Long non-coding RNA SNHG16 decreased SMAD4 to induce gemcitabine resistance in pancreatic cancer via EZH2-mediated epigenetic modification.

Gemcitabine resistance (GR) in pancreatic cancer (PC) results in poor patient outcomes. SMAD family member (Smad4) dysregulation is a significant role of GR in PC, and EZH2 is involved in Smad4 expression in tumor progression. Interestingly, lncRNA small nucleolar RNA host gene 16 (SNHG16) might interact with EZH2, indicating a potential pathway to overcome gemcitabine-resistant PC progression. We investigated the role of the SNHG16/EZH2/Smad4 pathway in gemcitabine-resistant PC cells (PANC-1/GR and SW1990/GR). First, we found that SNHG16 was upregulated both in wild-type PC cells and in gemcitabine-resistant PC cells. SNHG16 overexpression reduced gemcitabine cytotoxicity and apoptosis in PC cells. Meanwhile, SNHG16 upregulation caused p-Akt elevation and Smad4 reduction. However, SNHG16 silencing induced the opposite trend. Then, we found that EZH2 was enriched in SNHG16 based on RIP and RNA pulldown. In particular, SNHG16 overexpression promoted the interaction between EZH2 and the Smad4 promoter according to Chromatin immunoprecipitation-quantitative polymerase chain reaction. Finally, both EZH2 inhibition and Smad4 upregulation increased gemcitabine cytotoxicity and apoptosis in PC cells during SNHG16 overexpression. Moreover, both treatments decreased p-Akt and increased Smad4. Collectively, lncRNA SNHG16 decreased Smad4 to induce GR in PC via EZH2-mediated epigenetic modification.

Identification of key constituent of sugar-smoking pigment and its photobleaching, antioxidant and antiproliferative properties.

The key constituent(s) of sugar-smoking brown pigments were identified, the chemical and biological properties were subsequently investigated, and the possibility of obtaining it from nature was explored. A pigment was isolated, which was identified as condensation product of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) with glucose: 5-(α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyloxymenthyl)-2-furancarboxaldehyde (5-GGMF). The molecular weight was 450.00. The interaction between pigment with protein on chicken skin was mainly via hydrogen and carbon-hydrogen bonds. It was obtained by caramelization instead of Maillard reaction. Discoloration of sugar-smoking pigments were mainly due to cleavage of chromophores caused by the oxidation reaction followed by photobleaching mechanism. Both radical scavenging and antiproliferative capacity of sugar-smoking pigment were in a dose-dependent manner. However, the IC50 values for HepG2 and PANC-1 cells differed by more than ten times. Since 5-GGMF is also present in plant resources (like Rehmanniae Radix), it can be obtained as natural additive to produce clean label sugar-smoked meat products.

E3 ubiquitin ligase RBX1 drives the metastasis of triple negative breast cancer through a FBXO45-TWIST1-dependent degradation mechanism.

Triple-negative breast cancer (TNBC) patients are at high risk of recurrence and metastasis in the early stages, although receiving standard treatment. However, the underlying mechanism of TNBC remains unclear. Here, the critical effect of E3 ubiquitin ligase RBX1 in the metastasis of TNBC was reported for the first time. We discovered that RBX1 expression was evidently raised in the tissues of TNBC. Our clinical research displayed that high RBX1 expression was markedly related to poor distant invasion and survival. Functional analysis exhibited that RBX1 facilitated metastasis of TNBC cells through increasing EMT. Furthermore, we demonstrated that RBX1 knockdown increased the levels of the Twist family bHLH transcription factor 1 (TWIST1), is a significant regulator in the EMT process in some cancers. It can be observed an evident positive correlation between the TWIST1 and RBX1 levels, further confirming that EMT induced by RBX1 in TNBC cells is determined by TWIST1. Mechanistically, RBX1 modulates the expression of TWIST1 via modulating FBXO45, directly binding to FBXO45, and facilitating its degradation and ubiquitination. Briefly, our findings confirm that RBX1 is probably a new biomarker of TNBC carcinogenesis, thus suggesting that targeting the RBX1/FBXO45/TWIST1 axis may be an underlying strategy for TNBC treatment.

