D1 Protein Levels Research Articles

MiR‑483‑3p promotes the osteogenesis of human osteoblasts bytargeting Dikkopf 2 (DKK2) and the Wnt signaling pathway.

Osteoporosis is a systemic metabolic bone disease during which bone mass decreases and bone quality is reduced. Maintaining the bone formation capacity of osteoblasts is crucial for the treatment of osteoporosis. In the present study, bioinformatics analysis was performed on online microarray expression profiles to identify miRNA(s) related to osteoblast proliferation and bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation. The specific effects of candidate miRNAs on cell proliferation, osteogenic differentiation and Wnt signaling-related factors were examined. As regards the downstream mechanisms, online tools were employed to predict the downstream targets of candidate miRNAs and the predicted miRNA-mRNA binding was verified. Finally, the dynamic effects of miRNAs and mRNAs were examined. The results revealed that miR-483-3p expression was decreased in bone tissue samples from patients with osteoporosis. In miR-483-3p-overexpressing human osteoblasts, cell viability, DNA synthesis capacity and osteogenesis were promoted, and the protein levels of Wnt1, β-catenin and cyclin D1 were increased. However, the protein receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio and cell apoptotic rate were decreased. The Wnt signaling, antagonist Dikkopf 2 (DKK2), was targeted and negatively regulated by miR-483-3p. DKK2 knockdown exerted similar effects as miR-483-3p overexpression, while DKK2 overexpression inhibited cell viability, DNA synthesis capacity and osteogenesis. DKK2 overexpression also decreased the Wnt1, β-catenin, and cyclin D1 protein levels, whereas it promoted the the RANKL/OPG ratio and the apoptosis of human osteoblasts. DKK2 overexpression reversed the functions of miR-483-3p overexpression. On the whole, the findings of the present study demonstrate that the miR-483-3p/DKK2 axis modulates the bone formation process by affecting osteoblast proliferation, pre-osteoblast differentiation into mature osteoblasts and new bone matrix formation.

Carboplatin Inhibits the Progression of Retinoblastoma Through IncRNA XIST/miR-200a-3p/NRP1 Axis.

ObjectiveThis study was set out to explore the expression and related mechanism of XIST and miR-200a-3p in retinoblastoma (Rb).Patients and MethodsFifty-four children with Rb who came to our hospital for surgery from January 2018 to September 2019 were collected. In addition, Rb cells and human retinal epithelial cells were purchased. XIST-siRNA (si-XIST), XIST-shRNA (sh-XIST), empty vector plasmid (siRNA-NC), miR-200a-3p-mimics and miR −200a-3p-inhibition were transfected into Y79 cells. The expression of XIST and miR-200a-3p in the samples were determined by qRT-PCR. β-catenin, cyclin B1, cyclin D1, Bax, Caspase-3, N-cadherin, vimentin, Snail, E-Cadherin and ZO-1 protein levels were measured by WB. MTT, Transwell and flow cytometry were utilized to detect cell proliferation, invasion, and apoptosis, respectively.ResultsXIST was highly expressed while miR-200a-3p was lowly expressed in patients’ tissues, and the AUC of both was over 0.8. XIST and miR-200a-3p was related to differentiation degree in Rb patients. Y79 cells were selected for transfection. Compared with the siRNA-NC group, XIST was significantly reduced in the siRNA-XIST group, and it was significantly increased in the shRNA-XIST group (P<0.01). The proliferation capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated. The apoptosis rate of siRNA-XIST group was significantly up-regulated, while that of shRNA-XIST group was decreased (P<0.001). The invasive capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated (P<0.001). Silencing XIST and over-expressed miR-200a-3p could inhibit cell epithelial–mesenchymal transition (EMT), proliferation, invasion, and promote apoptosis. WB detection showed that Carboplatin + LncRNA XIST intervention group could more significantly inhibit β-catenin, cyclin B1, cyclin D1, N-cadherin, vimentin, Snail protein, and promote the up-regulation of Bax, Caspase-3, E-Cadherin and ZO-1 expression.ConclusionInhibition of XIST expression can up-regulate miR-200a-3p-mediated PI3K-Akt/MAPK-ERK signaling pathway and affect cell EMT, proliferation, invasion, and apoptosis, which is expected to be a potential therapeutic target for Rb.

