling , 66–68, ignaling , 70. Thu3 receptors as well as calcium influx through transient receptor potential (TRP) channels inducing axonal outgrowth. On the other hand, simultaneous association of WNT-5A with RYK and FZD releases calcium from TRP channels without involvement of IP3 receptors and induces axonal repulsion [WNT-5A activates intracellular calcium release to fine tune neuronal growth by axonal outgrowth and repulsion. WNT-5A signals via RYK leading to calcium release from stores through IPepulsion . WNT-5A epulsion , whereasepulsion .Recently, WNT-5A binding to an adhesion molecule CD146 has also been described, leading to the recruitment of DVL2 to the complex and activation of downstream JNK signaling cascade . CD146 h2, a receptor for both the WNTs, thereby inhibiting the WNT-3A-induced β-catenin signaling [4 and LRP5 [Interestingly, in addition to activating the β-catenin-independent WNT pathway, WNT-5A can also have positive or negative regulatory effects on WNT/β-catenin signaling depending on the receptor- and cell-context. Indeed, a study has shown that WNT-5A can both activate and inhibit β-catenin-dependent WNT signaling during mouse embryonic development . WNT-5A ignaling . The WNTignaling , 45. In ignaling . Similarignaling . Purifieand LRP5 , 46. Alsand LRP5 , 78 and and LRP5 . Similarand LRP5 .WNT-5A has been identified for its key involvement in defining the body outgrowths in addition to many other specific features. WNT-5A expression is most abundant during early embryonic developmental stages between 10–14 days post conception . ImportaSimilar to WNT-5A knock-out mice, WNT-5A transgenic mice show perinatal lethality when WNT-5A is induced early in development exhibiting severe deformities resembling the WNT-5A knock-out phenotype . OverexpFurther studies have looked into the organ-specific developmental roles of WNT-5A and have identified a crucial role for distal morphogenesis of internal organs. For instance, WNT-5A knock-out mice fail to develop the genital tubercle . PromineConvergent extension (CE) is the critical morphogenetic movement during gastrulation wherein the germ layers narrow down mediolaterally resulting in the elongation of embryo from head to tail and shaping of body axis . CE requLungs are complex organs with extensive branching, a large number of different types of specialized cells, and distinct P–D polarity. WNT-5A, as a major determinant of P–D polarity, is prominently expressed in the embryonic lungs , 22 wherWNT-5A expression is crucial for proper neuronal generation and axonal guidance during embryonic development and in post-natal life. WNT-5A knock-out mice show anomalies in the dopaminergic midbrain neuronal morphogenesis, organ innervation, and show increased neuronal apoptosis , 95. RobWNT-5A is also required for proper cardiac morphogenesis as WNT-5A knock-out mice show severe defects in the septation of the cardiac outflow tract (OFT) . OFT oriIn summary, WNT-5A signaling is crucial to the development of internal organs and the formation of skeletal structures. Of importance, WNT-5A cooperates with other WNTs  and several non-WNT morphogens involved in development including TGF-β, BMPs, FGFs, and SHH signaling , 99, 1002+ pathways have been linked with migration of cells. Several studies have elucidated the significance and molecular mechanisms of WNT-5A-induced cell migration  asymmetrically at the cell periphery in a DVL- and PKC-dependent manner in melanoma cells [CD146, an adhesion molecule, can also activate RhoA and has been shown to be involved in cell migration . Interesma cells . This coma cells . Of notema cells . Whether4-LRP6 binding. ARF6 releases membrane-bound β-catenin from N-cadherin increasing its cytosolic abundance and triggering β-catenin-dependent transcriptional program that induces invasion and metastasis [Besides non-canonical WNT signaling, WNT-5A can also activate β-catenin-dependent signaling to promote cell migration. In melanoma cells, WNT-5A activates small GTPase ADP-ribosylation factor 6 (ARF6) via FZDtastasis .3 activates the PI3K/AKT cascade in human dermal fibroblasts and promotes integrin-mediated adhesion of these cells [WNT-5A can also alter the adhesion properties of cells to regulate migration. For instance, WNT-5A binding to FZDse cells .Thus, WNT-5A exerts migratory effects in large number of cell and tissue types in physiological and pathological contexts.Owing to its property of regulating cell polarity, cell movement, and cell proliferation along with the antagonistic effects on WNT/β-catenin signaling, WNT-5A may play a critical role in modulating cell fate determination and differentiation of stem cells.Hematopoietic stem cells exhibit a shift from β-catenin-dependent to -independent WNT signaling with aging where high levels of WNT-5A are present in aged stem cells . InteresSimilarly, WNT-5A is also critical in mesenchymal stem cell (MSC) biology. MSCs can differentiate into multiple cell types such as adipocytes and osteocytes. Higher expression of WNT-5A is detected in MSCs as compared to committed preadipocytes which can only give rise to adipocytes . InteresIn line with its role in morphogenesis and stem cell differentiation, WNT-5A has recently been shown to be involved in tissue repair and regeneration after injury. A study demonstrated robust induction of WNT-5A-positive mesenchymal cells following an intestinal injury which are specifically localized in the wound bed . The pre3, FZD5, FZD7, and ROR2. Interestingly, WNT-5A is expressed and provided by the testicular stromal population—sertoli cells, where it promotes SSC maintenance and activity by inhibiting apoptosis in JNK-dependent manner [WNT-5A also regulates spermatogenesis by supporting self-renewal and survival of spermatogonial stem cells (SSC) . In contt manner .Thus, WNT-5A may exert a highly context-dependent cell-intrinsic and -extrinsic effects in regulation of stem cell biology, regeneration, and repair.Consistent with the broad functional effects of WNT-5A during embryonic and adult life, disrupted WNT-5A signaling leads to the development of various pathological conditions in humans. We here summarize the role of WNT-5A in human pathologies such as fibrosis, inflammation, and cancer.WNT-5A mRNA and protein expression is increased in fibroblasts obtained from lungs of usual interstitial pneumonia (UIP) patients . SimilarActivated hepatic stellate cells (HSCs) are keys to the development of fibrotic liver by contributing the extracellular matrix (ECM) and other fibrotic factors. WNT-5A is particularly enriched in the ECM deposited by activated HSCs  which ex2+-NFAT and JNK to induce ECM expression [Fibroblasts from pulmonary fibrosis patients and keloid regions show increased proliferation, survival, and expression of ECM proteins , 124. WNpression . While Tpression  underlinIn contrast of its profibrotic role, WNT-5A may be protective in diabetic renal nephropathy. High-glucose suppresses WNT-5A expression among other WNTs and promotes expression of fibrotic markers via TGF-β . Forced WNT-5A is associated with several inflammatory disorders where it not only mediates proinflammatory cytokine and chemokine production but also regulates migration and recruitment of various immune effector cells.Mycobacterium tuberculosis-infected lungs [Microbial pathogens , 126 anded lungs , in the ed lungs , sera aned lungs , the athed lungs , in humaed lungs , in circed lungs , and in ed lungs , 131.WNT-5A is associated with the maintenance of innate immune responses both in homeostasis and pathology. Basal WNT-5A expression by macrophages drives static IFN-β and -γ expression via a Rac1-NFκB pathway and also regulates expression of CD14 which is required for antigen recognition and innate immune responses during infection . In addiWNT-5A also contributes to the immune responses by regulating the differentiation of T cells . Mycobac2, FZD5, and FZD8 and treatment with WNT-5A induces the release of IL-8 and CCL2 via MAPK signaling, promoting neutrophil migration [Neutrophil recruitment to the region of infection or site of injury under the influence of various chemoattractants is another key event in innate immune response, whereas excessive neutrophilic inflammation has been linked to various diseases such as asthma and COPD. Human neutrophils express several WNT-5A receptors such as FZDigration . CCL2 isigration  and supeigration  emphasizWNT-5A has come under intense scrutiny for its role in neuroinflammatory disorders. WNT-5A induces upregulation of cyclooxygenase-2 (COX-2) expression and production of proinflammatory cytokines IL-1β, IL-6, and TNF-α in primary microglia . It has 2+-PKC-NFκB axis and increases vascular permeability and endothelial cell migration [The proinflammatory functions of WNT-5A are not only restricted to the immune cells. In human adipocytes, WNT-5A induces IL-6 and IL-1β expression . In boneigration . WNT-5A igration .In contrast to the proinflammatory role, WNT-5A can also have opposing effect on inflammation. It has been shown to negatively regulate LPS-induced inflammatory responses in microglia by inhibiting COX-2 upregulation . AnotherWNT/β-catenin signaling is closely associated with malignant disorders . WNT-5A,Aberrant expression of components of β-catenin-independent pathway, WNT/PCP, has also been reported in Chronic lymphocytic leukemia (CLL) . The stuIn contrast to CLL and ATL, WNT-5A may have tumor suppressive effects in ALL. Loss of WNT-5A expression