Protective effect of Ulinastatin on acute lung injury in diabetic sepsis rats.

This study explored the protective effect and its possible mechanism of ulinastatin (UTI) on acute lung injury (ALI) in type 2 diabetes mellitus (DM) sepsis rats. Following treatment with UTI, the wet/dry weight (W/D) ratio, pathological changes, hypoxia-inducible factor-1ɑlpha (HIF-1ɑ) protein and Toll-like receptor 4 (TLR4) mRNA expression of lung tissues, the expression levels of interleukin-1beta (IL-1ß), IL-18, and tumor necrosis factor-alpha (TNF-ɑ), the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected in type 2 DM sepsis rats. It was found that rats with type 2 DM and sepsis showed obvious damage in lung tissues with significantly increased inflammatory cells, necrosis, and swelling of alveolar epithelial cells, but UTI decreased the lung damage induced by DM and sepsis. In addition, compared with the control, the W/D ratio, serum IL-1ß, IL-18 and TNF-ɑ contents, HIF-1ɑ protein expression, TLR4 mRNA expression, pulmonary microvascular permeability, MDA content in serum in type 2 DM and sepsis groups were significantly increased in type 2 DM sepsis rats (p < 0.05). However, compared with the groups with type 2 DM sepsis, the W/D ratio, serum IL-1ß, IL-18, TNF-ɑ contents, HIF-1ɑ protein expression, TLR4 mRNA expression, and pulmonary microvascular permeability in UTI-treated group were significantly decreased, but the activity of SOD increased (p < 0.05). This study indicates that UTI can effectively reduce ALI induced by diabetic sepsis in rats through inhibiting inflammatory response, reducing oxidative stress, regulating hypoxia response pathway, and improving pulmonary microvascular permeability.

CYP26A1 Is a Novel Cancer Biomarker of Pancreatic Carcinoma: Evidence from Integration Analysis and In Vitro Experiments.

Background: CYP26A1 has been reported in multiple cancers. However, the role of CYP26A1 in pancreatic cancer (PC) has not been explored. Method: The public data used for this study was obtained from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE) cell lines. CCK8, colony formation, and EdU assay were used to assess the proliferation ability of cancer cells. Transwell and wound healing assays were used to evaluate the invasion and migration ability of cancer cells. qRT-PCR and western blot assays were used to analyze the RNA and protein level of genes. Survival package was used for prognosis analysis. Gene Set Enrichment Analysis (GSEA) was used to identify biological pathway differences between two groups. ssGSEA analysis was used to quantify the immune microenvironment in PC tissue. GDSC and TIDE analyses were used for sensitivity analysis of chemotherapy and immunotherapy. Results: Our results showed that CYP26A1 was overexpressed in PC tissue and cell lines. Meanwhile, metastatic PC cell lines tend to have a higher CYP26A1 level compared with the primary PC cell lines based on CCLE data. Moreover, CYP26A1 was associated with worse clinical features. Also, we found that CYP26A1 had a satisfactory efficiency in predicting overall survival, disease-specific survival, and progression-free interval of PC patients, independent of other clinical features. In vitro experiments indicated that CYP26A1 could significantly facilitate the proliferation, invasion, and migration ability of PC cells. GSEA showed that the pathways of angiogenesis, E2F target, MYC target, mTORC signaling, G2M checkpoint, and epithelial-mesenchymal transition were activated in high CYP26A1 patients. Immune infiltration analysis showed that CYP26A1 was positively correlated with macrophages, Th1 cells, and Treg cells, but negatively correlated with Th17 cells. TIDE analysis showed that non_responder patients had a higher CYP26A1 level compared with predicted responder patients of immunotherapy. Drug sensitivity analysis and assay showed that CYP26A1 could increase the chemotherapy sensitivity of gemcitabine. Conclusions: In summary, CYP26A1 promotes PC progression and is a novel biomarker of PC, with potential for clinical application.