Periostin inhibits hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts via p38 MAPK signaling pathway

To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved. In vitro cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low (25 ng/mL), moderate (50 ng/mL) or high (100 ng/mL) doses. MTT assay was used to measure the cell viability, and the cell apoptosis rate was determined using flow cytometry. The contents of IL-1β, IL-6 and TNF-α in the cells were determined with ELISA, and ROS levels were measured using a fluorescent plate reader. The intracellular SOD activity was detected using ELISA. The expressions of HIF-1α, P21, cyclin D1, Bax, cleaved caspase-3, Bcl-2, P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting. Hypoxia treatment significantly reduced the cell viability (P &lt; 0.05), increased P21, Bax, and cleaved caspase-3 protein levels (P &lt; 0.05), promoted cell apoptosis (P &lt; 0.05), and decreased cyclin D1 and Bcl-2 protein levels (P &lt; 0.05) in the cells. Compared with the hypoxic group, the cells treated with periostin at different concentrations showed significantly increased cell viability (P &lt; 0.05) with significantly lowered apoptotic rates (P &lt; 0.05) and decreased expression levels of Bax and cleaved caspase-3 (P &lt; 0.05) but significantly increased expression levels of cyclin D1 and Bcl-2 (P &lt; 0.05). Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1α and p-p38 MAPK (P &lt; 0.05) and increased levels of IL-1β, IL-6, TNF-α and ROS (P &lt; 0.05) but decreased SOD activity (P &lt; 0.05). Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1α and p-p38 MAPK (P &lt; 0.05) and the levels of IL-1β, IL-6, TNF-α and ROS (P &lt; 0.05) and significantly increased SOD activity in the hypoxic cells (P &lt; 0.05). Periostin promotes the proliferation, inhibits apoptosis, enhances cellular antioxidant capacity, and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.

Inhibition of ERKs/Akt-Mediated c-Fos Expression Is Required for Piperlongumine-Induced Cyclin D1 Downregulation and Tumor Suppression in Colorectal Cancer Cells.

BackgroundDeregulation of Cyclin D1 and cell cycle progression plays a critical role in tumorigenesis. The natural compound piperlongumine (PL) exhibits potential anticancer effects in various cancer models, but the underlying mechanism needs further elucidation.MethodsThe inhibitory effect of PL on colorectal cancer (CRC) cells was determined by anchorage-dependent and -independent assays. The protein level of Cyclin D1 was examined by immunoblot (IB) and immunohistochemical staining (IHC). The mRNA level was determined by qRT-PCR. Phosphorylation of histone H3 was analyzed by immunofluorescence (IF). The cell cycle was examined by flow cytometry. The in vivo antitumor effect was validated by the xenograft mouse model.ResultsCyclin D1 was overexpressed in CRC tissues and cells, and was required for maintaining cell growth, colony formation, and in vivo tumorigenesis. PL decreased the protein level of c-Fos, which eventually reduced the transcriptional activity of AP-1 and the mRNA level of Cyclin D1. Mechanism study showed that PL impaired EGF-induced activation of ERK1/2 and Akt signalings, which resulted in a reduction of c-Fos transcription. Furthermore, PL reduced the half-life of c-Fos and caused the ubiquitination-dependent degradation of c-Fos. Finally, the in vivo antitumor effect of PL on CRC cells was examined using a xenograft mouse model.ConclusionOur data indicate that PL is a promising antitumor agent that deserves further study for CRC treatment.

Uncovering the Modulatory Interactions of Brain Networks in Cognition with Central Thalamic Deep Brain Stimulation Using Functional Magnetic Resonance Imaging

Deep brain stimulation (DBS) is a promising treatment for neurological and psychiatric disorders. It acts by altering brain networks and facilitating synaptic plasticity. For enhancing cognitive functions, the central thalamus (CT) has been shown to be a potential DBS target. The network-level mechanisms contributing to the effect exerted by DBS on the CT (CT-DBS) remain unknown. Combining CT-DBS with functional magnetic resonance imaging (fMRI), this study explored brain areas activated while applying CT-DBS in rats, using a newly developed neural probe that was compatible with MRI and could minimize the image distortion and resolve safety issues. Results showed activation of the anterior cingulate cortex, motor cortex, primary and secondary somatosensory cortices, caudate putamen, hypothalamus, thalamus, and hippocampus, suggesting that the corticostriatal, corticolimbic, and thalamocortical brain networks were affected. Behaviorally, the CT-DBS group required a shorter time than sham controls to learn a water-reward lever-pressing task and made more correct choices in a T-maze task. Concurrent with enhanced learning performance, bilateral CT-DBS resulted in alteration in the functional connectivity of brain networks determined by resting-state fMRI. Western blot analyses showed that the protein level of both dopamine D1 and α4-nicotinic acetylcholine receptors was increased, and dopamine D2 receptor was decreased. These data suggest that CT-DBS can enhance cognitive performance as well as brain connectivity through the modulation of synaptic plasticity, such that CT is a target providing high potential for the remediation of acquired cognitive learning and memory disabilities.