is reported in acute myeloid and acute lymphoblast leukemia . WNT-5A Similarly, WNT-5A promoter hypermethylation is also observed in the esophageal squamous cell carcinoma (ESCC) tissues . EctopicWNT-5A expression is highly increased in gastric cancer and positively associates with tumor invasiveness, metastasis, and survival of the patients . AdminisWNT-5A expression is highly increased in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis , 162. To2+-dependent signaling leading to the activation of calpain protease which cleaves filamin A. Cleavage of filamin A induces cytoskeletal remodeling and cell motility [7- and RYK-mediated activation of prosurvival AKT signaling [Extensive WNT-5A expression is detected in human melanoma biopsies where it correlates with the formation of distant metastases and poor prognosis , 150. WNmotility . WNT-5A motility . This prignaling . Knock-dignaling .2+-CaMKII signaling [2 activates protein kinase D (PKD) and JNK to induce Matrixmetalloprotease 1 (MMP1) expression via JunD [WNT-5A regulates motility in prostate cancer cells as well by promoting actin remodeling via Caignaling . Prostatignaling , 168 proignaling . WNT-5A via JunD . MMP1 exvia JunD . Bone isvia JunD . The provia JunD . BMP-6, via JunD . Indeed,via JunD . This sivia JunD .2+- and CDC42-dependent process that requires cytoskeletal reorganization [Studies have suggested a broader function for WNT-5A in cancer than just cell growth and invasion. For instance, it can relay immunomodulatory and proangiogenic functions or modulate cell survival. WNT-5A induces the release of IL-6, MMP2, and vascular endothelial growth factor (VEGF) containing exosomes from melanoma cells in a Canization . Co-cultnization . WNT-5A nization . Interesnization . The sernization . WNT-5A nization . This isnization , 174. CoWhat drives increased expression of WNT-5A in cancer cells? A study has found that microRNA-26a expression is reduced in prostate cancer cells . miR-26aIn addition to tumor-promoting activity, WNT-5A also functions as tumor suppressor in few cancer types. In colorectal cancer (CRC), loss of WNT-5A is frequently observed and associated with poor prognosis and survival . In lineIn a contrasting study, increased WNT-5A expression is associated with poor prognosis in CRC patients and WNT-5A promoted directional cell migration and invasion in CRC cells. However, increased expression of WNT-5A is not sufficient to augment malignancy or metastasis in APC-driven intestinal tumor model  suggestiLoss of WNT-5A is observed in primary invasive breast cancers and is associated with higher histological grade and rapid appearance of distant metastases leading to shorter recurrence-free survival in these patients , 142. ThThe tumor suppressive function could also be attributed to adhesion promoting function of WNT-5A in certain cell types. WNT-5A could regulate mammary epithelial cell adhesion by phosphorylating Discoidin domain receptor 1 and activating its interaction with collagen thereby negatively regulating cell migration . SimilarIn contrast to the tumor-suppressor function of WNT-5A in breast cancer, studies have also suggested a cell migration-promoting role for WNT-5A. In a breast cancer cell line MDA-MB-231 which expresses very low endogenous WNT-5A, stimulation with WNT-5A activated a DVL2- and Daam1-dependent RhoA signaling inducing cell migration , whereasThe opposing roles for WNT-5A in cancer are intriguing and are matter of intense investigation. As WNT-5A antagonizes WNT/β-catenin signaling, it is tempting to speculate that it functions as tumor suppressor in WNT/β-catenin-dependent cancers provided it activates the downstream cascade involved in this antagonism. The pro-tumor activity might be attributed to the cell migratory, proliferative. and prosurvival effects of WNT-5A. Moreover, the differential role of WNT-5A could also be due to different properties of recently characterized WNT-5A isoforms. WNT-5A promoter generates two identical transcripts utilizing alternative transcription start sites—WNT-5A-L and WNT-5A-S –26. WhilWhile we still await a clear understanding of WNT-5A biology, development of certain WNT-5A mimicking molecules and their beneficial effects in animal models of diseases raise hopes for therapeutic targeting of WNT-5A for curing deadly diseases. Foxy5 is a WNT5A derived N-formylated hexapeptide which mimics tumor suppressive effects of WNT5A on breast cancer both in vitro , 191 and1/FZD2 antagonist with therapeutic benefit in reducing cardiac remodeling an animal model of myocardial infarction [Likewise, UM206, a oligopeptide with high homology to WNT-5A, functions as a FZDfarction . Althougfarction , 103.WNT-5A is a pleotropic growth factor with wide-ranging effects in different cells and tissues, regulating key functions throughout the human life span. While it is indispensable for proper embryonic development, it is equally critical for maintenance of tissue homeostasis in adult life. Simultaneously, derailed WNT-5A signaling results in various pathological disorders in humans. Understanding the mechanisms involved in the maintenance of WNT-5A homeostasis such as its inducers and signaling partners, both positive and negative modulators, is key for therapeutic targeting of this important WNT in various diseases.


Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required.A Wingless-type MMTV integration site family, member 1 ( Wnt-1), the first identified member of the Int-1 family, was named by Nusse in 1991  targeting.Wingless-type MMTV integration site, member 1 . The Wnt-1 primary antibody was obtained from Neomarkers Inc. , while the horseradish peroxidase-conjugated monoclonal goat anti-rabbit secondary antibody was purchased from ZhongShan Bio-Tech Co., Ltd. . High glucose Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Life Technologies and fetal calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. . The restriction enzymes , DNA markers, agarose gel DNA purification kit version 2.0, T4 DNA ligase Taq DNA polymerase and polymerase chain reaction (PCR) primers were purchased from Takara Bio, Inc. . Little mention plasmid kit, and propidium iodide (PI) and MTT reagents were purchased from Beyotime Institute of Biotechnology . M-MLV reverse transcriptase was purchased from Promega Corporation .U251 glioma cells were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology . The vectors used were as follows: pGPU6/green fluorescent protein (GFP)/Neo-short hairpin RNA (shRNA)-GAPDH served as a positive control and pGPU6/GFP/Neo-shNC served as a negative control. The pGPU6/GFP/Neo expression plasmid was purchased from Wuhan Genesil Biotechnology Co., Ltd . Transfections were performed using liposome LipofectamineWnt-1 gene sequence was retrieved from GenBank (NM_005430) and the siRNA design principles were obtained from Takara Biotechnology special siRNA design software , 1 μl dNTP (10 mM each), 100 units RNAsin, 10 units M-MLV reverse transcriptase and water, which was added to produce a total volume of 25 μl. The samples were incubated at 42°C for 60 min following a 5-min 95°C denaturalization step, subsequently placed in an ice bath for 3 min and stored at −20°C to conserve the RNA template for the PCR. The RNA template was then converted into cDNA for use in the RT-PCR, which was performed in a total reaction volume of 50 μl, containing 5 μl 10X buffer, 4 μl MgCl2 (25 mM), 1 μl dNTP (10 mM each), 0.5 μl primer (50 pmol/μl), 0.5 μl downstream primer (50 pmol/μl), 0.3 μl Taq enzyme (5 U/μl), 2 μl cDNA template and 30–50 μl paraffin oil. PCR reactions were performed as follows: 95°C denaturation for 5 min; 30 cycles of 94°C for 30 sec, 50°C annealing for 30 sec and 72°C for 1 min; with a final step at 72°C for 5 min. The products were subjected to 2% agarose electrophoresis and ethidium bromide staining. Images were captured using the IBM 586 computer-controlled Gel Doc 1000 imaging system .The primer design software Premier 5.0  was used to design the following primers: Sense, 5′-CTGCCTCTCTTCTTCCCCTT-3′ and antisense, 5′-TCACAGCTGTTCAATGGCTC-3′ for Western blot analysis was used to detect Wnt-1 protein expression prior to and following U251 transfection. Total cellular protein was determined by SDS-PAGE gel electrophoresis. The proteins were transferred to nitrocellulose membranes  for protein immunoassays and the primary monoclonal mouse anti-human proto-onocogene Wnt-1 antibody  was used at a dilution of 1:100, with the secondary monoclonal rabbit anti-mouse antibody  at 1:500.2 humidified atmosphere for 48 h. Each MTT well contained a 20-μl MTT solution (5 mg/ml), which was incubated for 4 h. At culture termination, the medium was carefully aspirated and the supernatant discarded. Each well contained 150 μl dimethyl sulfoxide and was agitated for 10 min to fully dissolve the crystals. ELISA was used to record the absorbance at 490 nm and the cellular proliferation rate was calculated as follows: Cellular proliferation rate (%) =  × 100.U251-transfected cells were subjected to trypsin digestion following 24 h. Single cell suspensions were prepared in culture medium containing 10% fetal calf serum, transferred into 96-well plates at a final volume of 200 μl and grown at 37°C in a 5% CO1/G0, G2/M and S phase fractions.Trypsinized cells were transfected with plasmids 48 h following digestion. The cell suspension was then centrifuged in a clean centrifuge tube using a high speed centrifuge  at 507 xg for 5 min. The