Erratum: RNF6 promotes the migration and invasion of breast cancer by promoting the ubiquitination and degradation of MST1.

[This corrects the article DOI: 10.3892/etm.2021.11041.].

Experimental Study on the Central Mechanism of Penehyclidine Hydrochloride against Relapse Behavior in Morphine-Dependent Rats.

OBJECTIVE: This article is mainly to study the central mechanism of penehyclidine hydrochloride against relapse behavior in morphine-dependent rats. METHODS: The rats were randomly divided into the blank control group (k), PHC low-dose group (LP according to a body weight of 0.22 mg/kg), middle-dose group (MP according to a body weight of 0.55 mg/kg), high-dose group (HP according to a body weight of 1.38 mg/kg), and administration group, with 40 rats in each group. Each group was randomly divided into 5 subgroups (n = 10): 4 h after administration, 7 h after administration, 13 h after administration, 25 h after administration (K48, LP48, MP48, and HP48), and 37 h after administration, and then, Morris water maze experiment and immunohistochemical detection of the rat brain hippocampus were carried out. RESULTS: 4 and 7 hours after administration, compared with group 1, the TchE activity increased and Ach level decreased in groups 2, 3, and 4 and the difference was significant (P < 0.05), so the principle of penehyclidine hydrochloride against morphine-dependent rats is that penehyclidine hydrochloride causes cognitive impairment in the brain of mice, thereby achieving antimorphine effects.

RNF6 promotes the migration and invasion of breast cancer by promoting the ubiquitination and degradation of MST1.

Ring finger protein 6 (RNF6), a member of E3 ubiquitin ligases, plays a potential role as a tumour promoter in numerous carcinomas. However, the role and expression of RNF6 in breast cancer (BC) remains to be elucidated. The present study showed that RNF6 upregulation was detected in BC tissues and was associated with short survival in patients with BC. Multivariate analysis also revealed that RNF6 overexpression is an independent predictor for poor outcome of patients with BC. Furthermore, migration and metastasis assay indicated that RNF6 silencing significantly inhibited the invasion and migration of BC cells in vivo and in vitro, and RNF6 suppression decreased YES-associated protein (YAP) expression. RNF6 promoted the metastatic ability of BC cells via YAP. Mechanistically, RNF6 interacts with mammalian STE20-like protein kinase 1 (MST1), a key factor that regulates YAP, and promoted its ubiquitination and degradation. Additionally, RNF6 regulated YAP signalling by promoting ubiquitination and degradation of MST1 in BC. Taken together, these data may highlight a role of RNF6 in BC, which could serve as a valuable prognostic indicator and potential therapeutic target for patients with BC.

Establish intelligent detection system to evaluate the sugar smoking of chicken thighs.

The objective of this study was to establish a standardized color detection method to achieve low-cost, rapid, nonintrusive and accurate characterization of the color change of smoked chicken thighs during the smoking process. This study was based on machine vision technology using the Mean algorithm, K-means algorithm and K-means algorithm + image noise reduction algorithm to establish 3 colorimetric cards for the color of sugar-smoked chicken thighs. The accuracy of the 3 colorimetric cards was verified by the K-medoids algorithm and sensory analysis, respectively. Results showed that all 3 colorimetric cards had significant color gradient changes. From the K-medoids algorithm, the accuracy of the colorimetric card produced by the Mean algorithm, K-means algorithm and K-means algorithm + image noise reduction algorithm was 87.2, 95.1, and 96.7%, respectively. Meanwhile, the verification results of the sensory analysis showed that the accuracy of the Mean algorithm, K-means algorithm and K-means algorithm + image noise reduction algorithm colorimetric card was 69.4, 80.9, and 79.2%, respectively. A comparative analysis found that the colorimetric cards produced by the K-means algorithm and K-means algorithm + image noise reduction have excellent accuracy. These 2 colorimetric cards could become a suitable method for rapid, low-cost, and accurate online color monitoring of smoked chicken.

