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FZD6 plays crucial roles in human tumorigenesis. However, its mechanism in regulating cancers has not been fully elucidated. In the study, we found that FZD6 repressed gastric cancer cell proliferation and migration via activating non-canonical wnt pathway. In addition, non-canonical wnt pathway ameliorated expression of canonical wnt pathway. We also demonstrated that the FZD6 was involved in miR-21-dependent effects in the canonical and non-canonical wnt pathways in gastric cancer. These findings provide a better understanding of the development and progression of gastric cancer and may be an important implication for future therapy.
Gastric cancer is the fifth most common cancer and the third most frequent cause of cancer death worldwide, and in spite of some therapeutic advances its prognosis is still unfavourable [1,2]. Genetic background, behavioural factors (eg, diet, smoking habit and alcohol consumption), and helicobactor pylori infection have been associated with the risk of developing gastric cancer [3,4]. However, the cascade of molecular events leading to gastric carcinogenesis are still largely unknown. An improved understanding of critical pathways involved in cancer will facilitate the development of effective targeted therapeutic strategies for gastric cancer. Many genes and pathways have been reported involved in gastric cancer formation and metastasis, such as canonical wnt/β-catenin , PI3K/AKT , p53  and stat3 . The canonical wnt/β-catenin pathway is involved in various differentiation events during embryonic development and associates with tumor formation when aberrantly activated. Besides canonical wnt/β-catenin pathway, the wnt pathway also includes non-canonical wnt pathway (the wnt/Ca2+ pathway, the planar cell polarity pathway), depending on the specific Wnt/Frz signal received . However, the effect of non-canonical wnt pathway on cancer and the association between canonical and non-canonical wnt pathways are still controversial.
FZD6 (frizzled class receptor 6) is a member of the “frizzled” gene family, which encode 7-transmembrane domain proteins that are receptors for non-canonical Wnt signaling proteins. Mounting evidences show that FZD6 is involved in cancer development. GM Caldwell et al reported that FZD6 was overexpressed in colorectal cancer . In contrast, FZD6 has also been shown reduced in leukemia cell lines . Recent studies indicate that FZD6 could be regulated by microRNAs. MiR-199a-5p targeted FZD6 to regulate growth of colorectal cancer . To date, the mechanism of FZD6 deregulation and its regulatory networks in gastric cancer remain elusive.
The microRNAs (miRNAs) are ~22 nucleotides noncoding RNAs and they can regulate tumorigenesis through targeting cellular function related mRNA . MiR-21 has been identified as the best hit in cancer related miRNAs by accumulating evidence . It was shown to promote proliferation and invasion of gastric cancer, hepatocellular cancer, colorectal cancer, breast cancer, glioma and other cancers by directly targeting PTEN, PDCD4, RECK and other signal transduction pathways [15-19].
In this study, we found that FZD6 represses gastric cancer cell proliferation and migration via activating non-canonical wnt pathway. In addition, non-canonical wnt pathway ameliorates expression of canonical wnt pathway. We also demonstrated that the FZD6 was involved in miR-21-dependent effects in the canonical and non-canonical wnt pathways in gastric cancer.
Human microRNA microarray analysis was performed by Ouyi Biotechnology (Shanghai). Total RNA was extracted from gastric cancer tissues and from normal gastric tissues, according to Trizol protocol (Takara, Japan). Total RNA was used for miRNA microarray analysis (Agilent human miRNA V19.0). This chip allows the simultaneous analysis of 723 human miRNAs (miRBase release 10.1). RNA labeling and hybridization were performed in accordance to the manufacturer’s indications. Agilent scanner and the Feature Extraction 10.7.1.1 software (Agilent Technologies) were used to obtain the microarray raw data. Microarray results were analyzed using the GeneSpring GX 12.5 software (Agilent Technologies). Differentially expressed miRNAs were identified by using a moderated t-test and Benjamini-Hochberg correction (adjusted P≤0.05 and fold change ≥2). Differentially expressed genes were measured similarity with miRNAs by the Pearson correlation and were analyzed in GO analysis, KEGG pathway analysis, and cluster analysis.