Comprehensive analysis of the HOXA gene family identifies HOXA13 as a novel oncogenic gene in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is one of the most common lethal cancers in the human urogenital system. As members of the Homeobox (HOX) family, Homeobox-A (HOXA) cluster genes have been reported to be involved in the development of many cancer types. However, the expression and clinical significance of HOXA genes in KIRC remain largely unknown. In this study, we comprehensively analyzed the mRNA expression and prognostic values of HOXA genes in KIRC using The Cancer Genome Atlas (TCGA) analysis databases online. Colony formation assay, flow cytometry and Western blot were used to detect cell proliferation, apoptosis, cell cycle, and protein level of the indicated gene. We found that the HOXA genes were differentially expressed in KIRC tissues when compared with normal tissues. The expression of HOXA4 and HOXA13 were significantly up-regulated, while HOXA7 and HOXA11 were down-regulated in KIRC. High mRNA levels of HOXA2, HOXA3 and HOXA13, and low level of HOXA7 predicted poor overall survival (OS) of KIRC patients. High mRNA level of HOXA13 further indicated a poor disease-free survival (DFS) of KIRC patients. Functionally, knockdown of HOXA13 significantly suppressed cell proliferation of KIRC in vitro, increased the protein level of p53 and decreased the protein level of cyclin D1 in KIRC cells. Over-expression of HOXA13 had the opposite effects on KIRC cells. Collectively, our findings suggest that HOXA13 functions as a novel oncogene in KIRC and may be a potential biomarker for this malignancy.

Integrated Bioinformatics Analysis of the Clinical Value and Biological Function of ATAD2 in Hepatocellular Carcinoma.

ATPase family AAA domain-containing protein 2 (ATAD2), a chromatin regulator and an oncogenic transcription cofactor, is frequently overexpressed in many cancers, particularly in hepatocellular carcinoma (HCC). By integrating open-access online mRNA datasets and our institutional tissue data on HCC, the clinical role and functions of ATAD2 were analyzed by bioinformatic algorithms. We systematically examined ATAD2 expression in HCC based on a large sample population, integrating data from our institution and the GEO, Oncomine, and TCGA datasets. Aberrant ATAD2 expression related to pathways was identified by bioinformatic algorithms. The effects of ATAD2 downregulation on the cycle cell were also determined. A pooled analysis from 28 datasets indicated that ATAD2 overexpression was found in HCC (SMD = 8.88, 95% CI: 5.96–11.81, P < 0.001) and was correlated with poor survival. Subgroup analysis of Asian patients with a serum alpha-fetoprotein (AFP) concentration < 200 ng/ml in stage I + II showed that the ATAD2-high group had a more unfavorable overall survival (OS) rate than the ATAD2-low group. The receiver operating characteristic curve indicated that the efficiency of ATAD2 for HCC diagnosis was considerable (area under the curve = 0.89, 95% CI: 0.86–0.91). Functional analysis based on bioinformatic algorithms demonstrated that ATAD2 participates in cell division, mitotic nuclear division, DNA replication, repair, and cell cycle processes. ATAD2 knockout in HCC cells downregulated cyclin C and cyclin D1 protein levels and resulted in G1/S phase arrest in vitro. The kinesin family member C1 (KIFC1), shugoshin 1 (SGO1), GINS complex subunit 1 (GINS1), and TPX2 microtubule nucleation factor (TPX2) genes were closely related to ATAD2 upregulation. ATAD2 may interact with TTK protein kinase (TTK) to accelerate HCC carcinogenesis. ATAD2 plays a vital role in HCC carcinogenesis by disturbing the interaction between chromatin proteins and DNA. Targeting ATAD2 represents a promising method for the development of therapeutic treatments for cancer.

Long Non-Coding RNA TTN-AS1 Serves as a Competing Endogenous RNA of miR-195 to Facilitate Clear Cell Renal Cell Carcinoma Progression.

