Levels Of Cell Cycle-related Proteins Research Articles

Anti-cancer activity and mechanism of flurbiprofen organoselenium compound RY-1-92 in non-small cell lung cancer.

Lung cancer is one of the malignancies with the highest incidence and mortality rates worldwide, and non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer types. In this study, the anti-cancer activities of a novel flurbiprofen organic selenium compound, RY-1-92, on NSCLC cells and a mouse model and the underlying molecular mechanisms were explored. We found that compound RY-1-92 can significantly inhibit the viability, colony formation and migration of A549, NCI-H460 lung cancer cells. Flow cytometry analysis showed that RY-1-92 also can lead to G2/M cell cycle arrest and apoptosis induced in lung cancer cells. Further, RY-1-92 can decrease the tumor size in the Lewis lung cancer tumor-bearing mouse model. The protein levels of cell cycle-related proteins CDK1/cyclinB1 were decreased, while the apoptosis-related protein BAX was increased dramatically after RY-1-92 treatment in vitro and in vivo. Impressively, it was found that TRPV1 might act as a potential molecular target of RY-1-92 using the SEA search server. Furthermore, down-regulation on TRPV1 and its downstream associated factors including p-AKT protein and MAPK signaling pathway-related proteins after RY-1-92 treatment was observed in A549, NCI-H460 lung cancer cells. Taken together, our findings shed light on the potential of RY-1-92 as a novel small molecular drug for NSCLC, and it is of great significance for its further in-depth research and development.

Regulation of Interferon-β-Modified Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes in Proliferation and Apoptosis of Prostate Cancer Cells

ABSTRACT Prostate cancer (PCa) poses a serious burden to men. Interferon-β (IFN-β) is implicated in cancer cell growth. This study hence explored the regulation of IFN-β-modified human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exos) in PCa cells. In vitro-cultured hUCMSCs were transfected with pcDNA3.1-IFN-β plasmid or IFN-β siRNA. hUCMSC-Exos were extracted by ultracentrifugation and identified. PCa cells (PC3 and LNCap) were treated with Exos. Cellular internalization of Exos by cells was detected by uptake assay. Cell proliferation, cycle, and apoptosis were evaluated by CCK-8, EdU staining, and flow cytometry. Levels of cell cycle-related proteins (cyclin D/cyclin E) were determined by Western blot. The effect of IFN-β-modified hUCMSC-Exos in vivo was analyzed. IFN-β-modified hUCMSC-Exos (Exooe- IFN −β or Exosi- IFN −β) were successfully isolated. IFN-β was encapsulated in Exos, and PCa cells could uptake Exos. After treating with Exooe- IFN −β, PCa cell proliferation was impeded, the percentage of cells in the G0/G1 phase, cyclin D/cyclin E levels, and cell apoptotic rate were elevated, while cells treated with Exooe- IFN −β exhibited contrary trends. IFN-β-modified hUCMSC-Exos reduced PCa tumor size and weight in vivo. Conjointly, IFN-β-modified hUCMSC-Exos suppress PCa cell proliferation and facilitate apoptosis.

WWP2 binds to NKRF, enhances the NF-κB signaling, and promotes malignant phenotypes of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is one of the hematological malignancies with a high recurrence rate. WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) is identified as a pivotal regulator of tumor progression. This study aimed to assess the possible role of WWP2 in AML. Analysis of the GEPIA database indicated an elevated WWP2 expression in AML. We established stable WWP2-overexpressed or WWP2-silenced cells using lentivirus loaded with cDNA encoding WWP2 mRNA or shRNA targeting WWP2. Notably, WWP2 overexpression facilitated cell proliferation and cell cycle progression, which was manifested as the increase of colony formation number, S-phase percentage and cell cycle related protein levels. As observed, WWP2 knockdown presented opposite effects, leading to inhibition of tumorigenicity. Strikingly, WWP2 knockdown induced apoptosis, accompanied by upregulation of pro-apoptosis proteins cleaved caspase-9, Bax and cleaved caspase-3 and downregulation of anti-apoptosis protein Bcl-2. Functionally, we further confirmed that WWP2 overexpression enhanced the NF-κB signaling and upregulated the levels of downstream genes, which may contribute to aggravating the development of AML. More importantly, by co-immunoprecipitation assay, we verified that WWP2 bound to NF-κB-repressing factor (NKRF) and promoted NKRF ubiquitylation. Dramatically, NKRF overexpression abolished the role of WWP2 in facilitating the process of AML. Overall, our observations confirm that WWP2 exerts a critical role in the tumorigenicity of AML, and NKRF is regarded as an essential factor in the WWP2-mediated AML progression. WWP2 may be proposed as a promising target of AML.