Quantifying the interaction of water and radiation use efficiency under plastic film mulch in winter wheat.

Local natural resources, (e.g., precipitation, solar radiation) are important for developing environmentally and scientifically sound management practices in dryland agroecosystem. Maximizing water use efficiency (WUE) in dryland farming systems remains a challenge. The objectives of this study were to assessing the robustness of radiation use efficiency (RUE) during different periods and investigate the interaction between RUE and WUE from water loss pattern and canopy development during wheat growth under different agricultural practices (non-mulched control, CK; transparent film mulching, TF; and black film mulching, BF) from 2013 to 2016 on the Loess Plateau, Northwest China. Results showed that RUE was mainly improved during post-anthesis under PM treatments. PM treatments contributed to elevated canopy photosynthesis and a delayed RUE peak during the reproductive period. Due to the increased spike number and ratio of plant transpiration to soil evaporation, TF and BF treatments had relatively stable photosynthetic activity relative to the CK treatment even those during dry periods. Initially, no relationship was found between WUE and RUE under the CK treatment. On the other hand, RUE and WUE were positively related in TF and BF treatments following a power function. RUE values increased with WUE rapidly to stabilize at a plateau value of 5.5 g MJ-1 under TF and BF treatments, and thus, the wheat WUE had a higher improvement potential than RUE as it did not have an apparent plateau value. PM treatments enhanced the wheat production by taking full advantage of local solar radiation and precipitation (improving RUE and WUE). This higher use efficiency of resources produced more photoassimilates for wheat than that under the CK management, increased source size (LAI) and sink size (spike number) during wheat growth seasons, and thus increased the final grain yield.

The significance of detecting glucose transporter 1 and calretinin in serous effusions to differentiate between carcinoma cells and reactive mesothelial cells.

BACKGROUND: The cytologic evaluation of serous effusions to distinguish malignant cells from reactive mesothelial cells (RMCs)was an enormous challenge. The purpose of this study was to investigate the diagnostic value of glucose transporter 1 (GLUT1) and calretinin (CR) in serous effusions of patients with malignant and in order to significantly ameliorate the diagnostic accuracy. METHODS: The expressions of GLUT1 and CR were measured by streptavidin-peroxidase (S-P) immunocytochemical technique in serous effusions of 183 patients with malignant and in 95 patients with benign diseases. RESULTS: The positive ratio of GLUT1 was 91.8% (168/183) in serous effusions from patients with malignant and 5.3% (5/95) in benign diseases, they had a significant difference (P < .01). CR was expressed 89.5% (85/95) in benign diseases and 6.6% (12/183) in malignant, it also showed an important difference (P < 0.01). The combination of GLUT1 + CR revealed the best efficiency: the sensitivity and specificity were 100% and 98.9%, respectively. CONCLUSION: Immunocytochemical staining for GLUT1 and CR may be used as a complementary tool for the detection of malignant effusions and help to make a distinction between cancer cells and RMCs. The combination of GLUT1 and CR with immunocytochemistry stained can be achieved a higher diagnostic performance.

Drought responses of profile plant-available water and fine-root distributions in apple (Malus pumila Mill.) orchards in a loessial, semi-arid, hilly area of China.

The number of apple (Malus pumila Mill.) orchards has increased substantially in hilly regions of the Loess Plateau of China, as a significant element of the large-scale 'Grain for Green' ecological rehabilitation program that aims to conserve soil and water while improving the regions economic prospects. However, the long-term effects of the orchard expansion and the adaptive responses of apple trees to drought are not known. Thus, using a space-for-time substitution approach, we investigated plant-available water and fine-root distribution in the 0-8 m soil profile in apple orchards of various ages in a dry year (2015, 392 mm rainfall) and the following year with normal precipitation (2016, 500 mm rainfall). We found that plant-available water gradually decreased with stand age in the dry year, but increased in the normal year, especially in the 0-2 m soil layer. Fine root (<2 mm diameter) distribution and biomass increased with stand age and decreased with increasing soil depth in all treatment plots, predominantly in the 0-2 m layer. In all treatment plots, most of the soil layers in the deep soil (>2 m) had soil moisture storage deficit. In the dry year (2015), the apple trees increased both the average depth (D50 and D95 values) and biomass of their fine-root systems in response to water stress, relative to the normal year (2016). Thus, the apple trees extracted water primarily from the shallow (<2 m) layers in the normal year, but from deeper soil layers in the dry year, to sustain growth. The results of this study will help to guide land and agricultural water management in rainfed apple orchards in hilly regions of the Loess Plateau and similar dryland regions.