IntroductionClear cell renal cell carcinoma (ccRCC) is an aggressive human malignancy. Long non-coding RNAs (lncRNAs) are critical regulators in many malignant tumors, including ccRCC. The aim of this study is to investigate the expression, functions and molecular mechanisms of lncRNA TTN-AS1 in ccRCC.MethodsA total of 145 paired cancer and normal tissues were collected from patients with ccRCC. The expression levels of TTN-AS1 and miR-195 in the tissues or cells were measured by RT-qPCR analysis. The expression levels of cyclin D1 protein were measured by Western blot analysis. Cell proliferation and cell cycle distribution were detected by MTT assay and flow cytometer analysis, respectively. The binding relationship between miR-195 and TTN-AS1 or cyclin D1 mRNA was validated by dual-luciferase reporter assay.ResultsOur results revealed that TTN-AS1 expression levels in human ccRCC tissues and cell lines were markedly increased. High expression of TTN-AS1 was closely associated with adverse clinicopathological characteristics of ccRCC patients. Gain- and loss-of-function experiments showed that TTN-AS1 overexpression promoted the proliferation and cell cycle transition of ccRCC cells, while the malignant traits were obviously suppressed after TTN-AS1 knockdown. Mechanistically, miR-195 was found to bind with and to be negatively regulated by TTN-AS1 in ccRCC cells. Further, we showed that cyclin D1 is a direct target of miR-195 in ccRCC, and rescue assays verified that restoration of miR-195 expression partially blocked the oncogenic effects of TTN-AS1 in ccRCC cells.ConclusionOur study provides a novel mechanism of TTN-AS1/miR-195/cyclin D1 regulatory axis in ccRCC, which may become a breakthrough for ccRCC therapy in the future.

Evodiamine Exerts Anticancer Effects Against 143B and MG63 Cells Through the Wnt/β-Catenin Signaling Pathway.

BackgroundOsteosarcoma is the most common primary malignant bone neoplasm and is associated with abysmal prognosis. There are limitations of current treatment methods. Therefore, developing new agents to treat osteosarcoma is exceptionally urgent.AimThis study aimed to evaluate the anticancer effects of evodiamine (EVO) on osteosarcoma cells and, meanwhile, to investigate the underlying mechanisms involved.Materials and MethodsThe effect of EVO on the proliferation of osteosarcoma was detected by MTT assay, crystal violet assay and colony formation assay. The effects of EVO on the migration and invasion of osteosarcoma were detected by wound-healing assay and transwell assay. The effect of EVO on apoptosis of osteosarcoma was measured by Hoechst 33258 staining and cell cycle assay. The protein expression levels were detected by Western blotting assay. The activity of Wnt/β-Catenin signaling pathway was detected by luciferase reporter assay and Western blotting assay.ResultsAccording to MTT, crystal violet and colony formation assay results, EVO significantly inhibited the cell proliferation in a dose-dependent manner. Hoechst 33258 staining assay revealed that EVO induced cell apoptosis in a concentration-dependent manner. Moreover, EVO inhibited the migration and invasion of the osteosarcoma cells. Mechanistic studies revealed that EVO suppresses metastatic through suppressing epithelial–mesenchymal transition (EMT) as indicated by elevating the expression of epithelial marker E‐cadherin and reducing the expression of mesenchymal markers N‐cadherin and vimentin, as well as EMT transcription factors Snail and MMPs. Subsequently, EVO induced cell cycle arrest at the G2/M phase that correlated with reduced levels of cyclin D1 protein, while the apoptotic effects of EVO were associated with the upregulation of Bax and Bad and a decrease in Bcl-2 protein levels. Furthermore, EVO exerted the anticancer effects by suppressing Wnt/β-catenin signal pathway in osteosarcoma cells.ConclusionIn summary, EVO exhibited potent anticancer effects against human osteosarcoma cells and promoted apoptosis through suppressing Wnt/β-catenin signaling pathway. These results indicated that EVO may be regarded as a new approach for osteosarcoma treatment.

MiR‑181b‑p53 negative feedback axis regulates osteosarcoma cell proliferation and invasion.

