Catenin Axis Research Articles

Small Nucleolar RNA Host Gene 9 Promotes the Development of Breast Cancer by Regulating the miR-326/Wnt5a/β-Catenin Axis

Breast cancer (BC) is a highly prevalent aggressive malignancy in women worldwide, and the search for key targets in its pathogenesis is a focus of research. Long non-coding RNAs (lncRNAs) play an important role in many cancers, including breast cancer. This study aimed to investigate the role of lncRNA SNHG9 in BC. The expression of SNHG9 in BC cells was found to be higher than that of human mammary epithelium. SNHG9 was found to inhibit the proliferation, migration, and invasion of cells and promote apoptosis. It was also found that SNHG9 regulates the miR-326/Wnt5a/β-catenin axis to promote the development of BC. Dual luciferase reporter and RNA pull-down assays confirmed the interaction between SNHG9, Wnt5a, and miR-326. Western blot analysis indicated that the expression of Wnt5a, β-catenin, c-myc, and cyclin D1 decreased significantly after the silencing of SNHG9 and the overexpression of miR-326. On the contrary, Wnt5a, β-catenin, c-myc, and cyclin D1 proteins were significantly up-regulated after inhibiting miR-326 expression. These findings suggest that SNHG9 is a promising target for BC therapy.

Ghrelin promotes cardiomyocyte differentiation of adipose tissue‑derived mesenchymal stem cells by DDX17‑mediated regulation of the SFRP4/Wnt/β‑catenin axis.

Adipose tissue‑derived mesenchymal stem cells (ADMSCs) differentiate into cardiomyocytes and may be an ideal cell source for myocardial regenerative medicine. Ghrelin is a gastric‑secreted peptide hormone involved in the multilineage differentiation of MSCs. To the best of our knowledge, however, the role and potential downstream regulatory mechanism of ghrelin in cardiomyocyte differentiation of ADMSCs is still unknown. The mRNA and protein levels were measured by reverse transcription‑quantitative PCR and western blotting. Immunofluorescence staining was used to show the expression and cellular localization of cardiomyocyte markers and β‑catenin. RNA sequencing was used to explore the differentially expressed genes (DEGs) that regulated by ghrelin. The present study found that ghrelin promoted cardiomyocyte differentiation of ADMSCs in a concentration‑dependent manner, as shown by increased levels of cardiomyocyte markers GATA binding protein 4, α‑myosin heavy chain (α‑MHC), ISL LIM homeobox 1, NK2 homeobox 5 and troponin T2, cardiac type. Ghrelin increased β‑catenin accumulation in nucleus and decreased the protein expression of secreted frizzled‑related protein 4 (SFRP4), an inhibitor of Wnt signaling. RNA sequencing was used to determine the DEGs regulated by ghrelin. Functional enrichment showed that DEGs were more enriched in cardiomyocyte differentiation‑associated terms and Wnt pathways. Dead‑box helicase 17 (DDX17), an upregulated DEG, showed enhanced mRNA and protein expression levels following ghrelin addition. Overexpression of DDX17 promoted protein expression of cardiac‑specific markers and β‑catenin and enhanced the fluorescence intensity of α‑MHC and β‑catenin. DDX17 upregulation inhibited protein expression of SFRP4. Rescue assay confirmed that the addition of SFRP4 partially reversed ghrelin‑enhanced protein levels of cardiac‑specific markers and the fluorescence intensity of α‑MHC. In conclusion, ghrelin promoted cardiomyocyte differentiation of ADMSCs by DDX17‑mediated regulation of the SFRP4/Wnt/β‑catenin axis.

Adipocytes-Derived Exosomal microRNA-1224 Inhibits M2 Macrophage Polarization in Obesity-Induced Adipose Tissue Inflammation via MSI2-Mediated Wnt/β-Catenin Axis.

