TCF4 and HuR mediated-METTL14 suppresses dissemination of colorectal cancer via N6-methyladenosine-dependent silencing of ARRDC4

Metastasis remains the major obstacle to improved survival for colorectal cancer (CRC) patients. Dysregulation of N6-methyladenosine (m6A) is causally associated with the development of metastasis through poorly understood mechanisms. Here, we report that METTL14, a key component of m6A methylation, is functionally related to the inhibition of ARRDC4/ZEB1 signaling and to the consequent suppression of CRC metastasis. We unveil METTL14-mediated m6A modification profile and identify ARRDC4 as a direct downstream target of METTL14. Knockdown of METTL14 significantly enhanced ARRDC4 mRNA stability relying on the “reader” protein YHTDF2 dependent manner. Moreover, we demonstrate that TCF4 can induce METTL14 protein expression, and HuR suppress METTL14 expression by directly binding to its promoter. Clinically, our results show that decreased METTL14 is correlated with poor prognosis and acts as an independent predictor of CRC survival. Collectively, our findings propose that METTL14 functions as a metastasis suppressor, and define a novel signaling axis of TCF4/HuR-METTL14-YHTDF2-ARRDC4-ZEB1 in CRC, which might be potential therapeutic targets for CRC.Subject terms: Cancer prevention, Post-translational modifications     

IGF2BP3 promotes cell metastasis and is associated with poor patient survival in nasopharyngeal carcinoma

Metastasis contributes to treatment failure in nasopharyngeal carcinoma (NPC) patients. Our study aimed at elucidating the role of insulin‐like growth factor 2 mRNA binding protein 3 (IGF2BP3) in NPC metastasis and the underlying mechanism involved. IGF2BP3 expression in NPC was determined by bioinformatics, quantitative polymerase chain reaction and immunohistochemistry analyses. The biological function of IGF2BP3 was investigated by using in vitro and in vivo studies. In this study, IGF2BP3 mRNA and protein levels were elevated in NPC tissues. In addition, IGF2BP3 exerted an oncogenic role by promoting epithelial‐mesenchymal transition (EMT), thereby inducing NPC cell migration and invasion. Further studies revealed that IGF2BP3 regulated the expression of key regulators of EMT by activating AKT/mTOR signalling, thus stimulating NPC cell migration and invasion. Remarkably, targeting IGF2BP3 delayed NPC metastasis through attenuating p‐AKT and vimentin expression and inducing E‐cadherin expression in vivo. Moreover, IGF2BP3 protein levels positively correlated with distant metastasis after initial treatment. Importantly, IGF2BP3 expression served as an independent prognostic factor in predicting the overall survival and distant metastasis‐free survival of NPC patients. This work identifies IGF2BP3 as a novel prognostic marker and a new target for NPC treatment.     

Methyltransferase like 3 promotes colorectal cancer proliferation by stabilizing CCNE1 mRNA in an m6A-dependent manner

m6A modification is the most prevalent RNA modification in eukaryotes. As the critical N6-methyladenosine (m6A) methyltransferase, the roles of methyltransferase like 3 (METTL3) in colorectal cancer (CRC) are controversial. Here, we confirmed that METTL3, a critical m6A methyltransferase, could facilitate CRC progression in vitro and in vivo. Further, we found METTL3 promoted CRC cell proliferation by methylating the m6A site in 3′-untranslated region (UTR) of CCNE1 mRNA to stabilize it. Moreover, we found butyrate, a classical intestinal microbial metabolite, could down-regulate the expression of METTL3 and related cyclin E1 to inhibit CRC development. METTL3 promotes CRC proliferation by stabilizing CCNE1 mRNA in an m6A-dependent manner, representing a promising therapeutic strategy for the treatment of CRC.     

METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial–mesenchymal transition of renal tubular cells in diabetic kidney disease

Histone deacetylase 5 (HDAC5) belongs to class II HDAC subfamily and is reported to be increased in the kidneys of diabetic patients and animals. However, little is known about its function and the exact mechanism in diabetic kidney disease (DKD). Here, we found that HDAC5 was located in renal glomeruli and tubular cells, and significantly upregulated in diabetic mice and UUO mice, especially in renal tubular cells and interstitium. Knockdown of HDAC5 ameliorated high glucose-induced epithelial–mesenchymal transition (EMT) of HK2 cells, indicated in the increased E-cadherin and decreased α-SMA, via the downregulation of TGF-β1. Furthermore, HDAC5 expression was regulated by PI3K/Akt signaling pathway and inhibition of PI3K/Akt pathway by LY294002 treatment or Akt phosphorylation mutation reduced HDAC5 and TGF-β1 expression in vitro high glucose-cultured HK2 cells. Again, high glucose stimulation downregulated total m6A RNA methylation level of HK2 cells. Then, m6A demethylase inhibitor MA2 treatment decreased Akt phosphorylation, HDAC5, and TGF-β1 expression in high glucose-cultured HK2 cells. In addition, m6A modification-associated methylase METTL3 and METTL14 were decreased by high glucose at the levels of mRNA and protein. METTL14 not METTL3 overexpression led to PI3K/Akt pathway inactivation in high glucose-treated HK2 cells by enhancing PTEN, followed by HDAC5 and TGF-β1 expression downregulation. Finally, in vivo HDACs inhibitor TSA treatment alleviated extracellular matrix accumulation in kidneys of diabetic mice, accompanied with HDAC5, TGF-β1, and α-SMA expression downregulation. These above data suggest that METTL14-regulated PI3K/Akt signaling pathway via PTEN affected HDAC5-mediated EMT of renal tubular cells in diabetic kidney disease.Subject terms: Epithelial-mesenchymal transition, Insulin signalling, Diabetes complications     

LncRNA DGUOK-AS1 facilitates non-small cell lung cancer growth and metastasis through increasing TRPM7 stability via m6A modification

BackgroundN6-methyladenosine (m6A) modification plays key roles in tumor progression. LncRNA deoxyguanosine kinase antisense RNA 1 (DGUOK-AS1) has been reported as a promoter in tumors, but its role and mechanism in non-small cell lung cancer (NSCLC) development remain uncertain.MethodsCell proliferation, migration, invasion and angiogenesis were investigated via CCK-8, colony formation, transwell, and tube formation assays, respectively. The location of DGUOK-AS1 was detected via FISH assay. The interaction relationship among DGUOK-AS1, IGF2BP2 and TRPM7 was confirmed by RIP and MeRIP assays. The effects of DGUOK-AS1 on NSCLC growth and metastasis in vivo were investigated using xenograft and pulmonary metastatic models.ResultsDGUOK-AS1 was upregulated in NSCLC. DGUOK-AS1 silencing inhibited NSCLC cell proliferation, migration, invasion and angiogenesis. DGUOK-AS1 was mostly expressed in cytoplasm, and positively regulated IGF2BP2. METTL3/IGF2BP2 axis could increase TRPM7 mRNA stability in m6A-dependent manner. TRPM7 overexpression reversed the inhibitive function of DGUOK-AS1 silencing on NSCLC development. DGUOK-AS1 knockdown suppressed NSCLC cell growth and metastasis in nude mice.ConclusionDGUOK-AS1 silencing restrains NSCLC cell growth and metastasis through decreasing TRPM7 stability via regulation of the METTL3/IGF2BP2-mediated m6A modification.     