Thirty-five pairs of snap-frozen gastric cancer tumor and matched adjacent non-tumor tissues were diagnosed histopathologically at the first affiliated hospital of Nanjing Medical University, Jiangsu Province, China from June 2013 to December 2013. The tissues were immediately stored in liquid nitrogen after surgery until further use. This protocol was approved by the Ethical Committee of the first affiliated hospital of Nanjing Medical University, and every patient had written informed consent.
Gastric cancer cell lines SGC-7901, AGS and immortal gastric epithelial cell line GES-1 were purchased from ATCC. Cell lines were maintained in RPMI 1640 (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, USA). Cells were cultured in a humidified incubator at 37°C in an atmosphere of 95% air and 5% carbon dioxide.
Real-time PCR was performed to determine the expression levels of microRNA and mRNA in human cell lines and tissues. Total RNA was extracted with a Trizol reagent (Takara, Japan) following the instruction. Reverse Transcription was carried out using miRNA First-Strand cDNA Synthesis Kit (Gencopoeia, Guangzhou) for miRNA and reverse transcription kit (Takara, Japan) for mRNA. Real-time PCR was employed in triplicate using the SYBR Green PCR Kit (Takara, Japan) on Applied Biosystems StepOne-Plus Real-Time PCR System. All mRNAs were normalized to GAPDH, and miRNAs were normalized to U6. Relative quantification expression was calculated according to the comparative method of 2-ΔΔCT. Primer sequences were listed in Table 2.
All 3’ UTR reporter vectors were prepared by amplifying the 3’ UTRs of FZD6, followed by insertion into the Pezx-MT01 vector (GeneCopoeia, Guangzhou). Site-specific mutants were generated by PCR in the Pezx-MT01 vector (GeneCopoeia, Guangzhou). SGC-7901 cells were seeded in the 24-well plates (1×105 cells per well) one day before transfection and then each well was transfected with a mixture of 100 ng 3’-UTR luciferase reporter vector and 50 pmol miRNA mimics, inhibitors or respective controls. Twenty four hours post transfection, the cells were lysed. Then, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA), with a Promega Glomax 2020 Single Tube Luminometer instrument (Promega, USA). Each experiment was performed in triplicate. The ratio of Renilla luciferase to Firefly luciferase was calculated for each well.
The total cellular protein was extracted with RIPA buffer containing a protease inhibitor. The protein concentration was detected by a BCA protein assay kit (Thermo Scientific, USA). Protein was loaded into 10% polyacrylamide SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk for 2 hour and incubated overnight at 4°C using the following antibodies: anti-FZD6 (Abcam, UK), anti-Actin (Abcam, UK). After three time washes with TBST (TBS with 0.1% tween), the membrane was incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG, Abcam, UK) at room temperature for 1 h. After another three washes with TBST, the signal was visualized through a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo, USA) and then exposed in Molecular Imager ChemiDoc XRS System (Bio-Rad, USA).
Cell viability was determined by CCK-8 (Dojindo, Japan). GC cells were seeded into 96-well plates at 1.0×104 cells per well in RPMI 1640 containing 10% FBS for 24 h and then transfected with mir-21 mimics, inhibitors, or respective controls, using Lipo2000 (Thermo, USA) according to manufacturer’s instruments. After 48 hours, CCK-8 solution (10 uL/well) was added 2 h before measurement. The absorbance at 450 nm of each well was recorded by microplate reader (Bio-Rad, USA).
Colonic gastric cancer cells were transfected with miR-21 mimic or miR-21 inhibitor or their respective controls. Then, triplicate samples of 400 cells from each cell line were plated on medium. The number of colonies was counted after 10 days.
We performed transwell migration assays in gastric cancer cell lines 48 hours after transfection with miR-21 mimic or miR-21 inhibitor or their respective controls. Assays were conducted according to manufacturer’s protocol, using 20% FBS as chemoattractant. Non-migrating cells on the top side of the membrane were removed while migrating cells were fixed with methanal and stained with crystal violet 24 h post seeding. In all assays, 10 fields per insert were scored and SE was calculated.
The data were presented as the mean ± s.e.m. Statistical analyses were performed with the use of SPSS 20.0 and Graphpad Prim 5. Student’s t test was used to examine the statistical difference. The correlation significance was determined by means of Spearman and Pearson correlation analyses. All used tests were two-tailed, and a P value of less than 0.05 was considered significant.
