Epidemiological studies suggest an association between folate deficiency (FD) and cervical squamous cell carcinoma (SCC) progression. However, the underlying mechanism is unclear. Our study showed that FD-driven downregulation of miR-375 promoted proliferation of SCC SiHa cells and progression of xenograft tumors developed from SiHa; however, the exact mechanism of this process remained unclear. The current study aimed to elucidate the underlying mechanisms by which FD promotes the progression of SiHa cells by downregulating miR-375 expression. The results showed that miR-375 acted as a suppressor of SCC and inhibited the proliferation, migration, and invasion of SiHa cells. The FZD4 gene was identified as a target gene of miR-375, which can reverse the anti-onco effect of miR-375 and promote the proliferation and migration of SiHa cells. Furthermore, the regulatory effects of miR-375 and FZD4 on SiHa cells may be achieved by activating the β-catenin signaling pathway. Moreover, FD may regulate the expression of miR-375 by regulating its DNA methylation level in the promoter region. In conclusion, our study reveals that FD regulates the miR-375/FZD4 axis by increasing the methylation of the miR-375 promoter, thereby activating β-catenin signaling to promote SiHa cells progression. This study may provide new insights into the role of folic acid in the prevention and treatment of SCC.

Lack of sensitive biomarkers in the early stages of endometriosis (EMs) results in delayed diagnosis and intervention. Long non-coding RNAs (lncRNAs) have prognostic and diagnostic values in various diseases. However, the prognostic and diagnostic effects of lncRNAs on EMs have rarely been discussed in EMs. In this study, we found that lncRNA C8orf49 was stably overexpressed in EMs tissues/plasma, and its expression greatly influenced dysmenorrhea (p = 2.2605E-9) and the revised American Society for Reproductive Medicine stage (p = 0.040765) of EMs. Multivariate logistic regression results revealed that C8orf49 expression was an independent risk factor for EMs [p = 6.4997E-17, 95% confidence interval (CI) = 0.000559-0.023853]. In primary endometrial stromal cells (ESCs), inhibition of C8orf49 could impede the proliferation and metastasis of ESCs. C8orf49 influenced the expression of PTEN/FZD4 by absorbing miR-1323, thus controlling ESCs activity. The results of a subcutaneous endometriosis animal model showed that the inhibition of C8orf49 restrained endometrial growth. Overall, C8orf49 functioned as an activator of EMs pathogenesis via the C8orf49/miR-1323/PTEN/FZD4 axis.

OBJECTIVE: To verify the role of YAP/WNT5A/FZD4 axis in stretch-induced osteogenic differentiation of hPDLCs.
BACKGROUND: During orthodontic tooth movement, differentiation of human periodontal ligament cells (hPDLCs) at the tension side of the periodontal ligament mediates new bone formation. WNT5A promotes osteogenesis and its regulator Yes-associated protein (YAP) is responsive to mechanical stimulation in hPDLCs. However, the mechanisms of YAP and WNT5A in alveolar bone remodeling remain unclear.
METHODS: Cyclic stretch was applied to hPDLCs to mimic the orthodontic stretching force. Osteogenic differentiation was determined by alkaline phosphatase (ALP) activity, Alizarin Red staining, qRT-PCR and western blotting. To detect activation of YAP and expression of WNT5A and its receptor Frizzled-4 (FZD4), western blotting, immunofluorescence, qRT-PCR and ELISA were performed. Verteporfin, Lats-IN-1, small interfering RNAs and recombinant protein were used to explore the relationship of YAP, WNT5A and FZD4, and the effect of their relationship on stretch-induced osteogenesis of hPDLCs.
RESULTS: WNT5A, FZD4 and nuclear localization of YAP were upregulated by cyclic stretch. YAP positively regulated WNT5A and FZD4 expression and osteogenic differentiation of hPDLCs under cyclic stretch by YAP inhibition or activation assay. Knockdown of WNT5A and FZD4 attenuated YAP-induced and stretch-induced osteogenic differentiation. Recombinant WNT5A rescued the suppressed osteogenic differentiation by YAP inhibitor in hPDLCs, whereas knockdown of FZD4 weakened the effect of WNT5A and amplified the suppression.
CONCLUSIONS: WNT5A/FZD4 could be positively regulated by YAP and the YAP/WNT5A/FZD4 axis mediated osteogenic differentiation of hPDLCs under cyclic stretch. This study provided further insight into the biological mechanism of orthodontic tooth movement.