Phenotypic switch of macrophage polarization in adipose tissue has been associated with obesity-induced adipose tissue inflammation (OATI). Therefore, this study aims to explore the possible mechanism of adipocytes-derived exosomes (ADEs) carrying microRNA-1224 (miR-1224) in M2 macrophage polarization of OATI. miR-1224-knockout (miR-1224-KO) mice for this study, and isolated primary adipocytes from high-fat diet (HFD) or normal diet (SD)-fed mice are developed. ADEs are extracted and cocultured with bone marrow-derived macrophages (BMDMs). The macrophagic crown-like structures (CLS) and M1 and M2 phenotype macrophages in epididymal white adipose tissue (epiWAT) are observed by immunohistochemistry and flow cytometry. The obtained data indicate that miR-1224 is highly expressed in adipose tissues and adipocytes of obese mice. miR-1224 knockout decreases CLS number and increases M2 macrophages polarization in epiWAT. In addition, miR-1224 can be transferred to BMDMs via ADEs, which targeted musashi RNA binding protein 2 (MSI2) expression and inactivated Wnt/β-catenin pathway, inhibiting macrophage M2 polarization and promoting inflammatory factor release. Exosomal miR-1224 derived by adipocytes targets MSI2 and blocks the Wnt/β-catenin pathway, which inhibits macrophage M2 polarization and promotes inflammatory factor release, ultimately promoting OATI.

EMX1 functions as a tumor inhibitor in spinal cord glioma through transcriptional suppression of WASF2 and inactivation of the Wnt/β-catenin axis.

BackgroundGliomas are the most frequent and aggressive cancers in the central nervous system, and spinal cord glioma (SCG) is a rare class of the gliomas. Empty spiracles homobox genes (EMXs) have shown potential tumor suppressing roles in glioma, but the biological function of EMX1 in SCG is unclear.MethodsThe EMX1 expression in clinical tissues of patients with SCG was examined. SCG cells were extracted from the tissues, and altered expression of EMX1 was then introduced to examine the role of EMX1 in cell growth and invasiveness in vitro. Xenograft tumors were induced in nude mice for in vivo validation. The targets of EXM1 were predicted via bioinformatic analysis and validated by luciferase and ChIP‐qPCR assays. Rescue experiments were conducted to validate the involvements of the downstream molecules.ResultsEMX1 was poorly expressed in glioma, which was linked to decreased survival rate of patients according to the bioinformatics prediction. In clinical tissues, EMX1 was poorly expressed in SCG, especially in the high‐grade tissues. EMX1 upregulation significantly suppressed growth and metastasis of SCG cells in vitro and in vivo. EMX1 bound to the promoter of WASP family member 2 (WASF2) to suppress its transcription. Restoration of WASF2 blocked the tumor‐suppressing effect of EMX1. EMX1 suppressed Wnt/β‐catenin signaling activity by inhibiting WASF2. Coronaridine, a Wnt/β‐catenin‐specific antagonist, blocked SCG cell growth and metastasis induced by WASF2.ConclusionThis study elucidates that EMX1 functions as a tumor inhibitor in SCG by suppressing WASF2‐dependent activation of the Wnt/β‐catenin axis.

Pyruvate Kinase M2: a novel regulator of adipogenesis

Obesity is the cornerstone of many other conditions, with far‐reaching impacts on numerous body systems and quality of life. Obesity is closely and likely causally associated with metabolic syndrome and consequent atherosclerosis, heart disease, stroke, and type 2 diabetes as well as diseases such as cancer and osteoarthritis. Recent studies suggest exploring the possibility of activating brown adipose tissue as a potential anti‐obesity strategy. However, understanding the mechanisms underlying the processes of brown adipogenesis and browning is foundational for effectively promoting this strategy as a primary, secondary, and/ or tertiary preventive measure against obesity. Additionally, recent studies have identified the glycolytic enzyme pyruvate kinase M2 (PKM2), as a novel regulator of white and brown adipogenesis. Yet, the molecular mechanisms remain to be elucidated. In the current study, we investigated the effects of PKM2 deficiency on white and brown fat adipogenesis using shRNA‐mediated lentiviral approach. We also investigated the underlying molecular mechanisms. Preliminary findings demonstrate that deletion of PKM2 enhances brown fat adipogenesis via modulation of the β‐catenin signaling pathway. Additionally, disruption of β‐catenin activity altered adipocytes differentiation and mimicked the effects of PKM2 deficiency on adipogenesis. Taken together, our study suggests that targeting PKM2/β‐catenin axis may serve as a strategy to promote brown fat adipogenesis, enhance whole body energy expenditure, and prevent obesity and its associated complications.