Downregulation of lncRNA NEAT1 interacts with miR-374b-5p/PGAP1 axis to aggravate the development of osteoarthritis

BackgroundOsteoarthritis (OA), characterized by inflammation and articular cartilage degradation, is a prevalent arthritis among geriatric population. This paper was to scrutinize the novel mechanism of long noncoding RNA (lncRNA) NEAT1 in OA etiology.MethodsA total of 10 OA patients and 10 normal individuals was included in this study. Cell model of OA was built in human normal chondrocytes induced by lipopolysaccharide (LPS). An OA Wistar rat model was established through intra-articular injection of L-cysteine and papain mixtures (proportion at 1:2) into the right knee. Quantitative reverse transcription-polymerase chain reaction was employed to ascertain the expression levels of NEAT1, microRNA (miR)-374b-5p and post-GPI attachment to protein 1 (PGAP1), while dual-luciferase reporter experiments were used for the validation of target relationship among them. Cell cycle and apoptosis were calculated by flow cytometry analysis. CCK-8 assay was done to evaluate the proliferative potentials of chondrocytes. The levels of cell cycle-related proteins (Cyclin A1, Cyclin B1 and Cyclin D2) and pro-apoptotic proteins (Caspase3 and Caspase9) were measured by western blotting. Tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 levels were determined via ELISA. Hematoxylin & eosin (HE) Staining was used for pathological examination in OA rats.ResultsPronounced downregulation of NEAT1 and PGAP1 and high amounts of miR-374b-5p were identified in OA patients, LPS-induced chondrocytes and OA rats. NEAT1 targeted miR-374b-5p to control PGAP1 expression. Loss of NEAT1 or upregulation of miR-374b-5p dramatically accelerated apoptosis, led to the G1/S arrest and promoted the secretion of inflammatory cytokines in LPS-induced chondrocytes, while ectopic expression of PGAP1 exhibited the opposite influences on chondrocytes. Additionally, we further indicated that upregulation of miR-374b-5p attenuated the effects of PGAP1 overexpression on LPS-induced chondrocytes.ConclusionsReduced NEAT1 induces the development of OA via miR-374b-5p/PGAP1 pathway. This suggests that the regulatory axis NEAT1/miR-374b-5p/PGAP1 is a novel and prospective target for OA treatment.

Design of ion channel blocking, toxin-like Kunitz inhibitor peptides from the tapeworm, Echinococcus granulosus, with potential anti-cancer activity

Over-expression of K+ channels has been reported in human cancers and is associated with the poor prognosis of several malignancies. EAG1, a particular potassium ion channel, is widely expressed in the brain but poorly expressed in other normal tissues. Kunitz proteins are dominant in metazoan including the dog tapeworm, Echinococcus granulosus. Using computational analyses on one A-type potassium channel, EAG1, and in vitro cellular methods, including major cancer cell biomarkers expression, immunocytochemistry and whole-cell patch clamp, we demonstrated the anti-tumor activity of three synthetic small peptides derived from E. granulosus Kunitz4 protease inhibitors. Experiments showed induced significant apoptosis and inhibition of proliferation in both cancer cell lines via disruption in cell-cycle transition from the G0/G1 to S phase. Western blotting showed that the levels of cell cycle-related proteins including P27 and P53 were altered upon kunitz4-a and kunitz4-c treatment. Patch clamp analysis demonstrated a significant increase in spontaneous firing frequency in Purkinje neurons, and exposure to kunitz4-c was associated with an increase in the number of rebound action potentials after hyperpolarized current. This noteworthy component in nature could act as an ion channel blocker and is a potential candidate for cancer chemotherapy based on potassium channel blockage.