PDGF-BB-induced MT1-MMP expression regulates proliferation and invasion of mesenchymal stem cells in 3-dimensional collagen via MEK/ERK1/2 and PI3K/AKT signaling

Mesenchymal stem cells (MSCs) mobilize membrane type-1 matrix metalloproteinase (MT1-MMP) to traffic through both 3-dimensional (3D) collagen as well as basement membrane barriers, but factors capable of regulating the expression and activity of the protease remain unidentified. Herein, we report that the MT1-MMP-dependent invasive activities of rat MSCs are controlled by PDGF-BB. Furthermore, PDGF-BB also stimulates MSC proliferation in 3D type I collagen via an MT1-MMP-dependent process that is linked to pericellular collagen degradation. PDGF-BB stimulates MT1-MMP expression at both the mRNA and protein levels in concert with ERK1/2 and PI3K/AKT activation. Inhibition of ERK1/2 or PI3K/AKT activity potently suppresses both MT1-MMP-dependent invasive and proliferative activities. Basement membrane invasion is likewise stimulated by PDGF-BB in an MT1-MMP-dependent manner via ERK1/2 and PI3K/AKT signaling. Taken together, these data serve to identify PDGF-BB as an important MSC agonist that controls invasive and proliferative activities via MT1-MMP-dependent processes that are regulated by the ERK1/2 and PI3K/AKT signaling pathways.     

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.     

METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.     

KRASG12D- and BRAFV600E-Induced Transformation of Murine Pancreatic Epithelial Cells Requires MEK/ERK-Stimulated IGF1R Signaling

Mutation of KRAS is a common initiating event in pancreatic ductal adenocarcinoma (PDAC). Yet, the specific roles of KRAS-stimulated signaling pathways in the transformation of pancreatic ductal epithelial cells (PDEC), putative cells of origin for PDAC, remain unclear. Here, we show that KRAS(G12D) and BRAF(V600E) enhance PDEC proliferation and increase survival after exposure to apoptotic stimuli in a manner dependent on MEK/ERK and PI3K/AKT signaling. Interestingly, we find that activation of PI3K/AKT signaling occurs downstream of MAP-ERK kinase (MEK), and is dependent on the autocrine activation of the insulin-like growth factor (IGF) receptor (IGF1R) by IGF2. Importantly, IGF1R inhibition impairs KRAS(G12D)- and BRAF(V600E)-induced survival, whereas ectopic IGF2 expression rescues KRAS(G12D)- and BRAF(V600E)-mediated survival downstream of MEK inhibition. Moreover, we show that KRAS(G12D)- and BRAF(V600E)-induced tumor formation in an orthotopic model requires IGF1R. Interestingly, we show that while individual inhibition of MEK or IGF1R does not sensitize PDAC cells to apoptosis, their concomitant inhibition reduces survival. Our findings identify a novel mechanism of PI3K/AKT activation downstream of activated KRAS, illustrate the importance of MEK/ERK, PI3K/AKT, and IGF1R signaling in pancreatic tumor initiation, and suggest potential therapeutic strategies for this malignancy. Mol Cancer Res; 10(9); 1228-39. ?2012 AACR.     

METTL3-mediated m6A modification of STEAP2 mRNA inhibits papillary thyroid cancer progress by blocking the Hedgehog signaling pathway and epithelial-to-mesenchymal transition

Papillary thyroid cancer (PTC) is a common endocrine system malignancy all over the world. Aberrant expression of six transmembrane epithelial antigen of the prostate 2 (STEAP2) has been functionally associated with cancer progression in many cancers. Nevertheless, its biological function in PTC is still unclear. Here, we found that PTC tissues had preferentially downregulated STEAP2 as compared with noncancerous tissues. Low STEAP2 expression correlated with aggressive clinicopathological characteristics and dismal prognosis in patients with PTC. We performed gain- and loss-of-function experiments, including cell proliferation assay (Cell Counting Kit-8 assay), EdU (5-ethynyl-2′-deoxyuridine) and colony formation assays, transwell migration, and invasion assays, and constructed a nude mouse xenograft tumor model. The results demonstrated that STEAP2 overexpression inhibited PTC cell proliferation, migration, and invasion in vitro and inhibited lung metastasis and tumorigenicity in vivo. Conversely, silencing STEAP2 yielded the opposite results in vitro. Mechanistically, bioinformatics analysis combined with validation experiments identified STEAP2 as the downstream target of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification. METTL3 stabilized STEAP2 mRNA and regulated STEAP2 expression positively in an m6A-dependent manner. We also showed that m6A-mediated STEAP2 mRNA translation initiation relied on a pathway dependent on the m6A reader protein YTHDF1. Rescue experiments revealed that silencing STEAP2 partially rescued the tumor-suppressive phenotype induced by METTL3 overexpression. Lastly, we verified that the METTL3–STEAP2 axis functions as an inhibitor in PTC by suppressing epithelial–mesenchymal transition and the Hedgehog signaling pathway. Taken together, these findings strongly suggest that METTL3-mediated STEAP2 m6A modification plays a critical tumor-suppressive role in PTC progression. The METTL3–STEAP2 axis may be a potential therapeutic molecular target against PTC.Subject terms: Metastasis, Prognostic markers     