LncRNA LINC00284 promotes nucleus pulposus cell proliferation and ECM synthesis via regulation of the miR‑205‑3p/Wnt/β‑catenin axis.

Intervertebral disc degeneration (IDD) is a leading cause of degenerative spinal disease. Long non-coding RNA (lncRNA) LINC00284 is overexpressed in multiple types of cancer and promotes cancer cell proliferation and inhibits apoptosis; however, its role in human IDD and nucleus pulposus (NP) remain unclear. In the present study, intervertebral disc (IVD) tissues were collected from IDD patients for detection of LINC00284 expression using reverse transcription-quantitative PCR, the binding effect between miR-205-3p and LINC00284 was validated by dual-luciferase reporter assay. miR-205-3p and small interfering RNA (siRNA) was used for LINC00240 knockdown to investigate the proliferation, apoptosis of cells in the NP cells measured by Cell Counting Kit (CCK)-8 assay and Annexin V-FITC/Propidium Iodide (PI) staining with flow cytometry receptivity. IDD animal models were constructed for in vivo study of the role LINC00284 in IDD improvement. The results showed that LINC00284 expression was upregulated in IDD tissue and IL-1β-induced NP cells. LINC00284 knockdown resulted in an increase in IL-1β-induced NP cell proliferation, a decrease in apoptosis and matrix metalloproteinase-3 expression and an increase in expression of extracellular matrix (ECM) markers aggrecan and collagen II. In vivo experiments and histomorphometric analysis confirmed the protective effect of LINC00284 knockdown in IDD. LINC00284 was also shown to be a target of microRNA (miR)-205-3p, and there was a negative correlation between LINC00284 and miR-205-3p levels in IDD tissue. Additionally, LINC00284 knockdown or miR-205-3p upregulation resulted in inhibition of Wnt/β-catenin signaling and subsequent degradation of the ECM. The present study demonstrated that LINC00284 activated the Wnt/β-catenin signaling via sponging miR-205-3p, resulting in inhibition of NP cell proliferation and ECM synthesis. These results suggested that targeting LINC00284 to rescue miR-205-3p expression may be a potential method for IDD management.

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR-34c-5p/LGR4/β-catenin axis activity.

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real‐time PCR (qRT‐PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK‐8, wound‐healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual‐luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non‐HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR‐34c‐5p sponge to upregulate the expression of leucine‐rich repeat‐containing G protein‐coupled receptor 4 (LGR4), activating the β‐catenin pathway, and finally facilitating HCC progression. Additionally, a β‐catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour‐promoting role in HCC cells by regulating the miR‐34c‐5p/LGR4/β‐catenin axis, which may be a potential target for HCC therapy.

LncRNA metastasis-associated lung adenocarcinoma transcript-1 promotes osteogenic differentiation of bone marrow stem cells and inhibits osteoclastic differentiation ofMø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/β-catenin axis.