culture medium was discarded and washed three times with cold PBS. The cells were resuspended by adding 75% alcohol, fixed for 30 min in ethanol and centrifuged at 1,917 xg for 5 min, followed by washing three times with PBS. The cells were then resuspended in 100 μl PBS and 2.5 μl RNase (10 mg/ml), followed by 25 μl PI pyridine (10 mg/ml) and staining for 30 min. The cells were examined using a FACSAria flow cytometer  to determine the GSPSS 10.0  was used for analysis of variance. Data are presented as the mean ± standard deviation. The means between two groups were compared using Student’s t-test, while multiple comparisons were analyzed using the F-test. P<0.05 was considered to indicate a statistically significant difference.BamHI, but not PstI confirmed construction of the recombinant plasmid vector . Furthermore, the number of replicating cells decreased, which indicated that U251 proliferation was inhibited , which indicated that the plasmid and transfection reagents were not toxic. The positive controls transfected with pGPU6/GFP/Neo-shRNA-nhibited .Wnt signaling pathways are important in cellular differentiation and proliferation in a variety of species, and are involved in the formation of certain types of tumor (Wnt/β-catenin signaling pathway is considered to be a classical signaling pathway, which has been investigated in-depth (Wnt signal transduction mediates other processes, including Wnt/Ca2+ signaling, planar cell polarity, spindle orientation and asymmetric cell growth (Wnt binding of the Frizzled receptor inhibits the effect of glycogen synthase kinase 3-β, axin, and adenomatous polyposis coli protein complexes that are involved in β-catenin phosphorylation and degradation. Non-phosphorylated β-catenin is not degraded, however, accumulates in the cytoplasm, prior to translocation to the nucleus. β-catenin then binds to the lymphocyte enhancer factor/T cell-specific transcription factor, activating the target genes, c-myc, cyclin D and MMP7, leading to cellular proliferation and tumor formation. Therefore, certain studies have hypothesized that targeted therapy of Wnt signaling may inhibit tumor formation and growth (The of tumor –13. The in-depth . Wnt sigl growth ,14. Wnt d growth .Wnt/β-catenin signaling cascade is an important signal transduction pathway in human cancer (Wnt family consists of 19 members that regulate cell proliferation, differentiation, motility and fate during embryonic development and tumorigenesis. Wnt members bind to the cell surface receptors, Frizzled and low density lipoprotein receptor-related protein, and transduce their signals through β-catenin-dependent and -independent intracellular signaling pathways (Wnt-1 was one of the first signaling molecules to be identified and it is considered to be the first step in the classical Wnt/β-catenin signaling pathway (Wnt/β-catenin and PI3K/AKT signaling cascades is involved in tumor development and progression. However, the mechanism of this in glioma is not well understood (Wnt-1 expression (Wnt-1 gene expression is increased in glioma, with little or weak expression identified in normal brain tissue. Furthermore, Wnt-1 expression showed a positive correlation with the glioma grade (Wnt-1 gene expression by RNAi knockdown inhibits the growth of human glioma cells. This demonstrates that glioma growth may be closely correlated with Wnt-1 protein expression. Malignant glioma growth may involve activation of the Wnt-1 signaling pathway (Gliomas are the most common type of primary intracranial tumor and more effective therapeutic strategies are required to stop glioma progression. The n cancer . The humpathways . Wnt-1 w pathway ,19. Incrderstood . Althougpression , abnormama grade . The pre pathway , however pathway .Wnt signaling pathway may be ideal for targeted drug development. However, few studies have used Wnt-1 gene RNAi. Certain studies have used fragments of chemically synthesized siRNA in transfected cells (Wnt-1 into SH-SY5Y neuroblastoma cells using LipofectamineTM 2000 (Wnt-1 and β-catenin protein expression decreased following transfection with siRNA, as did the cell number, and was accompanied by abundant floating dead cells. Additionally, the SH-SY5Y cells transfected with siRNA targeting Wnt-1 showed less viability.Current RNAi methods are important for investigating gene function ,23,24 aned cells ,26, or ped cells , which ceTM 2000 . Wnt-1 aWnt-1 was used to investigate which region of the Wnt-1 gene may be targeted as a novel hotspot (In conclusion, the current study used a pGPU6/GFP/Neo carrier plasmid containing a GFP reporter, which detects cellular expression efficiency and Kanamycin/G418 resistance, into stable cell lines. Therefore, siRNA targeting  hotspot . The res