Functional Mechanism of MicroRNA-25-3p in Hilar Cholangiocarcinoma Cell Proliferation and Migration Through Regulation of Dual Specificity Phosphatase 5

Objective Hilar cholangiocarcinoma (HCCA) is a highly aggressive biliary tract tumor. microRNAs (miRs) exert dual actions in various cancers. This paper seeks to expound on the functional mechanisms of miR-25-3p/dual specificity phosphatase 5 (DUSP5) in HCCA cell proliferation and migration. Methods HCCA-related data were downloaded from GEO database to screen out differentially-expressed genes. The potential target miR (miR-25-3p) and its expression in HCCA were analyzed on Starbase. The binding relation between miR-25-3p and DUSP5 was confirmed by dual-luciferase assay. Levels of miR-25-3p and DUSP5 in FRH-0201 cells and HIBEpics were determined by RT-qPCR and Western blot. miR-25-3p and DUSP5 levels were intervened with to explore their effects on FRH-0201 cells. The apoptosis, proliferation, migration, and invasion of FRH-0201 cells were evaluated by TUNEL, CCK8, scratch healing, and Transwell assays. Flow cytometry was conducted to assess FRH-0201 cell cycle. Levels of cell cycle-related proteins were determined by Western blot. Results DUSP5 was weakly-expressed and miR-25-3p was highly-expressed in HCCA samples and cells. miR-25-3p targeted DUSP5. miR-25-3p suppressed FRH-0201 cell apoptosis and increased cell proliferation, migration, and invasion. DUSP5 overexpression partially abrogated miR-25-3p overexpression-exerted effects on FRH-0201 cells. miR-25-3p stimulated G1/S phase transition of FRH-0201 cells by targeting DUSP5. Conclusion miR-25-3p regulated HCCA cell cycle and facilitated cell proliferation and migration by targeting DUSP5.

Mechanism of the HIF-1α/VEGF/VEGFR-2 pathway in the proliferation and apoptosis of human haemangioma endothelial cells.

Haemangiomas (HAs) are prevalent vascular endothelial cell tumours. With respect to the possible involvement of HIF-1α in HAs, we have explored its role in haemangioma endothelial cell (HemEC) proliferation and apoptosis. shRNA HIF-1α and pcDNA3.1 HIF-α were manipulated into HemECs. HIF-α, VEGF, and VEGFR-2 mRNA and protein levels were assessed by qRT-PCR and Western blotting. Cell proliferation and viability, cell cycle and apoptosis, migration and invasion, and ability to form tubular structures were assessed by colony formation assay, CCK-8, flow cytometry, Transwell assay, and tube formation assay. Cell cycle-related protein levels, and VEGF and VEGFR-2 protein interaction were detected by Western blot and immunoprecipitation assays. An Haemangioma nude mouse model was established by subcutaneous injection of HemECs. Ki67 expression was determined by immunohistochemical staining. HIF-1α silencing suppressed HemEC neoplastic behaviour and promoted apoptosis. HIF-1α facilitated VEGF/VEGFR-2 expression and the VEGF had interacted with VEGFR-2 at protein - protein level. HIF-1α silencing arrested HemECs at G0/G1 phase, diminished Cyclin D1 protein level, and elevated p53 protein level. VEGF overexpression partially abrogated the effects of HIF-1α knockdown on inhibiting HemEC malignant behaviours. Inhibiting HIF-1α in nude mice with HAs repressed tumour growth and Ki67-positive cells. Briefly, HIF-1α regulated HemEC cell cycle through VEGF/VEGFR-2, thus promoting cell proliferation and inhibiting apoptosis.

HERPUD1, a Member of the Endoplasmic Reticulum Protein Quality Control Mechanism, may be a Good Target for Suppressing Tumorigenesis in Breast Cancer Cells.