Activated cMET and IGF1R-Driven PI3K Signaling Predicts Poor Survival in Colorectal Cancers Independent of KRAS Mutational Status

Background

Oncogenic mutational analysis provides predictive guidance for therapeutics such as anti-EGFR antibodies, but it is successful only for a subset of colorectal cancer (CRC) patients.

Method

A comprehensive molecular profiling of 120 CRC patients, including 116 primary, 15 liver metastasis, and 1 peritoneal seeding tissue samples was performed to identify the relationship between v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) WT and mutant CRC tumors and clinical outcomes. This included determination of the protein activation patterns of human epidermal receptor 1 (HER1), HER2, HER3, c-MET, insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositide 3-kinase (PI3K), Src homology 2 domain containing (Shc), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) kinases using multiplexed collaborative enzyme enhanced reactive (CEER) immunoassay.

Results

KRAS WT and mutated CRCs were not different with respect to the expression of the various signaling molecules. Poor prognosis in terms of early relapse (<2 years) and shorter disease-free survival (DFS) correlated with enhanced activation of PI3K signaling relative to the HER kinase pathway signaling, but not with the KRAS mutational status. KRAS WT CRCs were identified as a mixed prognosis population depending on their level of PI3K signaling. KRAS WT CRCs with high HER1/c-MET index ratio demonstrated a better DFS post-surgery. c-MET and IGF1R activities relative to HER axis activity were considerably higher in early relapse CRCs, suggesting a role for these alternative receptor tyrosine kinases (RTKs) in driving high PI3K signaling.

Conclusions

The presented data subclassified CRCs based on their activated signaling pathways and identify a role for c-MET and IGF1R-driven PI3K signaling in CRCs, which is superior to KRAS mutational tests alone. The results from this study can be utilized to identify aggressive CRCs, explain failure of currently approved therapeutics in specific CRC subsets, and, most importantly, generate hypotheses for pathway-guided therapeutic strategies that can be tested clinically.     

Increased IGF2BP3 expression promotes the aggressive phenotypes of colorectal cancer cells in vitro and vivo

Previous literatures reported insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a poor prognostic marker for colorectal cancer (CRC) patients. However, basic research on the effect and biological role of IGF2BP3 in CRC was still scare. Real-time quantitative polymerase chain reaction and western blot analysis were used to examine IGF2BP3 expression level in tumors and paired normal tissues from CRC patients. Tissue microarrays with 192 CRC patients were subjected to immunohistochemical staining to analyze the prognostic value of IGF2BP3. Proliferation assays, migration assays, and xenograft tumor formation in nude mice were performed to assess the biological role of IGF2BP3 in CRC cells. IGF2BP3 expression was significantly upregulated in tumor tissues compared with the matched normal tissues both in messenger RNA and protein level and was associated with worse prognosis. IGF2BP3 knockdown made cell cycle arrest to impair the proliferation ability of CRC cells and further inhibited the xenograft tumor growth in nude mice, also inhibited the migration ability of CRC cells via inducing epithelial–mesenchymal transition. Therefore, the research demonstrated that increased IGF2BP3 expression promoted the aggressive phenotypes of CRC cells. Targeted IGF2BP3 could be a novel and effective gene therapy for CRC patients to make a better prognosis.     