Osteosarcoma (OS) is one of the most common malignant tumors in young adults and as a high distant metastasis rate. The p53 protein, a potent prognostic biomarker for patients with OS, is altered in ~50% of OS cases. p53 was reported to exert its effects through regulating the transcription of microRNAs (miRNAs/miRs) and other genes. In the present study, the expression of miR-181b, a critical OS oncomiR, was shown to be significantly upregulated whereas p53 expression was downregulated within OS tissues and cells; in tissue samples, miR-181b and p53 were negatively correlated. p53 inhibited the transcription of miR-181b via targeting its promoter region, whereas miR-181b bound the TP53 3′-untranslated region (UTR) to inhibit p53 expression. miR-181b silencing considerably increased p53, p21, and epithelial-Cadherin protein levels but decreased Cyclin D1 protein levels in OS cells. In addition, miR-181b inhibition reduced OS cell proliferation and invasion. In contrast, p53 knockdown had the opposite effects on these proteins and OS cell proliferation and invasion. Above all, p53 knockdown significantly attenuated the effects of miR-181b inhibition. Moreover, OS cell xenograft assays further confirmed the roles of the miR-181b/p53 axis in OS growth. In conclusion, miR-181b and p53 are negatively regulated by one another and therefore form a negative feedback axis that regulates the proliferation and invasion abilities of OS cells. Targeting miR-181b to inhibit its abnormal upregulation might be a potent strategy for OS treatment.

LncRNA TTTY15 regulates hypoxia-induced vascular endothelial cell injury via targeting miR-186-5p in cardiovascular disease.

Dysfunction of vascular endothelial cells was associated with diverse human diseases, including cardiovascular disease. Long noncoding RNAs (LncRNAs) were involved in the regulation of cell injury. We aimed to investigate the role of lncRNA testis-specific transcript, Y-linked 15 (TTTY15) in hypoxia-induced cell injury in human umbilical vein endothelial cells (HUVECs). Cell counting kit-8 (CCK8) assay was used to check cell viability. Lactate dehydrogenase (LDH) assay kit and flow cytometry were utilized to evaluate the leakage rate of LDH and cell apoptosis, respectively. The protein levels of Cyclin D1 and B-cell lymphoma-2-Associated X (Bax) in hypoxia-induced HUVECs were measured by Western blot. Quantitative Real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression levels of TTTY15 and miR-186-5p in hypoxia-induced HUVECs. The starBase was utilized to predict the binding sites between TTTY15 and miR-186-5p and the dual-luciferase reporter assay was performed to verify the interaction. Hypoxia inhibited cell viability and promoted the release of LDH and cell apoptosis in HUVECs. Besides, hypoxia significantly decreased the protein level of Cyclin D1 and increased the protein level of Bax in HUVECs. In addition, the expression level of TTTY15 was obviously upregulated in hypoxia-induced HUVECs, opposite to the level of miR-186-5p. Meanwhile, knockdown of TTTY15 or upregulation of miR-186-5p mitigated hypoxia-induced cell injury in HUVECs. Further studies suggested that TTTY15 targeted miR-186-5p and regulated hypoxia-induced cell injury via interacting with miR-186-5p in HUVECs. Downregulation of TTTY15 ameliorated hypoxia-induced cell injury by targeting miR-186-5p in HUVECs.

5-(Carbamoylmethylene)-oxazolidin-2-ones as a Promising Class of Heterocycles Inducing Apoptosis Triggered by Increased ROS Levels and Mitochondrial Dysfunction in Breast and Cervical Cancer.

Oxazolidinones are antibiotics that inhibit protein synthesis by binding the 50S ribosomal subunit. Recently, numerous worldwide researches focused on their properties and possible involvement in cancer therapy have been conducted. Here, we evaluated in vitro the antiproliferative activity of some 5-(carbamoylmethylene)-oxazolidin-2-ones on MCF-7 and HeLa cells. The tested compounds displayed a wide range of cytotoxicity on these cancer cell lines, measured by MTT assay, exhibiting no cytotoxicity on non-tumorigenic MCF-10A cells. Among the nine tested derivatives, four displayed a good anticancer potential. Remarkably, OI compound showed IC50 values of 17.66 and 31.10 µM for MCF-7 and HeLa cancer cells, respectively. Furthermore, we assessed OI effect on the cell cycle by FACS analysis, highlighting a G1 phase arrest after 72 h, supported by a low expression level of Cyclin D1 protein. Moreover, mitochondrial membrane potential was reduced after OI treatment driven by high levels of ROS. These findings demonstrate that OI treatment can inhibit MCF-7 and HeLa cell proliferation and induce apoptosis by caspase-9 activation and cytochrome c release in the cytosol. Hence, 5-(carbamoylmethylene)-oxazolidin-2-ones have a promising anticancer activity, in particular, OI derivative could represent a good candidate for in vivo further studies and potential clinical use.