Breast cancer is the most frequently diagnosed cancer type and the second leading cause of cancer-related death in women. Recent studies have highlighted the importance of the endoplasmic reticulum (ER) protein quality control mechanism for the survival of many cancers. It has also been recommended as a good target for the treatment of many cancer types. Homocysteine inducible ER protein with ubiquitin-like domain 1 (HERPUD1) functions as one of the main components of ER-associated degradation, which is an ER-resident protein quality mechanism. Today, the association of HERPUD1 with breast carcinogenesis is still not fully understood. Herein, we evaluated the possibility of HERPUD1 as a potential therapeutic target for breast cancer. The effects of HERPUD1 silencing on epithelial-mesenchymal transition (EMT), angiogenesis, and cell cycle proteins were analyzed by immunoblotting studies. To test the role of HERPUD1 on tumorigenic features, WST-1-based cell proliferation assay, wound-healing assay, 2D colony formation assay, and Boyden-Chamber invasion assay were performed in human breast cancer cell line MCF-7. The statistical significance of the differences between the groups was determined by Student's t-test. Our results displayed that suppressing HERPUD1 expression reduced the cell cycle-related protein levels, including cyclin A2, cyclin B1, and cyclin E1 in MCF-7 cells. Also, silencing of HERPUD1 remarkably decreased expression levels of EMT-related N-cadherin and angiogenesis marker vascular endothelial growth factor A. Moreover, we determined that cell proliferation, migration, invasion, and colony formation of MCF-7 cells were significantly limited by silencing of HERPUD1. Present data suggest that HERPUD1 may be an effective target for biotechnological and pharmacological strategies to be developed to treat breast cancer.

MiR-302a-3p Promotes Radiotherapy Sensitivity of Hepatocellular Carcinoma by Regulating Cell Cycle via MCL1.

Background The relationship between tumor suppressor gene miR-302a-3p and radiotherapy for hepatocellular carcinoma (HCC) remains unclear. This study intended to illustrate the molecular mechanism how miR-302a-3p regulated radiotherapy sensitivity of HCC. Methods miR-302a-3p expression in HCC tissues and cells was examined by qRT-PCR. The effect of miR-302a-3p on HCC radiotherapy sensitivity were detected by CCK-8, colony formation, and flow cytometry assays. The expression levels of cell cycle-related proteins were detected by Western blot. The influence of miR-302a-3p on radiotherapy sensitivity of HCC was further investigated via cell cycle inhibitor (Caudatin) treatment. The target gene (MCL1) of miR-302a-3p was obtained by bioinformatics analysis, and their binding relationship was confirmed by RNA-binding protein immunoprecipitation assay. The mechanisms of miR-302a-3p regulating cell cycle and affecting radiotherapy sensitivity of HCC cells through MCL1 were further explored through the rescue experiments. Results miR-302a-3p expression was remarkably reduced in radiotherapy-resistant tissues and cells of HCC. miR-302a-3p overexpression restored sensitivity of radiotherapy-resistant HCC cells to radiotherapy. Treatment with cell cycle inhibitor Caudatin could reverse suppressive effect of miR-302a-3p downregulation on sensitivity of HCC to radiotherapy. Additionally, miR-302a-3p could restrain MCL1 expression. In vitro cell assays further revealed that miR-302a-3p/MCL1 axis could enhance radiotherapy sensitivity of HCC cells by inducing G0/G1 arrest. Conclusions miR-302a-3p facilitated radiotherapy sensitivity of HCC cells by regulating cell cycle via MCL1, which provided a new underlying target for radiotherapy resistance of HCC patients.

DFNA5 inhibits colorectal cancer proliferation by suppressing the mTORC1/2 signaling pathways via upregulation of DEPTOR

ABSTRACT The human deafness, autosomal dominant 5 gene (DFNA5), a newly discovered executor of pyroptosis, has been strongly implicated in the tumorigenesis of several human cancers. However, an understanding of the functional role of DFNA5 in the development and progression of colorectal cancer (CRC) is limited. In this study, we demonstrated that DFNA5 was downregulated in CRC tissues. Ectopic expression of DFNA5 inhibited tumor cell growth in vitro, retarded tumor formation in vivo, and blocked a cell-cycle transition from the G0/G1 to the S phase, whereas a DFNA5 knockdown promoted cell proliferation. Western blotting showed that the levels of cell cycle-related proteins, including cyclin D1, cyclin E, CDK2, and p21, were accordingly altered upon DFNA5 overexpression or DFNA5 knockdown. Mechanistic studies indicated that DFNA5 exerted its tumor suppressor functions by antagonizing mTORC1/2 signaling via upregulation of DEPTOR. In addition, blockage of mTORC1/2 signaling by Torin-1 abolished the accelerative proliferation by DFNA5 knockdown. In conclusion, these results indicated that DFNA5 inhibits the proliferation and tumor formation of colon cancer cells by suppressing mTORC1/2 signaling.