Osteoporosis is defined as a skeletal disorder characterized by impairment in bone strength. The potential application of lncRNAs as therapeutic targets for osteoporosis has been unveiled. This study investigated the regulatory mechanism of lncRNA MALAT1 in the differentiation of bone marrow stem cells (BMSCs) and macrophages (Mø) in osteoporosis. MALAT1 expression in peripheral blood of elderly osteoporosis patients and healthy volunteers was detected. BMSCs and mononuclear Mø were isolated and cultured. Osteogenic differentiation of BMSCs and osteoclastic differentiation of Mø were induced. BMSCs and Mø were transfected with si-MALAT1, miR-124-3p mimics, miR-124-3p inhibitor, or pcDNA IGF2BP1, followed by detection of cell differentiation. The target microRNAs (miRs) and downstream genes and signaling pathways of MALAT1 were examined. The ovariectomy-induced mouse model of osteoporosis was established, and the mice were injected with pcDNA-MALAT1. MALAT1 was downregulated in osteoporosis patients, increased in BMSCs after osteogenic differentiation, and diminished in Mø after osteoclastic differentiation. Downregulation of MALAT1 repressed osteogenic differentiation of BMSCs and facilitated osteoclastic differentiation of Mø. MALAT1 upregulated IGF2BP1 expression by competitively binding to miR-124-3p. miR-124-3p silencing reversed the effect of si-MALAT1 on BMSCs and Mø differentiation, and IGF2BP1 upregulation averted the effect of overexpressed-miR-124-3p by activating the Wnt/β-catenin pathway. Upregulation of MALAT1 activated the Wnt/β-catenin pathway and attenuated bone injury in mice. In conclusion, lncRNA MALAT1 promoted the osteogenic differentiation of BMSCs and inhibited osteoclastic differentiation of Mø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/β-catenin axis.

Down-regulated LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR-212-5p/FZD5/Wnt/β-catenin axis.

Prostate cancer is the second most frequent malignancy in men worldwide, and its incidence is increasing. Therefore, it is urgently required to clarify the underlying mechanisms of prostate cancer. Although the long non‐coding RNA LINC00115 was identified as an oncogene in several cancers, the expression and function of LINC00115 in prostate cancer have not been explored. Our results showed that LINC00115 was significantly up‐regulated in prostate cancer tissues, which was significantly associated with a poor prognosis for prostate cancer patients. Functional studies showed that knockdown LINC00115 inhibited cell proliferation and invasion. In addition, LINC00115 served as a competing endogenous RNA (ceRNA) through sponging miR‐212‐5p to release Frizzled Family Receptor 5 (FZD5) expression. The expression of miR‐212‐5p was noticeably low in tumour tissues, and FZD5 expression level was down‐regulated with the knockdown of LINC00115. Knockdown LINC00115 inhibited the Wnt/β‑catenin signalling pathway by inhibiting the expression of FZD5. Rescue experiments further showed that LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR‐212‐5p/ FZD5/ Wnt/β‐catenin axis. The present study provided clues that LINC00115 may be a promising novel therapeutic target for prostate cancer patients.

Salidroside inhibits chronic myeloid leukemia cell proliferation and induces apoptosis by regulating the miR-140-5p/wnt5a/β-catenin axis.

Salidroside, an active ingredient of Rhodiola rosea, exhibits antitumor effects in various types of cancer. However, the role of salidroside in chronic myeloid leukemia (CML) has not been elucidated. In the presents study, cell viability was assessed by CCK-8 assay, while apoptosis was detected by flow cytometry. Reverse transcription-quantitative PCR analysis was used to examine the expression levels of miR-140-5p in human CML cell lines. The expression levels of apoptosis and cell cycle-associated proteins and of the wnt5a/β-catenin signaling pathway were determined by western blot analysis. Bioinformatic analysis and luciferase reporter assays were employed to investigate the association between miR-140-5p and wnt5a. The results revealed that exposure of CML cells to salidroside (80 µM) inhibited cell proliferation and promoted apoptosis. In addition, salidroside treatment led to the upregulation of miR-140-5p expression. Furthermore, the inhibition of wnt5a/β-catenin signaling pathway and the pro-apoptotic effects induced by salidroside were attenuated by miR-140-5p silencing. Notably, wnt5a was revealed to be a direct target of miR-140-5p. The present findings indicated that salidroside exerted anti-CML effects through regulating miR-140-5p by suppressing the wnt5a/β-catenin signaling pathway. The present study provided evidence of the therapeutic role of salidroside in CML.