Skeletal muscle can undergo a regenerative process in response to injury or disease to maintain muscle quality and function. Myogenesis depends on the proliferation and differentiation of myoblasts, and miRNAs can maintain the balance between them by precisely regulating many key factors in the myogenic network. Here, we found that miR-136-5p was significantly upregulated during the proliferation and differentiation of C2C12 cells. We demonstrate that miR-136-5p acts as a myogenic negative regulator during the development of mouse C2C12 myoblasts. In terms of mechanism, miR-136-5p inhibits the formation of β-catenin/LEF/TCF DNA-binding factor transcriptional regulatory complex by targeting FZD4, a gating protein in the Wnt signaling pathway, thereby enhancing downstream myogenic factors and finally promoting myoblast proliferation and differentiation. In addition, in BaCl2 -induced muscle injury mouse model, miR-136-5p knockdown accelerated the regeneration of skeletal muscle after injury, and further led to the improvement of gastrocnemius muscle mass and muscle fiber diameter, while being suppressed by shFZD4 lentivirus infection. In summary, these results demonstrate the essential role of miR-136-5p/FZD4 axis in skeletal muscle regeneration. Given the conservation of miR-136-5p among species, miR-136-5p may be a new target for treating human skeletal muscle injury and improving the production of animal meat products.

UNASSIGNED: This study sought to explore the role and molecular mechanism of circ_0049271 in hypoxia-reoxygenation (H/R)-induced cardiomyocyte injury.
UNASSIGNED: Significantly upregulated circular ribonucleic acids (circRNAs) in Gene Expression Omnibus (GEO) data sets were identified using a Venn diagram. A H9c2 (rat cardiomyocytes) cell model of acute myocardial infarction (AMI) was induced by 1% H/R. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression levels of circ_0049271, miR-17-3p, and FZD4 in clinical blood samples and cells, and Cell Counting Kit-8 (CCK-8) was used to determine the proliferation rate of the cells in each group. Next, flow cytometry and Western blot were used to evaluate cell apoptosis. Biochemical tests and enzyme-linked immunosorbent assays (ELISAs) were then used to determine the activities/levels of the cell damage markers [i.e., creatine kinase (CK) and lactate dehydrogenase (LDH)], oxidative stress substances [i.e., malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD)], and inflammatory factors [i.e., interleukin (IL)-1β, IL-6, and IL-8]. In addition, intermolecular interactions were verified using dual-luciferase reporter and RNA pull-down experiments.
UNASSIGNED: Circ_0049271 was significantly upregulated in both the blood of the AMI patients and the H/R-induced H9c2 cells. The knockdown of circ_0049271 increased the cell proliferation rate, decreased the apoptosis rate, inhibited oxidative stress (ROS and MDA were upregulated, and SOD was downregulated) and inflammatory responses (IL-1, IL-6, and IL-8 were downregulated), and relieved cell damage. However, the overexpression of circ_0049271 promoted H/R-induced H9c2 cell damage. Further experiments showed that miR-17-3p was a target of circ_0049271, and miR-17-3p was negatively correlated with circ_0049271 in the AMI blood samples. Additionally, miR-17-3p was found to target FZD4. A further exploration also revealed that miR-17-3p knockdown or FZD4 overexpression reversed the effects of si-circ_0049271 on the H/R-induced H9c2 cells; that is, miR-17-3p knockdown or FZD4 overexpression promoted H/R-induced injury in the H9c2 cells.
UNASSIGNED: Circ_0049271 promoted cellular function damage (e.g., proliferation inhibition, apoptosis, oxidative stress, and inflammation) in H/R-induced H9c2 cardiomyocytes via the miR-17-3p/FZD4 signaling axis.

BACKGROUND: Accumulating evidence highlights the critical roles of circular RNAs (circRNAs) in the malignant progression of cancers. In this study, we investigated the expression pattern of a newly identified circRNA (hsa_circ_0017109) in non-small cell lung cancer (NSCLC), and examined its downstream molecular targets.
METHODS: Quantitative real-time PCR (qRT-PCR) and Western blotting (WB) were conducted to quantify gene and protein expression. In vitro functional assays such as colony formation assay, cell counting kit-8 (CCK-8) and flow cytometry were used to study cell proliferation and apoptosis. RNA pull-down assay, luciferase reporter assay and RNA immunoprecipitation were performed to validate molecular interaction. Mouse xenograft model of NSCLC cells was used to assess the role of circ_0017109 in tumorigenesis.
RESULTS: Circ_0017109 was upregulated in NSCLC tumor samples and cells. Silencing circ_0017109 impaired cell proliferation and promoted apoptosis in NSCLC cells, and circ_0017109 knockdown suppressed in vivo tumorigenesis of NSCLC cells in mouse xenograft model. MiR-671-5p was identified as a target of circ_0017109, and circ_0017109 negatively impacted on miR-671-5p expression. MiR-671-5p downregulated FZD4 and dampened the activity of Wnt/β-catenin signaling pathway. Circ_0017109 modulated FZD4 expression by suppressing miR-671-5p activity.
CONCLUSIONS: Elevated circ_0017109 expression promotes tumor progression of NSCLC by modulating miR-671-5p/FZD4/β-catenin axis.