Abstract PD2-02: Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: In the PALOMA-3 trial, the combination of the CDK4/6 inhibitor palbociclib (P) with fulvestrant (F) led to a significant improvement in clinical outcomes, compared to F+Placebo, in endocrine-pretreated patients (pts) with ER+/HER2- metastatic breast cancer (BC). Not all pts, however, benefit from F+P, and virtually all ultimately progress. Elucidating the resistance mechanisms would assist in identifying biomarkers to select pts who may most benefit from F+P, as well as novel therapeutic targets for combating resistance. Materials and Methods: The ER+ MCF7 BC cell lines made resistant (R) to long-term estrogen deprivation (EDR) or tamoxifen (TamR), and the 600MPE de novo TamR cells were tested for sensitivity to F, P and F+P. MCF7 EDR and MCF7 TamR cells were exposed to increasing concentrations of F+P to generate R derivatives (FPR, cells grow in the presence of F 100nM + P 100nM). The transcriptomic and proteomic profiles of MCF7 EDR, TamR, EDR/FPR and TamR/FPR were determined by RNA-seq and reverse-phase protein arrays (RPPA), respectively. Pathway analysis was performed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). mRNA and protein levels of select markers were assessed by RT-PCR and Western blot (WB), respectively. Cell growth, colony formation, and migratory and invasive potential were assessed by methylene blue staining, clonogenic assay, and in vitro migration and invasion assays. Results: Treatment of MCF7 EDR and TamR cells with P resulted in a dose-dependent inhibition of cell growth [IC50 = 149nM and 89nM, respectively] and colony formation [clonogenic inhibition at 100nM &amp;gt;85% and &amp;gt;95%, respectively]. In both MCF7 endocrine-R models, P (100nM) or F (100nM) monotherapy significantly inhibited cell growth and reduced phosphorylated (p)-Rb and the E2F target CDK2 protein levels compared to vehicle control. However, F+P resulted in an enhanced cell growth inhibition (&amp;gt;90%) and a more pronounced reduction in p-Rb and CDK2 expression compared to each agent alone. In the FPR lines, ‘oncogenic signatures’ representative of altered KRAS and MEK signaling were highly enriched compared to their sensitive counterparts, as revealed by GSEA analysis of the transcriptomic profiles. IPA analysis of the proteomic profiles confirmed the activation of the MAPK signaling pathway in FPR cells and identified EGFR as one of the top ranked upstream regulators. Increased EGFR mRNA and protein levels in FPR models were confirmed by RT-PCR and WB analysis. Additionally, gene signatures of cyclin D1 hyperactivation and increased cyclin D1 protein levels were enriched in FPR cell lines. Importantly, the EGFR inhibitor gefitinib (1µM), the pan-HER inhibitor neratinib (250nM), and the MEK1 inhibitor selumetinib (S) (1µM) selectively or more greatly inhibited the cell growth and reduced Cyclin D1 protein levels in FPR cells compared to their sensitive counterparts. Further, we found a significant enrichment of the ‘hallmark’ epithelial-mesenchymal transition gene signature in FPR models, which is in line with their increased migratory and invasive capabilities. Treatment with S reduced the migration and invasion of the FPR cells. Finally, the aggressive 600MPE de novo TamR cells harboring hyperactive MEK/MAPK signaling due to the KRAS and MAP2K4 activating mutations were significantly less sensitive to F, P, and F+P. However, the addition of S to F+P significantly increased the cell growth inhibition in this model. Conclusion: Acquired and intrinsic resistance to F+P is associated with hyperactivation of the EGFR/RAS/MEK signaling pathway, which sustains cell proliferation and promotes an aggressive phenotype. The EGFR/RAS/MEK pathway may serve as a predictive biomarker for F+P resistance and offer therapeutic targets to overcome resistance in pts with ER+/HER2- BC. Citation Format: Carmine De Angelis, Maria Letizia Cataldo, Agostina Nardone, Jamunarani Veeraraghavan, Xiaoyong Fu, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-02.

Salicylic Acid Protects Photosystem II by Alleviating Photoinhibition in Arabidopsis thaliana under High Light.

Salicylic acid (SA) is considered to play an important role in plant responses to environmental stresses. However, the detailed protective mechanisms in photosynthesis are still unclear. We therefore explored the protective roles of SA in photosystem II (PSII) in Arabidopsis thaliana under high light. The results demonstrated that 3 h of high light exposure resulted in a decline in photochemical efficiency and the dissipation of excess excitation energy. However, SA application significantly improved the photosynthetic capacity and the dissipation of excitation energy under high light. Western blot analysis revealed that SA application alleviated the decrease in the levels of D1 and D2 protein and increased the amount of Lhcb5 and PsbS protein under high light. Results from photoinhibition highlighted that SA application could accelerate the repair of D1 protein. Furthermore, the phosphorylated levels of D1 and D2 proteins were significantly increased under high light in the presence of SA. In addition, we found that SA application significantly alleviated the disassembly of PSII-LHCII super complexes and LHCII under high light for 3 h. Overall, our findings demonstrated that SA may efficiently alleviate photoinhibition and improve photoprotection by dissipating excess excitation energy, enhancing the phosphorylation of PSII reaction center proteins, and preventing the disassembly of PSII super complexes.