Graphene quantum dots rescue angiogenic retinopathy via blocking STAT3/Periostin/ERK signaling

BackgroundPathological retinal angiogenesis resulting from a variety of ocular diseases including oxygen induced retinopathy, diabetic retinopathy and ocular vein occlusion, is one of the major reasons for vision loss, yet the therapeutic option is limited. Multiple nanoparticles have been reported to alleviate angiogenic retinopathy. However, the adverse effect cannot be ignored due to the relatively large scale. Graphene quantum dots (GQDs) have shown potential in drug delivery and have been proved biocompatible. In this study, Graphene quantum dots are extensively investigated for their application in angiogenic retinopathy therapy.ResultsWe showed that GQDs were biocompatible nanomaterials in vitro and in vivo. The nanoparticles have a dose-dependent inhibitory effect on proliferation, migration, tube formation and sprouting of human umbilical vein endothelial cells (HUVECs). Further data show that GQDs could inhibit pathological retinal neovascularization in an oxygen-induced retinopathy (OIR) model. The data of RNA sequencing suggested that periostin is involved in this process. GQDs inhibit the expression of periostin via STAT3, and further regulated cell cycle-related protein levels through ERK pathway. The signaling pathway was conformed in vivo using OIR mouse model.ConclusionsThe present study indicated that GQDs could be a biocompatible anti-angiogenic nanomedicine in the treatment of pathological retinal neovascularization via disrupting periostin/ERK pathway and subsequent cell cycle.Graphical

Long non-coding RNA SPRY4-IT1 promotes proliferation and metastasis in nasopharyngeal carcinoma cell.

BackgroundLong non-coding RNA SPRY4 intronic transcript 1 (Lnc RNA SPRY4-IT1) was aberrant-expressed in various kinds of cancer. Increasing evidence demonstrated that lnc RNAs involved in tumorigenesis and metastasis. In this study, we aimed to explore the biological role of SPRY4-IT1 on the phenotype of nasopharyngeal carcinoma (NPC) in vitro and in vivo.MethodsThe expression level of SPRY4-IT1 in NPC cell lines were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and colony formation assay were used to detect cell proliferation. Wound-healing assay, transwell assay and animal experiment were performed to evaluate the ability of cell migration and metastasis. Cell cycle distribution and apoptosis were determined by flow cytometry. Western blotting and immunofluorescence were employed to identify protein expression.ResultsSPRY4-IT1 was significantly up-regulated in several NPC cell lines (6-10B, CNE-2, and HONE-1) compared with human immortalized nasopharyngeal epithelial cell (NP69). Silencing of SPRY4-IT1 inhibited proliferation, migration, and metastasis, and induced significant G2/M phase arrest and apoptosis. Western blotting showed that the expression levels of cell cycle-related proteins (cyclin B1, cdc2 and p-cdc2) were down-regulated and apoptosis-associated proteins (PARP, cleaved PARP and cleaved caspase-3) were up-regulated after knockdown of SPRY4-IT1. The expression level of E-cadherin was increased and the expression of Vimentin, Snail and Twist1 were decreased after the SPRY4-IT1 knockdown.ConclusionlncRNA SPRY4-IT1 played a significant role in NPC proliferation, migration and metastasis, suggesting that SPRY4-IT1 might be a potential therapeutic target for the treatment of NPC.

5-Bromo-3,4-dihydroxybenzaldehyde Promotes Hair Growth through Activation of Wnt/β-Catenin and Autophagy Pathways and Inhibition of TGF-β Pathways in Dermal Papilla Cells.