IGHG1 upregulation promoted gastric cancer malignancy via AKT/GSK-3\u03b2/\u03b2-Catenin pathway

BackgroundDespite current advances in gastric cancer treatment, disease metastasis and chemo-resistance remain as major hurdles against better overall prognosis. Previous studies indicated that IGHG1 as well as -Catenin serve as important regulators of tumor cellular malignancy. Therefore, understanding detailed molecular mechanism and identifying druggable target will be of great potentials in future therapeutic development.MethodsSurgical tissues and gastric cancer cell lines were retrieved to evaluate IGHG1 expression for patients with or without lymph node/distal organ metastasis. Functional assays including CCK8 assay, Edu assay, sphere formation assay and transwell assay, wound healing assay, etc. were subsequently performed to evaluate the impact of IGHG1/-catenin axis on tumor cell proliferation, migration and chemo-resistance.ResultsGastric cancer tissues and tumor cell lines demonstrated significantly higher level of IGHG1. Functional study further demonstrated that IGHG1 promoted proliferative and migration as well as chemo-resistance of gastric cancer tumor cells. Further experiments indicated that IGHG1 activated AKT/GSK-3/-Catenin axis, which played crucial role in regulation of proliferative and chemo-resistance of gastric cancer cells.ConclusionThis study provided novel evidences that IGHG1 acted as oncogene by promotion of gastric cancer cellular proliferation, migration and chemo-resistance. Our research further suggested that IGHG1/AKT/GSK-3β/β-Catenin axis acted as novel pathway which regulated gastric cancer cellular malignant behavior. Our research might inspire future therapy development to promote overall prognosis of gastric cancer patients.

MiR-221 regulates proliferation, invasion, apoptosis and progression of prostate cancer cells by modulating E-cadherin/Wnt/β catenin axis

MicroRNA-221 (miR-221) is aberrantly expressed in various tumours including prostate carcinoma and can act as either tumour suppressor or oncogene depending on the tumour system and stage. Epithelial-mesenchymal transition (EMT) is a plastic transition in tumour progression characterized by decreased expression of epithelial markers and increased expression of mesenchymal markers and has been known to play a critical role in promoting metastases. In this study, we have demonstrated the functional role of miR-221 in regulating epithelial-mesenchymal transition in prostate cancer. Expression of miR-221 was found to be downregulated in androgen-independent prostate cancer cells and was found to be inversely correlated with EMT. Ectopic expression of miR-221 potentially inhibited invasion and migration of androgen-independent prostate cancers cells and inhibition of miR-221 expression rescued the anti-invasive effects of miR-221. Flow cytometric analysis further revealed that miR-221 could induce cell cycle arrest with simultaneous induction of apoptosis. Additionally, transfection with miR-221 mimic upregulated negative regulators of Wnt/β catenin pathway such as GSK-3β thereby inhibiting it and downregulated mesenchymal markers viz., N-cadherin and Vimentin with simultaneous upregulation of epithelial marker E-cadherin as revealed by real-time PCR and Western blot analysis. A study using xenograft model further confirmed the anti-tumorigenic and anti-metastatic role of miR-221 in prostate cancer. We believe that his study is the first report documenting the miR-221- Wnt/β catenin signaling-EMT regulatory circuit in prostate cancer.

Chemerin located in bone marrow promotes osteogenic differentiation and bone formation via Akt/Gsk3β/β-catenin axis in mice.

Chemerin, a secreted protein mainly produced by adipocytes and hepatocytes, plays a variety of roles in endocrine or paracrine signaling. As reported in human epidemiology, chemerin was correlated with osteoporosis. And the previous in vitro study found that chemerin knockdown promoted osteogenesis and inhibited adipogenesis. However, the function of chemerin in bone metabolism and the underlying mechanism remains unclear. In this study, we uncovered the in vivo function of chemerin in bone homeostasis. We discovered that in obese mice, chemerin was increased in serum, while decreased in the bone marrow; and the chemerin expression in bone tissue was positively correlated with osteogenic genes. To further investigate the function of chemerin in bone metabolism, we generated chemerin deficiency and overexpression mice. We found bone mass and osteogenesis were decreased in chemerin deficiency mice, while were increased in chemerin overexpression mice. Furthermore, we observed that the chemerin expression increased during osteogenic differentiation of MSCs. Besides, we verified that chemerin promoted osteogenic differentiation in C3H10T1/2 cells and BMSCs through Akt/Gsk3β/β-catenin axis. Treatment with Akt inhibitor (MK2206) abolished the promoting effect of chemerin on osteogenic differentiation and active β-catenin. Together, our results suggest chemerin in bone marrow, not in serum, promotes osteogenic differentiation and bone formation via Akt/Gsk3β/β-catenin axis. Chemerin may serve as a therapeutic strategy for osteoporosis.

Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/β-catenin axis.

ObjectivesIt is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose‐derived stem cells (ADSCs) will be probably promoted by exosomes.Materials and MethodsBy means of lentiviral transfection, miR‐130a‐3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis‐related genes, to verify the effect of miR‐130a‐3p on the osteogenic differentiation of ADSCs. CCK‐8 was used to detect the effect of miR‐130a‐3p on the proliferation of ADSCs. The target binding between miR‐130a‐3p and SIRT7 was verified by dual‐luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR‐130a‐3p was further verified by detecting osteogenic‐related genes and proteins and alkaline phosphatase activity.Results(a) Overexpression of miR‐130a‐3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR‐130a‐3p. (b) Our study further found that overexpression of miR‐130a‐3p leads to down‐regulation of SIRT7 expression with up‐regulation of Wnt signalling pathway‐associated protein. (c) Overexpression of miR‐130a‐3p inhibited proliferation of ADSCs, while knockdown promoted it.ConclusionsThe obtained findings indicate that exosomal miR‐130a‐3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/β‐catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.

The role of miRNA-133b and its target gene SIRT1 in FAP-derived desmoid tumor.

Signaling pathways have a key role in driving the uncontrolled development of familial adenomatous polyposis (FAP)- associated and sporadic desmoid tumors (DTs).The relationship between the Wnt/b-catenin signaling pathway and DTs has been extensively studied, but no reliable biomarkers able to detect their histological subtype have been identified for the accurate diagnosis.In this study we studied the differences in miRNA expression between sporadic (20 patients) and FAP-associated DTs (7 patients) using microarray confirmed by quantitative PCR (qPCR). The analysis showed 19 dysregulated miRNAs. Among them miR-133b levels were significantly lower in FAP-associated DT than in sporadic DT. Therefore, two mRNAs, associated to miR-133b and β-catenin expression, the SIRT1 and ELAVL1were analyzed.The qPCR analysis showed that SIRT1 mRNA levels were significantly up-regulated in FAP-associated DT than in sporadic DT, whereas no differences in ELAVL1 expression was observed between these two DT types. In addition, a negative correlation was observed between miR-133b and SIRT1 in FAP-associated DTs, but not in sporadic DTs.The miR-133b-SIRT1-β-catenin axis may represent a novel mechanism underlying progression of FAP-associated DT.

Cross talk between RNA N6-methyladenosine methyltransferase-like 3 and miR-186 regulates hepatoblastoma progression through Wnt/β-catenin signalling pathway.

ObjectivesN6‐methyladenosine (m6A) is a ubiquitous epigenetic RNA modification that plays a pivotal role in tumour development and metastasis. In this study, we aimed to investigate the expression profiling, clinical significance, biological function and the regulation of m6A‐related genes in hepatoblastoma (HB).Materials and MethodsThe mRNA and protein expression levels of m6A‐related genes were analysed using Gene Expression Omnibus (GEO) and tissue microarray (TMA) cohort. Kaplan‐Meier analysis was performed to evaluate the prognostic value of m6A‐related genes in HB. Knockdown of m6A‐related genes was conducted to analyse its function on cell proliferation, migration and invasion. Furthermore, bioinformatics analysis and experimental verification were used to explore the potential molecular mechanism and signalling pathway.ResultsWe found that most m6A‐related genes were significantly upregulated in HB tumour tissues. High levels of methyltransferase‐like 3 (METTL3, P = .013), YTHDF2 (P = .037) and FTO (P = .032) indicated poor clinical outcomes, and the upregulation of METTL3 was an independent prognostic factor in HB patients. Functional assays showed that knockdown of METTL3 could dramatically suppress the proliferation, migration and invasion of HB cells. In addition, METTL3 was identified to be a direct target of microRNA‐186 (miR‐186). Consistently, miR‐186 was low expressed in HB tumour tissues. Moreover, overexpression of miR‐186 significantly inhibited cell aggressive phenotype both in vitro and in vivo, while the inhibitory effect could be reversed by METTL3 overexpression. Mechanism study indicated that miR‐186/METTL3 axis contributed to the progression of HB via the Wnt/β‐catenin signalling pathway.ConclusionsM6A‐related genes were frequently dysregulated in HB. miR‐186/METTL3/Wnt/β‐catenin axis might serve as novel therapeutic targets and prognostic biomarkers in HB.