Effect of CASC19 on proliferation, apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression

Objective: To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells. Methods: (1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p. Results: (1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased (F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased (F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression level of cyclin D1 protein was significantly reduced (P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased (P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased (P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased (P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05). Conclusion: CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.

Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts

Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.

Circ-Smad5 retards the G1/S transition of cell cycle via inhibiting the activity of wnt/lef/cyclind1 signaling in JB6 cells

Circular RNAs are a large class of noncoding RNAs. Smad5 functions in cell differentiation, cell proliferation and metastasis. It has been reported that lnc-Smad5 can inhibit the proliferation of diffuse large B cell lymphoma. However, the function of circ-Smad5 has not yet been reported. Lentivirus vectors were constructed to establish circ-Smad5 upregulated and circ-Smad5 downregulated cell models. A CCK-8 assay was used to detect the proliferation of JB6 cells. FACS was used to analyze the cell cycle in the cell models. Western blot, immunofluorescence staining and TOP/FOP flash dual luciferase activity assays were used to determine the activity of the Wnt signaling pathway. The results revealed that the expression level of circ-Smad5 in JB6 cells was significantly lower than the expression level of linearized-Smad5. Compared with the control group, the percentage of S phase cells and the expression level of cyclin D1 protein were significantly higher in the sh-circ-Smad5 group. In the sh-circ-Smad5 group, β-catenin and LEF-1 were significantly increased, p-β-catenin was significantly decreased, and the relative activity of the TOP/FOP reporter gene was higher compared to the control group levels. These phenomena could be reversed by treating with Wnt signaling inhibitor PNU-74654. We conclude that the circ-Smad5 retards the proliferation and the cell cycle progression of JB6 cells. Thus, circ-Smad5 may function by inhibiting the activation of Wnt/β-catenin/Lef 1 signaling, which inhibits the expression of cyclin D1. To the best of our knowledge, we are the first to report the function of circ-Smad5.

Identification of a novel microRNA-141-3p/Forkhead box C1/β-catenin axis associated with rheumatoid arthritis synovial fibroblast function in vivo and in vitro.

Rationale: Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis in which synovial fibroblasts (SFs) play key roles in cartilage and bone destruction through tumor-like proliferation, migration, invasion and inflammation. This study aimed to research forkhead box protein C1 (FoxC1) and microRNA (miR)-141-3p, which modulate pathological changes in the synovial membrane, to find possible strategies for treating RA.Methods: FoxC1, β-catenin and miR-141-3p gene expression in synovial tissues and SFs was quantified by real-time PCR; FoxC1 and β-catenin protein levels were evaluated by immunohistochemistry, immunofluorescence, and Western blotting. We transiently transfected human SFs with FoxC1 and β-catenin overexpression and silencing vectors and assessed proliferation, migration, invasion and inflammation by cell function and enzyme-linked immunosorbent assays. We also assessed downstream signaling activation using immunofluorescence, real-time PCR and Western blotting. Double luciferase, coimmunoprecipitation and chromatin immunoprecipitation assays were used to verify miR-141-3p, FoxC1 and β-catenin gene and protein combinations. Finally, the therapeutic effects of FoxC1 silencing and miR-141-3p overexpression were evaluated in type II collagen-induced arthritis (CIA) rats.Results: We found that FoxC1 expression was significantly upregulated in synovium and SFs in both RA patients and rats with collagen-induced arthritis (CIA). FoxC1 overexpression increased β-catenin messenger RNA (mRNA) and protein levels and upregulated cyclin D1, c-Myc, fibronectin and matrix metalloproteinase 3 (MMP3) mRNA and protein expression in RA SFs (RASFs). In contrast, FoxC1 knockdown reduced β-catenin mRNA and protein levels as well as cyclin D1, c-Myc, and fibronectin mRNA and protein levels in RASFs. Furthermore, altering FoxC1 expression did not significantly change GSK3β and pGSK3β levels. FoxC1 overexpression promoted proliferation, migration, invasion and proinflammatory cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)) production and reduced anti-inflammatory cytokine (IL-10) levels in RASFs. FoxC1 bound to the β-catenin promoter, and β-catenin mediated the FoxC1-induced pathological changes. We also observed downregulated microRNA (miR)-141-3p expression in SFs from both RA patients and CIA rats and further found that miR-141-3p bound to the FoxC1 3′UTR and suppressed FoxC1 expression. Intra-ankle miR-141-3p agomir or FoxC1-specific siRNA injection hindered CIA development in rats.Conclusions: FoxC1 and miR-141-3p participate in RA pathogenesis by mediating inflammation and SF proliferation, migration, and invasion and thus could be novel targets for RA therapy as a nonimmunosuppressive approach.