Various studies addressing the increasing problem of hair loss, using natural products with few side effects, have been conducted. 5-bromo-3,4-dihydroxybenzaldehyde (BDB) exhibited anti-inflammatory effects in mouse models of atopic dermatitis and inhibited UVB-induced oxidative stress in keratinocytes. Here, we investigated its stimulating effect and the underlying mechanism of action on hair growth using rat vibrissa follicles and dermal papilla cells (DPCs), required for the regulation of hair cycle and length. BDB increased the length of hair fibers in rat vibrissa follicles and the proliferation of DPCs, along with causing changes in the levels of cell cycle-related proteins. We investigated whether BDB could trigger anagen-activating signaling pathways, such as the Wnt/β-catenin pathway and autophagy in DPCs. BDB induces activation of the Wnt/β-catenin pathway through the phosphorylation of GSG3β and β-catenin. BDB increased the levels of autophagic vacuoles and autophagy regulatory proteins Atg7, Atg5, Atg16L, and LC3B. We also investigated whether BDB inhibits the TGF-β pathway, which promotes transition to the catagen phase. BDB inhibited the phosphorylation of Smad2 induced by TGF-β1. Thus, BDB can promote hair growth by modulating anagen signaling by activating Wnt/β-catenin and autophagy pathways and inhibiting the TGF-β pathway in DPCs.

Dichloromethane fractions of Calystegiasoldanella induce S‑phase arrest and apoptosis in HT‑29 human colorectal cancer cells.

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT-29 was examined. First, five solvent fractions [n-hexane, dichloromethane (DCM), ethyl acetate, n-butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose- and time-dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT-29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose-dependent manner, which was associated with the protein expression levels of apoptosis-related proteins, including increased Fas, Bad and Bax, and decreased pro-caspase-8, Bcl-2, Bcl-xL, pro-caspase-9, pro-caspase-7 and pro-caspase-3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose-dependent manner in HT-29 cells treated with DCM. In addition, cell cycle analysis confirmed S-phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle-related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT-29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S-phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT-29 colorectal cells. Further research on the composition of the DCM fraction is warranted.

Anti-tumor effect of ginkgetin on human hepatocellular carcinoma cell lines by inducing cell cycle arrest and promoting cell apoptosis

ABSTRACT This study explored the anti-tumor effect of ginkgetin, an extract from ginkgo biloba, on human hepatocellular carcinoma cell lines and the underlying mechanisms. Cell viability was measured by MTT assay. Apoptotic cell morphology was observed under an inverted microscope after Hoechst 33,258 staining, and the ratio of apoptotic and necrotic cells was examined by flow cytometry after FITC/PI staining. Cell cycle changes were analyzed using flow cytometry. Cytochrome c release and caspase 3 and 8 activities were monitored using the relevant reagent kits. The levels of cell cycle-related proteins were detected by Western blot. MTT results indicated that ginkgetin significantly reduced HepG2 cell viability in a dose-dependent manner. Cellular morphology observation revealed that ginkgetin induced typical apoptotic morphological features of HepG2 cells, such as increased apoptotic bodies and cell shrinkage. Cell cycle analysis showed that ginkgetin increased the proportion of cells in the S phase. S-phase cell accumulation could be attributed to the decreased expression of cell cycle regulatory factors. Similarly, ginkgetin also induced the apoptosis and S phase cell accumulation of another human HCC cell line SK-HEP-1. Furthermore, ginkgetin treatment increased caspase-3 activity and cytochrome c release but not caspase-8 activity, implying that ginkgetin might mediate cell apoptosis through the mitochondrial pathway. In addition, the tumor formation experiment in nude mice showed that ginkgetin administration inhibited tumor growth. These results suggest that ginkgetin could be a cell apoptosis stimulator by affecting the balance between cell proliferation and apoptosis, suggesting that ginkgetin might be suitable for human HCC treatment.

Effect of MiR-142-3p Targeting HOXA5 on Proliferation, Cycle Arrest and Apoptosis of Acute B Lymphocytic Leukemia Cells