LncRNA TUG1 contributes to ESCC progression via regulating miR-148a-3p/MCL-1/Wnt/β-catenin axis in vitro.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Latest studies report that long noncoding RNAs (LncRNAs) play an essential role in diversified pathological processes of ESCC, although the mechanism by which they do so remains unknown. This study aimed to explore the parts of lncRNA taurine upregulated gene 1 (TUG1) in ESCC tissues and cells, its biofunctional effect and its underlying regulatory mechanism in ESCC.MethodsThe levels of TUG1 and miR‐148a‐3p were detected by quantitative real‐time polymerase chain reaction (qRT‐PCR) in ESCC cells and tissues. The biofunctional effects were examined by MTT, flow cytometry, and transwell assay. The protein expression levels of epithelial‐mesenchymal transition (EMT)‐related proteins and MCL‐1 were determined by western blot analysis. The binding sites between miR‐148a‐3p and TUG1 or MCL‐1 were predicted by online software starBase and confirmed by dual luciferase reporter assay.ResultsThe mRNA expression of TUG1 was significantly upregulated in ESCC tissues or cells, and was negatively correlated to miR‐148a‐3p expression in tissues. Knockdown of TUG1 inhibited the proliferation, migration, and invasion, promoted apoptosis, and relieved the EMT progression in EC9706 and OE19 cells. Besides, knockdown of miR‐148a‐3p inverted positive effects from TUG1 deletion on ESCC cells. Besides, MCL‐1 reversed the inhibitive effects from TUG1 deletion on expression of EMT‐associated proteins (Wnt1, C‐myc, CyclinD1, and β‐catenin) above subsequently.ConclusionTUG1 regulated the biofunction and EMT progression of ESCC by mediating miR‐148a‐3p/MCL‐1/Wnt/β‐catenin axis in vitro.

MicroRNA-331-3p inhibits epithelial-mesenchymal transition by targeting ErbB2 and VAV2 through the Rac1/PAK1/β-catenin axis in non-small-cell lung cancer.

MicroRNAs have been reported to play critical roles in the regulation of non‐small‐cell cancer (NSCLC) development, but the role of microRNA (miR)‐331‐3p in NSCLC is still unclear. In this study, the expression levels of miR‐331‐3p in NSCLC tumor tissues and adjacent normal tissues were examined by quantitative RT‐PCR, and the relationship between miR‐331‐3p expression and patient clinicopathological characteristics was analyzed. The effects of miR‐331‐3p on epithelial‐mesenchymal transition (EMT), migration, and metastasis of NSCLC cells were determined in vitro and vivo. Direct functional targets of miR‐331‐3p were identified by luciferase reporter assay, western blot assay, immunohistochemical staining, and rescue assay. The downstream pathway regulated by miR‐331‐3p was identified by immunofluorescence, immunoprecipitation, and Rac1 activity examination. Our results showed that miR‐331‐3p was significantly downregulated in NSCLC tumor tissues and was correlated with clinicopathological characteristics, and miR‐331‐3p could be an independent prognostic marker for NSCLC patients. Furthermore, miR‐331‐3p significantly suppressed EMT, migration and metastasis of NSCLC cells in vitro and in vivo. Both ErbB2 and VAV2 were direct functional targets of miR‐331‐3p. The activities of Rac1, PAK1, and β‐catenin were regulated by miR‐331‐3p through ErbB2 and VAV2 targeting. These results indicated that miR‐331‐3p suppresses EMT, migratory capacity, and metastatic ability by targeting ErbB2 and VAV2 through the Rac1/PAK1/β‐catenin axis in NSCLC.