Toll-like receptor 4 deficiency ameliorates ß2-microglobulin induced age-related cognition decline due to neuroinflammation in mice.

Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that ß2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline.

LncRNA SNHG16 induces the SREBP2 to promote lipogenesis and enhance the progression of pancreatic cancer.

Aim: Blocking lipogenesis could significantly inhibit the progression of pancreatic cancer. Exploring the regulatory mechanisms of lipogenesis by lncRNA SNHG16 might be of great significance to control the development of pancreatic cancer. Methods: The proliferation, migration, invasion and lipogenesis were determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, transwell and Oil Red O staining assays, respectively. The interactions among lncRNA SNHG16, miR-195 and SREBP2 were analyzed by dual luciferase reporter assays. Results: Both the knock down of lncRNA SNHG16 and SREBP2 and overexpression of miR-195 suppressed the proliferation, migration, invasion and lipogenesis in pancreatic cancer cells. LncRNA SNHG16 directly sponged miR-195 to modulate the lipogenesis via regulating the expression of SREBP2. Conclusion: LncRNA SNHG16 accelerated the development of pancreatic cancer and promoted lipogenesis via directly regulating miR-195/SREBP2 axis.

FAT10 promotes the invasion and migration of breast cancer cell through stabilization of ZEB2.

FAT10, an ubiquitin-like protein, functions as a potential tumor promoter in several caners. However, the function and clinical significance of FAT10 in breast cancer (BC) remains unclear. Here, we found that high FAT10 expression was detected frequently in primary BC tissues, and was closely associated with malignant phenotype and shorter survival among the BC patients. Multivariate analyses also revealed that FAT10 overexpression was independent prognostic factors for poor outcome of patients with BC. Function assay demonstrated that FAT10 knockdown significantly inhibited the metastasis abilities and the epithelial-mesenchymal transition (EMT) of breast cancer cell. Further investigation revealed that FAT10 directly bound ZEB2 and decreased its ubiquitination to enhance the protein stability of ZEB2 in BC cells. Moreover, our data shown that the pro-metastasis effect of FAT10 in BC is partially dependent on ZEB2 enhancement. Collectively, our data suggest that FAT10 plays a crucial oncogenic role in BC metastasis, and we provide a novel evidence that FAT10 may be serve as a prognostic and therapeutic target for BC patients.

PSMB5 is associated with proliferation and drug resistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by advanced disease stage and poor prognosis. Moreover, due to the lack of therapeutic markers, TNBC patients can't benefit fully from currently available targeted therapies. METHODS: To fully understand the molecular basis of TNBC, we used gene set enrichment analysis (GSEA) to screen out the most altered functional module in TNBC, from publicly available microarray data and studied the association of the candidate gene with TNBC development. RESULTS: We found that the proteasome was significantly activated in TNBC. As compared with other breast cancer subtypes and normal tissue, proteasome subunit beta 5 (PSMB5), the key regulator of proteasome function, was overexpressed in TNBC tissue and predictive of poor prognosis. Moreover, we also found that PSMB5 knockdown induced TNBC apoptosis and significantly enhanced cancer cell sensitivity to the chemotherapeutic agents bortezomib and paclitaxel. CONCLUSIONS: Our results suggest a potential role for PSMB5 as a biomarker and therapeutic target for TNBC.

Applications of high pressure to pre-rigor rabbit muscles affect the water characteristics of myosin gels.