To investigate the effect and molecular mechanism of miR-142-3p to the proliferation, cycle and apoptosis of acute B lymphocytic leukemia (B-ALL) cells by regulating the homeobox gene 5 (HOXA5) expression. Real-time fluorescence quantitative PCR was used to detect the expression levels of miR-142-3p and HOXA5 in human B-ALL cell Nalm6 cell line and human B lymphoblast Hmy2-cir cells. Nalm6 was transfected by using liposome transfection technology, miR-142-3p mimic, pcDNA-HOXA5 overexpression plasmid, miR-142-3p mimic+pcDNA-HOXA5 overexpression plasmid, and control. The binding site of HOXA5 and miR-142-3p was predicted according to microRNA.org, and the targeting relationship between miR-142-3p and HOXA5 gene was detected by double luciferase reporter gene experiment. The effect of miR-142-3p to the proliferation of Nalm6 cells was detected using the Cell Counting Box-8 (CCK-8) method and cell clone formation experiments. Flow cytometry was used to detect the effects of miR-142-3p to cell cycle distribution and apoptosis of Nalm6 cells. The expression levels of cell cycle-related proteins, including G1 /S-specific cyclin-D1 (CyclinD1), Cyclin-dependent kinase 4 (CDK4) and B-cell lymphoma/ leukemia-2 protein (BCL-2), BCL-2 related X protein (Bax), cysteine-aspartate-specific protease (Caspase-3) were detected by Western blot. Compared with Hmy2-cir cells, miR-142-3p showed low expression in Nalm6 cells and HOXA5 showed high expression (P<0.05). MiR-142-3p and HOXA5 3'-UTR showed complementary binding regions, the luciferase activity of miR-142-3p mimic and wild-type HOXA5 3'-UTR was significantly lower than that of miR-142-3p negative control and wild-type HOXA5 3'-UTR (P<0.05). The proliferation of Nalm6 cells and the number of cell clones could be inhibited by miR-142-3p mimic after 48 and 72 hours of transfection (P<0.05), which causing G1 phase arrest of Nalm6 cells and inhibiting the expression of CyclinD1 and CDK4 protein (P<0.01), promoting the apoptosis of Nalm6 cells, and inhibiting the expression of BCL-2 protein, moreover, promoting the expression of Bax and Caspase-3 protein (P<0.05). MiR-142-3p can inhibit the proliferation of Nalm6 cells by targeting down-regulation the expression of HOXA5, arrest the G1 phase of cells, and promote apoptosis of the cells.

Wilms' tumor 1-associating protein contributes to psoriasis by promoting keratinocytes proliferation via regulating cyclinA2 and CDK2

BackgroundWilms’ tumor 1-associating protein (WTAP) is a ubiquitously expressed nuclear protein, and involved in multiple pathophysiological processes, including cell cycle, RNA splicing and stabilization, N6-methyladenosine RNA modification, cell proliferation, and apoptosis as well as embryonic development. Here, we investigated the specific role of WTAP in the pathogenesis of psoriasis and its underlying mechanism. MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analyses and multi-spectrum immunohistochemistry were applied to evaluate the level of WTAP expression in psoriatic skin and normal skin. HaCaT cells was stably transfected with WTAP small interfering (si)RNA and plasmid using Lipofectamine®2000 and proliferation was determined by CCK8. Apoptosis and cell cycle analysis were conducted by flow cytometry. Western blot assay was used to explore the expression levels of cell cycle-related proteins in HaCaT cells after WTAP overexpression or inhibition. Furthermore, HaCaT cells were stimulated with proinflammatory cytokines (ie, IL-17A, IL-22, IL-1a, oncostatin M, and TNF-a) to assess WTAP expression. ResultsWe demonstrated that the mRNA and protein levels of WTAP were significantly increased in lesional skins of psoriasis patients and psoriatic cell model compared with normal controls. WTAP was highly expressed in epidermis rather than dermis. Overexpression of WTAP promoted keratinocytes proliferation, which might be related to the up-regulation of cyclinA2 and CDK2. ConclusionsThese results indicate that overexpression of WTAP may contribute to the pathogenesis of psoriasis by regulating cell cycle progression and highlight WTAP as a potential therapeutic target for psoriasis treatment.

Sauchinone: a prospective therapeutic agent-mediated EIF4EBP1 down-regulation suppresses proliferation, invasion and migration of lung adenocarcinoma cells.