We found that FZD6 was down-regulated in gastric cancer tissues and gastric cancer cell lines SGC7901 and AGS compared with normal gastric tissues and normal gastric epithelial cell line GES-1 (Figure 1A and and1B).1B). In order to evaluate the effects of FZD6 on malignant phenotypes in gastric cancer cells, we transfected SGC7901 cells with either pc-control or pc-FZD6 and AGS cells with either si-control or si-FZD6. Transfection efficiency was confirmed through real-time PCR (both P<0.05, Figure 1C and and1D).1D). CCK-8 assays revealed that pc-FZD6 transfected cells exhibited significantly decreased growth rate than pc-control transfected cells (P<0.05, Figure 1E). Colony formation assays also showed that promoting FZD6 expression resulted in significant tumor growth inhibition (P<0.05, Figure 1F). We next examined the effect of FZD6 on migration ability of gastric cancer cells by transwell assay. Increased FZD6 expression significantly reduced migration of gastric cancer cells (P<0.05, Figure 1G). Inversely, si-FZD6 transfected gastric cancer cells displayed increased cell proliferation and migration compared to cells transfected with si-control. These results suggested that FZD6 could suppress gastric cancer cell proliferation and migration.
We first isolated and compared the miRNA expression profile in 6 matched pairs of gastric cancer and control noncancerous gastric tissues using the Agilent human miRNA V19.0 (Figure 2A and and2B).2B). Twenty-seven miRNAs (17 up-regulated and 10 down-regulated) were expressed differentially in gastric cancer according to class comparison analysis of fold change ≥2 and p≤0.05 (Table 1). The group of miRNA that were altered significantly in expression may contribute to the pathogenesis or phenotypic behavior of gastric cancer. Moreover, these miRNAs also are potential markers that may be useful for the diagnosis of gastric cancer.
To establish the molecular link between FZD6 and miRNAs, we identified related miRNAs of FZD6 by computational prediction. Among hundreds of miRNAs predicted by online miRNA target prediction website (starBase, http://starbase.sysu.edu.cn/), we forcused our attention on miR-21 [20,21]. According to the results of our microarray, miR-21 was highly significantly increased in gastric cancer tissues with the lowest p value of 1.24E-04. We found that miR-21 is significantly up-regulated in gastric cancer tissues (Figure 2C) and FZD6 was inversely correlated with miR-21 status (Figure 2D). What’s more, Quantitative real time PCR (qRT-PCR) and western bolt revealed significantly different expression of FZD6 in cells transfected with miR-21 mimic or inhibitor relative to respective controls (Figure 3A-E).
To assess whether miR-21 can directly alter the expression of FZD6 in GC cells, a fragment of the 3’ UTR of FZD6 mRNA, containing the putative miR-21 binding sequence, was cloned into a firefly luciferase reporter construct, which contained both luciferase and renilla expression mRNA, along with miR-21 mimic, inhibitor or respective controls. Co-transfection of miR-21 mimic and the WT FZD6 3’-UTR construct resulted in a significant inhibition of the luciferase activity compared with cells co-transfected with negative control. An increase in relative luciferase activity was noted with miR-21 inhibitor, indicating that miR-21 could modulate gene expression directly at the FZD6 3’ UTR (Figure 3F and and3G).3G). In addition, we constracted mutated luciferase reporter with mutations of putative miR-21-bingding sites in these 3’-UTR regions and found cotransfecting with miR-21 mimic and inhibitor abrogated the inhibiting and promoting effects (Figure 3H). Taken together, these results demonstrate that FZD6 is the direct target of miR-21-5p.
It is known that mir-21 promotes GC via regulating wnt pathway . Wnt parthway can be divided into canonical and non-canonical pathways. The representative molecular in canonical pathway includes MMP2, MMP9 and CCND1 and non-canonical pathway includes RHOA and JNK . We studied the influence of mir-21 and FZD6 for wnt by real time quantitative PCR. Up regulation of MMP2, MMP9, CCND1 and downregulation of RHOA and JNK were noticed when cells were transfected with mir-21 and si-FZD6 (P<0.05, Figure 4A and and4B).4B). In contrast, when cells were transfected with mir-21 inhibitor or pc-FZD6, the phenomenon is quite opposite (P<0.05, Figure 4C and and4D).4D). In combination, these studies defined an important role for FZD6 in canonical and non-canonical wnt pathways in human gastric cancer cells (Figure 4E).