To explore the clinical manifestations and search for the variants of six related genes (LRP5, FZD4, TSPAN12, NDP, KIF11 and ZNF408) in Chinese patients with familial exudative vitreoretinopathy (FEVR), and investigate the correlation between the genetic variants and the clinical characteristics.
Clinical data, including the retinal artery angle, acquired from wide-field fundus imaging, structural and microvascular features of the retina obtained from optical coherence tomography (OCT) and OCT angiography (OCTA) were collected from 33 pedigrees. Furthermore, mutation screening was performed. Variants filtering, bioinformatics analysis and Sanger sequencing were conducted to verify the variants.
Twenty-one variants were successfully detected in 16 of 33 families, of which 10 variants were newly identified. The proportion of variants in LRP5, FZD4, TSPAN12, NDP and KIF11 was 38.1% (8/21), 33.3% (7/21), 19.1% (4/21), 4.8% (1/21) and 4.8% (1/21), respectively. Three new variants were considered to be pathogenic or likely pathogenic. The FEVR group tended to exhibit a smaller retinal artery angle, higher incidence of foveal hypoplasia and lower vascular density compared to the control group. Patients who harboured variants of FZD4 exhibited greater severity of FEVR than those with LRP5 variants. However, those who harboured LRP5 variants tended to possess lower foveal vascular density.
Six known pathogenic genes were screened in 33 pedigrees with FEVR in our study, which revealed 10 novel variants. These findings enrich the clinical features and mutation spectrum in Chinese patients with FEVR, revealing the genotype-phenotype relationship, and contributing to the diagnosis and treatment of the disease.Key messagesWe identified 21 variants in 5 genes (LRP5, FZD4, TSPAN12, NDP and KIF11) associated with FEVR, 10 of which are novel (three were pathogenic or likely pathogenic).The proportion of variants was the highest for the LRP5 gene.FZD4 variants may be responsible for greater FEVR severity than LRP5 variants.

MicroRNAs are small, non-coding RNAs that regulate the expression of target genes. Previous research has demonstrated that microRNA-200a regulates cell apoptosis, tumor progression, and autoimmune disease. Preliminary studies found that microRNA-200a was differently expressed in the skin of Cashmere goats of various coat colors. However, the role of microRNA-200a in skin pigmentation remained poorly understood. In the current study, we investigated the effect of microRNA-200a on pigmentation in Cashmere goats. The expression of target genes was detected by real-time quantitative PCR, western blot, and immunohistochemistry staining both in vivo and in vitro. Luciferase reporter assays were used to demonstrate the relationship between microRNA-200a and its target genes Wnt family member 5A and frizzled class receptor 4 (WNT5A and FZD4) in HEK293T cells. BALB/c mice were injected with antagomiR-200a to detect melanin content and the expression of microRNA-200a and its target genes. The results demonstrated that the expression of microRNA-200a was significantly higher in brown tissue. Luciferase reporter assays confirmed that microRNA-200a targeted WNT5A and FZD4. The expression of WNT5A and FZD4 in the skin of brown Cashmere goats was significantly lower than that in white Cashmere goats by the detection of mRNA and protein levels. Overexpression/inhibition of microRNA-200a in keratinocytes decreased/increased the mRNA and protein expression of WNT5A and FZD4, respectively. In addition, the expression of WNT5A and FZD4 increased in the skin of BALB/c mice injected with antagomiR-200a, but the melanin content decreased. In summary, this study indicated that microRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/β-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/β-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/β-catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/β-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

BACKGROUND: Accumulating articles have suggested the important regulatory roles of circular RNAs in human cancers, including oral squamous cell carcinoma (OSCC). However, the role of circ_0001971 in OSCC progression remains to be determined.
METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays were conducted to analyze cell proliferation ability. Cell migration and invasion abilities were assessed by transwell assays. Dual-luciferase reporter assay was conducted to confirm the target relation between miR-107 and circ_0001971 or FZD4. Xenograft tumor model was established to analyze the biological role of circ_0001971 in regulating tumor growth in vivo.
RESULTS: Circ_0001971 was markedly up-regulated in OSCC tissues and cell lines. Circ_0001971 knockdown inhibited the growth of xenograft tumors in vivo. miR-107 was confirmed as a direct target of circ_0001971, and circ_0001971 depletion-mediated anti-tumor effects in OSCC cells could be largely alleviated by silencing miR-107. miR-107 directly targeted the 3\' untranslated region of FZD4, and FZD4 overexpression largely reversed the anti-tumor effects of circ_0001971 in OSCC cells. Circ_0001971 could positively regulate FZD4 expression by targeting miR-107 in OSCC cells.
CONCLUSIONS: Circ_0001971 promoted the proliferation, migration, and glycolysis of OSCC cells through mediating miR-107/FZD4 axis. Circ_0001971 might be a new effective target for OSCC treatment in future.