Myosin was extracted immediately after high-pressure treatment (HP, 100-300MPa for 15 or 180s) to pre-rigor rabbit muscles (PRRMs) for evaluating the influences of HP-treatment on gel properties, using untreated muscles as Controls. Assessment of myosin yields, water-holding capacity (WHC), water mobility and distribution demonstrated that HP modified myosin before its extraction. Myosin gels subjected to HP at 100MPa 180s and 200MPa 15s had enhanced WHC compared with Controls. Also, the highest proportion of immobile-water was observed in myosin gels treated at 200MPa for 15s. HP-treatment of PRRMs affected their physicochemical properties as evidenced by alterations in tertiary, secondary conformations and rheological properties during subsequent heating. These modifications appear to induce various degrees of exposure of hydrophobic and sulfhydryl groups, resulting in different gelation rates. These alterations partly explain the various gel qualities obtained and indicate the potential of HP for pre-rigor muscles.

Applications of high pressure to pre-rigor rabbit muscles affect the functional properties associated with heat-induced gelation.

High pressure (HP) was applied to pre-rigor rabbit muscles (PRRMs) for the manufacture of value-added gel-type products. A 2×3 factorial design was adopted for HP-treatment (100, 200 and 300MPa for 15 or 180s) to PRRMs. The HP-treated meat was used for gel preparation and determination of water mobility properties and textural profiles. Salt-soluble meat proteins (SSMP) were then extracted from meat for estimating the tertiary conformational changes (hydrophobic and sulfhydryl groups). The water-holding capacity of treated samples increased significantly (P<0.05) except for those treated at 300-180 (MPa-s). The hardness, cohesiveness and chewiness were significantly improved (P<0.05) by moderate HP-treatments (100-200MPa for 15 or 180s). HP-induced the de-polymerization of some protein components, together with unfolding and refolding of protein tertiary conformations, partly explaining the observed functionality changes. In conclusion, application of moderate HP to PRRM modified the protein conformation in a manner that improved the functional properties of gels.

AMPK activators suppress breast cancer cell growth by inhibiting DVL3-facilitated Wnt/ß-catenin signaling pathway activity.

Adenosine monophosphate-activated protein kinase (AMPK) is a principal regulator of metabolism and the conservation of energy in cells, and protects them from exposure to various stressors. AMPK activators may exhibit therapeutic potential as suppressors of cell growth; however, the molecular mechanism underlying this phenomenon in various cancer cells remains to be fully elucidated. The present study investigated the effects of AMPK activators on breast cancer cell growth and specified the underlying molecular mechanism. In the present study, the AMPK activator metformin impaired breast cancer cell growth by reducing dishevelled segment polarity protein 3 (DVL3) and ß­catenin levels. Western blotting and immunohistochemistry demonstrated that DVL3 was recurrently upregulated in breast cancer cells that were not treated with metformin, and was significantly associated with enhanced levels of ß­catenin, c­Myc and cyclin D1. Overexpression of DVL3 resulted in upregulation of ß­catenin and amplification of breast cancer cell growth, which confirmed that Wnt/ß­catenin activation via DVL3 is associated with breast cancer oncogenesis. To elucidate the underlying mechanism of these effects, the present study verified that metformin resulted in a downregulation of DVL3 and ß­catenin in a dose­dependent manner, and induced phosphorylation of AMPK. Compound C is an AMPK inhibitor, which when administered alongside metformin, significantly abolished the effects of metformin on the reduction of DVL3 and activation of the phosphorylation of AMPK. Notably, the effects of metformin on the mRNA expression levels of DVL3 remain to be fully elucidated; however, a possible interaction with DVL3 at the post­transcriptional level was observed. It has previously been suggested that the molecular mechanism underlying AMPK activator­induced suppression of breast cancer cell growth involves an interaction with, and impairment of, DVL3 proteins. The results of the present study are of future clinical importance and advocate the use of metformin as a potential therapeutic agent against breast cancer.