IGF2BP3-EGFR-AKT axis promotes breast cancer MDA-MB-231 cell growth

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an emerging prognostic indicator, and its elevated expression correlates with malignancy in a broad spectrum of cancers. However, its regulatory networks have not yet been reported. In this study, we identified the regulatory targets of IGF2BP3 in breast cancer MDA-MB-231 cells using RNA immunoprecipitation sequencing (RIP-seq) and high-throughput RNA-sequencing (RNA-seq). We discovered that these targets were enriched in the inflammatory response, endoplasmic reticulum stress, cell cycle, and cancer-related pathways, providing a new perspective for better understanding the functional mechanisms of IGF2BP3. Moreover, we identified that the epidermal growth factor receptor (EGFR), a downstream target, is regulated by IGF2BP3. IGF2BP3 binds to and protects EGFR mRNA from degradation and facilitates cell proliferation via the EGFR/AKT pathway in MDA-MB-231 cells. In addition, IGF2BP3 expression was robust and could not be altered by stimulation with EGF and anti-EGFR siRNA or EGFR signaling pathway inhibitors (gefitinib, LY294002 and SL-327). These results demonstrate that IGF2BP3, as a stubborn oncogene, promotes triple-negative breast cancer MDA-MB-231 cell proliferation by strengthening the role of the EGFR-AKT axis.     

Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation,Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways

Background

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126.

Methods

The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay.

Results

We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein.

Conclusions

MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.     

Positive regulation of TNFR1 signaling via SH3 recognition motif

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the “death domain”, it also activates IKKα/β, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.     

BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.     

A positive feedback loop of lncRNA-RMRP/ZNRF3 axis and Wnt/β-catenin signaling regulates the progression and temozolomide resistance in glioma

Drug resistance strikingly limits the therapeutic effect of temozolomide (TMZ) (a common drug for glioma). Long non-coding RNA (lncRNA) RMRP has been found to be implicated in glioma progression. However, the effect of RMRP on TMZ resistance along with related molecular mechanisms is poorly defined in glioma. In the present study, RMRP, ZNRF3, and IGF2BP3 were screened out by bioinformatics analysis. The expression levels of lncRNAs and mRNAs were measured by RT-qPCR assay. Protein levels of genes were detected by western blot and immunofluorescence assays. ZNRF3 mRNA stability was analyzed using Actinomycin D assay. Cell proliferative ability and survival rate were determined by CCK-8 assay. Cell apoptotic pattern was estimated by flow cytometry. The effect of RMRP knockdown on the growth of TMZ-treated glioma xenograft tumors was explored in vivo. The relationships of IGF2BP3, RMRP, and ZNRF3 were explored by bioinformatics prediction analysis, RNA immunoprecipitation, luciferase, and RNA pull-down, and chromatin immunoprecipitation assays. The results showed that RMRP was highly expressed in glioma. RMRP knockdown curbed cell proliferation, facilitated cell apoptosis and reduced TMZ resistance in glioma cells, and hindered the growth of TMZ-treated glioma xenograft tumors. RMRP exerted its functions by down-regulating ZNRF3 in glioma cells. IGF2BP3 interacted with RMRP and ZNRF3 mRNA. IGF2BP3 knockdown weakened the interaction of Argonaute 2 (Ago2) and ZNRF3. RMRP reduced ZNRF3 expression and mRNA stability by IGF2BP3. RMRP knockdown inhibited β-catenin expression by up-regulating ZNRF3. The inhibition of Wnt/β-catenin signaling pathway by XAV-939 weakened RMRP-mediated TMZ resistance in glioma cells. β-catenin promoted RMRP expression by TCF4 in glioma cells. In conclusion, RMRP/ZNRF3 axis and Wnt/β-catenin signaling formed a positive feedback loop to regulate TMZ resistance in glioma. The sustained activation of Wnt/β-catenin signaling by RMRP might contribute to the better management of cancers.Subject terms: Drug regulation, Long non-coding RNAs