8-Chloro-Adenosine Inhibits Proliferation of MDA-MB-231 and SK-BR-3 Breast Cancer Cells by Regulating ADAR1/p53 Signaling Pathway

8-Chloro-adenosine (8-Cl-Ado) has been shown to exhibit its antitumor activity by inducing apoptosis in human lung cancer A549 and H1299 cells or autophagy in chronic lymphocytic leukemia, and MDA-MB-231 and MCF-7 breast cancer cells. Adenosine deaminases acting on RNA 1 (ADAR1) is tightly associated with cancer development and progression. The aim of this study was to investigate the role of ADAR1 in the proliferation of MDA-MB-231 and SK-BR-3 breast cancer cell lines after 8-Cl-Ado exposure and its possible mechanisms. After 8-Cl-Ado exposure, CCK-8 assay was performed to determine the cell proliferation; flow cytometry was used to analyze the cell cycle profiles and apoptosis; and the protein levels of ADAR1, p53, p21, and cyclin D1 were measured by western blotting. The results showed that the cell proliferation was greatly inhibited, G1 cell cycle was arrested, and apoptosis was induced after 8-Cl-Ado exposure. ADAR1 and cyclin D1 protein levels were dramatically decreased, while p53 and p21 levels were increased after 8-Cl-Ado exposure. Moreover, the cell growth inhibition was rescued, apoptosis was reduced, and p53 and p21 protein levels were downregulated, while cyclin D1 was upregulated when cells were transfected with plasmids expressing ADAR1 proteins. More importantly, RNA-binding domain of ADAR1 is critical to the cell growth inhibition of breast cancer cells exposed to 8-Cl-Ado. Together, 8-Cl-Ado inhibits the cell proliferation, induces G1 phase arrest and apoptosis at least by targeting ADAR1/p53/p21 signaling pathway. The findings may provide us with insights into the role of ADAR1 in breast cancer progression and help us better understand the effects of 8-Cl-Ado in the treatment of breast cancer.

STK31 regulates the proliferation and cell cycle of lung cancer cells via the Wnt/β‑catenin pathway and feedback regulation by c‑myc.

Lung cancer, which is a leading cause of cancer-related deaths, is diagnosed at a male to female ratio of 2.1:1. Serine-threonine kinase 31 (STK31) is a novel cancer/testis (CT)-related gene that is highly expressed in several types of cancers, such as lung and colorectal cancer, and plays crucial roles in cancer. In the present study, increased expression of STK31 and β-catenin was observed in lung cancer tissues and cell lines. Downregulation of STK31 expression in lung cancer cells significantly inhibited their proliferation by arresting the cell cycle in the G1 phase concurrent with decreased β-catenin, c-myc and cyclin D1 protein levels, while upregulation of STK31 had the opposite effects. In addition, STK31-induced lung cancer cell viability, proliferation, cell cycle progression, and expression of related genes were completely attenuated by a Wnt/β-catenin inhibitor (XAV939). Similar to XAV939, a c-myc inhibitor (10058-F4) also significantly attenuated STK31-induced proliferation and cell cycle progression in lung cancer cells. Inhibiting c-myc and TRRAP significantly decreased the expression of STK31, and a chromatin immunoprecipitation (ChIP) assay confirmed that c-myc directly bound to the STK31 promoter. These results indicated that STK31 may act as an oncogene in lung cancer and that c-myc may be the transcription factor that promotes STK31 expression. Moreover, the results suggested that c-myc can also regulate STK31 expression in a positive feedback loop, and the downregulation of STK31 in lung cancer cells had an inhibitory effect on cell viability, cell proliferation and cell cycle progression, likely by inactivating the Wnt/β-catenin pathway and positive feedback regulation by c-myc.