Although many genes and pathways in cancer have been proposed, the molecular mechanisms of tumor proliferation and migration are not fully known. We show that FZD6, a gene involved in non-canonical wnt pathway, suppresses gastric cancer proliferation and migration. Moreover, it is targeted by cancer promoter miR-21 and associated with miR-21-dependent effects in the canonical and non-canonical wnt pathway. The identification of FZD6 as an important regulator of tumor cell proliferation and migration in vitro provides insight into gastric oncogenesis and molecular target treatment.
Wnt signaling pathway is closely associated with tumor development, including gastric cancer . Wnts have been classified into two functional groups with separate downstream signaling pathways, canonical and non-canonical wnt pathway. FZD6 is involved in non-canonical wnt pathway and RHOA and JNK are critical downstream molecules of non-canonical wnt patheway . However, the function of FZD6 in tumor is still controversial. Some report that aberrant activation of the FZD6 leads to more malignant phenotypes, such as abnormal tissue polarity, invasion, and metastasis . However, Piga et al silenced FZD6 in non-tumor human type II bronchial epithelial BEAS-2B cells and found enrichment of the Gene Ontology terms “oncogenes”, “cell proliferation” and “cell cycle”, which reveals FZD6 may inhibit tumor formation  Moreover, Beatriz et al reported that WNT4 ligand regulated the cell growth of leukemia derived cells by arresting cells in the G1 cell cycle phase in an FZD6-independent manner, possibly through antagonizing the canonical WNT/β-catenin signaling pathway . In addition, FZD6 was reported to activate the kinase-NEMO-like kinase pathway that blocks TCF/lymphoid enhancer, thereby inhibiting the ability of canonical wnt/β-catenin pathway .
miR-21 has been shown to be overexpressed in many different solid tumors, including gastric cancer. Therefore, it is highly likely that miR-21 plays a fundamental role in malignant transformation and targets tumor suppressor mRNA in tumor cells. A number of target genes have been identified [27,28]. Dan et al reported that miR-21 regulates lung cancer via wnt pathway . In our study, we found inversely expression level of mir-21 and FZD6 via real time quantitative PCR and western blot and confirmed the direct targeting of mir-21 to FZD6 via luciferase. What’s more, we confirmed miR-21 can promote canonical wnt pathway and inhibit non-canonical wnt pathway via FZD6.
Although we found FZD6 and miR-21 expression levels had the tendency of being inversely correlated, the p value is more than 0.05. The reason may be the limited number of tissues and more clinical tissues are needed. In addition, when cells were transfected with miR-21 mimic, the changes of MMP2 and MMP9 were not significantly. We analyzed that the phenomenon may be led by high expression of miR-21 in gastric cancer cells. This also suggest that the regulation of miR-21 and its targets is very complex and other studies explaining the mechanism are still needed.
Proliferation and metastasis are associated with a poor prognosis, and therefore targeting these mechanisms may lead to more effective treatment for gastric cancer patients. There is considerable interest in microRNA-mediated therapy. Firstly, microRNAs readily cross cell membranes . Secondly, microRNAs are not immunogenic because of lacking poly-A tail . Thirdly, these molecules are fairly stable both in vitro and in vivo, although they can be further modified to prolong their biological half-life [31,32]. Therefore, we are hopeful that this study provides enticing data to support the use of microRNAs inhibitor, specifically miR-21, for the treatment of gastric cancer and other cancers.
In summary, our results demonstrate that FZD6 suppresses gastric cancer proliferation and migration and is involved in miR-21-dependent effects in the canonical and non-canonical wnt pathway. Exploring gastric cancer mechanism can provide a more effective treatment for gastric cancer patients.
This work was supported by National Natural Science Foundation of China (No. 81270476 and 81470830), Jiangsu standard diagnosis and treatment research for key diseases (BE2015716), the Priority Academic Program Development of Jiangsu Higher Education Institutions (JX10231802) and Jiangsu postgraduate scientific research and innovation projects (KYZZ15_0265).