Lung adenocarcinoma (LUAD) is the top prevalent histological kind of lung cancer worldwide. Recent evidences have demonstrated that Sauchinone plays an anticancer role in tumor cell invasion and migration. Therefore, we performed this investigation to explain the potential role of Sauchinone in LUAD as well as the potential mechanism involved. Cell counting kit 8 (CCK-8) and transwell experiments were implemented to measure the proliferative, invasive and migratory abilities of LUAD cells. qRT-PCR and Western blot were performed to detect the transfection efficiency of si-EIF4EBP1s. Additionally, Western blot was also implemented to evaluate the effect of Sauchinone on EIF4EBP1 expression level as well as cell cycle-related proteins. Our findings showed that Sauchinone remarkably suppressed the proliferative ability of LUAD cells in a dose-dependent and time-dependent manner. EIF4EBP1 was a candidate target gene of Sauchinone. EIF4EBP1 expression was increased in LUAD tissues, and its high expression induced a poorer prognosis of LUAD patients. EIF4EBP1 expression was positively associated with cell cycle in LUAD. Sauchinone treatment attenuated EIF4EBP1 expression and cell cycle-related protein levels. Knockdown of EIF4EBP1 repressed the proliferation, invasion and migration of LUAD cells; furthermore, Sauchinone stimulation enforced its inhibitory effect. Meanwhile, the treatment of Sauchinone intensified the arrest of cell cycle induced by EIF4EBP1 knockdown. To sum up, our discovery indicated that Sauchinone exerts an anticancer role through down-regulating EIF4EBP1 and mediating cell cycle in LUAD.

Mechanism of psoralen in aggravating hepatotoxicity induced by CCl_4 by delaying liver regeneration

This study aimed to investigate whether psoralen can aggravate hepatotoxicity induced by carbon tetrachloride(CCl_4) by inducing hepatocyte cycle arrest and delaying liver regeneration. Female C57 BL/6 mice aged 6-8 weeks were randomly divided into control group, model group(CCl_4 group), combined group(CCl_4+PSO group) and psoralen group(PSO group). CCl_4 group and CCl_4+PSO group were given CCl_4 intraperitoneally at a dose of 100 μL·kg~(-1) once; olive oil of the same volume was given to control group and PSO group intraperitoneally; 12 h, 36 h and 60 h after CCl_4 injection, PSO group and CCl_4+PSO group were administrated with PSO intragastrically at a dose of 200 mg·kg~(-1); 0.5% CMC-Na of the same volume was administrated to control group and PSO group intragastrically. The weight of mice was recorded every day. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were measured at 36 h, 60 h and 84 h after CCl_4 injection. Mice were sacrificed after collection of the last serum samples. Liver samples were collected, and liver weight was recorded. Histopathological and morphological changes of liver were observed by haematoxylin and eosin staining. The mRNA levels of HGF, TGF-β, TNF-α, p53 and p21 in liver were detected by RT-qPCR. Western blot was used to detect the levels of cell cycle-related proteins. According to the results, significant increase of serum ALT and AST and centrilobular necrosis with massive inflammatory cell infiltration were observed in CCl_4+PSO group. After PSO administration in CCl_4 model, the mRNA levels of HGF(hepatocyte growth factor) and TNF-α were reduced, while the mRNA expressions of TGF-β, p53 and p21 was up-regulated. The expression of PCNA(proliferating cell nuclear antigen) was significantly increased in CCl_4 and CCl_4+PSO group, while the relative protein level in CCl_4+PSO group was slightly lower than that in CCl_4 group. Compared with control and CCl_4 group, the expression of p27(cyclic dependent kinase inhibitor protein p27) was prominently increased in CCl_4+PSO group. These results indicated that hepatotoxicity induced by CCl_4 could be aggravated by intraperitoneal administration with PSO, and the repair process of liver could be delayed. The preliminary mechanism may be related to the inhibition of PCNA and regulation of some cell cycle-associated protein by psoralen, in which the significant up-regulation of p27, p53 and p21 may play important roles.

RNA m6 A methylation regulates uveal melanoma cell proliferation, migration, and invasion by targeting c-Met.

N6 -methyladenosine (m6 A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6 A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6 A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6 A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6 A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation-quantitative polymerase chain reaction identified c-Met as a direct target of m6 A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c-Met, p-Akt, and cell cycle-related protein levels in UM cells. Taken together, our results demonstrate that METTL3-mediated m6 A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c-Met. Such a modification acts as a critical oncogenic regulator in UM development.