LncRNA MALAT1 Depressed Chemo-Sensitivity of NSCLC Cells through Directly Functioning on miR-197-3p/p120 Catenin Axis.

This study was aimed to explore if lncRNA MALAT1 would modify chemo-resistance of non-small cell lung cancer (NSCLC) cells by regulating miR-197-3p and p120 catenin (p120-ctn). Within this investigation, we totally recruited 326 lung cancer patients, and purchased 4 NSCLC cell lines of A549, H1299, SPC-A-1 and H460. Moreover, cisplatin, adriamycin, gefitinib and paclitaxel were arranged as chemotherapies, and half maximal inhibitory concentration (IC50) values were calculated to evaluate the chemo-resistance of the cells. Furthermore, mice models of NSCLC were also established to assess the impacts of MALAT1, miR-197-3p and p120-ctn on tumor growth. Our results indicated that MALAT1 and miR-197-3p were both over-expressed within NSCLC tissues and cells, when compared with normal tissues and cells (P < 0.05). The A549, H460, SPC-A-1 and SPC-A-1 displayed maximum resistances to cisplatin (IC50 = 15.70 μg/ml), adriamycin (IC50 = 5.58 μg/ml), gefitinib (96.82 μmol/L) and paclitaxel (141.97 nmol/L). Over-expression of MALAT1 and miR-197-3p, or under-expression of p120-ctn were associated with promoted viability and growth of the cancer cells (P < 0.05), and they could significantly strengthen the chemo-resistance of cancer cells (P < 0.05). MALAT1 Wt or p120-ctn Wt co-transfected with miR-197-3p mimic was observed with significantly reduced luciferase activity within NSCLC cells (P < 0.05). Finally, the NSCLC mice models were observed with larger tumor size and weight under circumstances of over-expressed MALAT1 and miR-197-3p, or under-expressed p120-ctn (P < 0.05). In conclusion, MALAT1 could alter chemo-resistance of NSCLC cells by targeting miR-197-3p and regulating p120-ctn expression, which might assist in improvement of chemo-therapies for NSCLC.

Diminished membrane recruitment of Akt is instrumental in alcohol-associated osteopenia via the PTEN/Akt/GSK-3β/β-catenin axis.

Alcohol is considered a leading risk factor for osteopenia. Our previous research indicated that the Akt/GSK-3β/β-catenin pathway plays a critical role in the ethanol-induced antiosteogenic effect in bone mesenchymal stem cells (BMSCs). PI3K/Akt is negatively regulated by the phosphatase and tensin homolog (PTEN) phosphatase. In this study, we found that ethanol increased PTEN expression in the BMSCs and bone tissue of ethanol-treated Sprague-Dawley rats. PTEN upregulation impaired Akt recruitment to the plasma membrane and suppressed Akt phosphorylation at Ser473, thereby inhibiting Akt/GSK3β/β-catenin signaling and the expression of COL1 and OCN in BMSCs in vitro and in vivo. The results of in vivo assays indicated that PTEN inhibition protected bone tissue against ethanol. Interestingly, our data revealed that following ethanol stimulation, PTEN and PTEN pseudogene 1 (PTENP1) mRNA expression was increased in a time-dependent manner, resulting in an increased PTEN protein level. In addition, ethanol upregulated PTEN expression and decreased PTEN phosphorylation (p-PTEN), indicating an increase in functional PTEN levels. In summary, the ethanol-mediated transcriptional and post-transcriptional regulation of PTEN impaired downstream Akt/GSK3β/β-catenin signaling and BMSC osteogenic differentiation. Therefore, we propose that Akt/GSK3β/β-catenin activation via PTEN inhibition may be a potential therapeutic approach for preventing the development of alcohol-induced osteopenia.