The role of D3-type cyclins is related to cytokinin and the bHLH transcription factor SPATULA in Arabidopsis gynoecium development.

MAIN CONCLUSION: We studied the D3-type cyclin function during gynoecium development in Arabidopsis and how they are related to the hormone cytokinin and the transcription factor SPATULA. Growth throughout the life of plants is sustained by cell division and differentiation processes in meristematic tissues. In Arabidopsis, gynoecium development implies a multiphasic process where the tissues required for pollination, fertilization, and seed development form. The Carpel Margin Meristem (CMM) is a mass of undifferentiated cells that gives rise to the gynoecium internal tissues, such as septum, ovules, placenta, funiculus, transmitting tract, style, and stigma. Different genetic and hormonal factors, including cytokinin, control the CMM function. Cytokinin regulates the cell cycle transitions through the activation of cell cycle regulators as cyclin genes. D3-type cyclins are expressed in proliferative tissues, favoring the mitotic cell cycle over the endoreduplication. Though the role of cytokinin in CMM and gynoecium development is highly studied, its specific role in regulating the cell cycle in this tissue remains unclear. Additionally, despite extensive research on the relationship between CYCD3 genes and cytokinin, the regulatory mechanism that connects them remains elusive. Here, we found that D3-type cyclins are expressed in proliferative medial and lateral tissues. Conversely, the depletion of the three CYCD3 genes showed that they are not essential for gynoecium development. However, the addition of exogenous cytokinin showed that they could control the division/differentiation balance in gynoecium internal tissues and outgrowths. Finally, we found that SPATULA can be a mechanistic link between cytokinin and the D3-type cyclins. The data suggest that the role of D3-type cyclins in gynoecium development is related to the cytokinin response, and they might be activated by the transcription factor SPATULA.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function.

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 µM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 µM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 µg/mL of LPS, with or without 2 µM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 µg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 µg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Effect of Ferroptosis Inducers and Inhibitors on Cell Proliferation in Acute Leukemia.

BACKGROUND/AIM: Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation. MATERIALS AND METHODS: Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated. RESULTS: Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines. CONCLUSION: Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL.

Incidental Detection of TFEB-Amplified Renal Cell Carcinoma by Colocated Gene Amplification of CCND3 (6p21): A Case Report and Review of the Literature.

TFEB-amplified renal cell carcinoma (RCC), which belongs to the MITF family of RCC, is characterized by genomic amplification at the 6p21.1 locus where the TFEB gene is located. The vascular endothelial growth factor A and cyclin D3 genes are also located at this same locus. When tumors lack classic morphologic features, they may be classified as "RCC not otherwise specified (NOS)." However, it is increasingly important to accurately diagnose the RCC subtype to define the patient's individual prognosis and select the subsequent therapeutic modalities, which now include targeted agents. Therefore, knowledge of the diagnostic features of TFEB-altered RCCs, such as t(6;11) RCCs and TFEB-amplified RCCs, is critical for identifying these tumors. Herein, we present an interesting case of TFEB-amplified RCC that was initially diagnosed as RCC NOS on biopsy of a renal tumor in a community practice setting with available molecular findings demonstrating CCND3 amplification. The genetic abnormality was "accidentally" detected due to the amplification of the colocated CCND3 gene at the 6p21 locus of the TFEB gene on a limited genetic sequencing panel. This case highlights the importance of molecular tests in accurately diagnosing RCC and carefully interpreting molecular findings in the context of histomorphologic features.

The Cyclin D3 Protein Enforces Monogenic TCRß Expression by Mediating TCRß Protein-Signaled Feedback Inhibition of Vß Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vß-to-DßJß rearrangements in noncycling double-negative thymocytes, TCRß protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αß T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αß T cells that express TCRß proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRß expression, we used our mouse lines with enhanced rearrangement of specific Vß segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αß T cells that display proteins from RSS-augmented Vß segments on both alleles. By assaying mature αß T cells, we find that cyclin D3 deficiency increases the levels of Vß rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRß protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRß expression and resulting uniform specificity of individual αß T cells.

Lipopolysaccharides of Brucella suis S2 Impaired the Process of Decidualization in Early Pregnancy in Mice.

Brucellosis is a notorious zoonotic disease caused by Brucella, which can lead to reproductive diseases in humans and animals, such as infertility and abortion. Lipopolysaccharides (LPS) are the main virulence factor of Brucella. LPS derived from Brucella are different and non-classical and are less toxic and less active than LPS isolated from E. coli. However, the effects and possible mechanisms of Brucella LPS-caused pregnancy loss remain to be revealed. In the present study, we investigated the effects of Brucella suis S2 LPS on early pregnancy loss in mice. The results indicated that embryo implantation failure was induced by Brucella LPS treatment in a dose-dependent manner. The injection of Brucella LPS mainly resulted in fibrinolysis in the decidual area of the uterus on the 6th day post coition (dpc), infiltration of large granular cells among the decidual cells near the embryo on the 8th dpc, a large number of gaps in the decidual area, and cell necrosis around the embryo. In addition, the expression of Cyclin D3 mRNA in the uterus on the 7th and 8th dpc and IGFBP-1 mRNA and the progesterone receptor in the uterus on the 6th and 7th dpc were also inhibited. Moreover, the expression of decidualization marker Cyclin D3 and decidualization prolactin-associated protein (dPRP) in endometrial stromal cells were also inhibited by Brucella LPS treatment in vitro. In summary, Brucella LPS affect the process of endometrial decidualization in mice by affecting the structure of the decidua and the expression of decidual marker factors in endometrial stromal cells.

BRD4 PROTAC degrader MZ1 exhibits anti-B-cell acute lymphoblastic leukemia effects via targeting CCND3.

INTRODUCTION: B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent malignant tumor affecting children. While the majority of B-ALL patients (90%) experience successful recovery, early relapse cases of B-ALL continue to exhibit high mortality rates. MZ1, a novel inhibitor of Bromodomains and extra-terminal (BET) proteins, has demonstrated potent antitumor activity against hematological malignancies. The objective of this study was to examine the role and therapeutic potential of MZ1 in the treatment of B-ALL. METHODS: In order to ascertain the fundamental mechanism of MZ1, a sequence of in vitro assays was conducted on B-ALL cell lines, encompassing Cell Counting Kit 8 (CCK8) assay, Propidium iodide (PI) staining, and Annexin V/PI staining. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to examine protein and mRNA expression levels. Transcriptomic RNA sequencing (RNA-seq) was utilized to screen the target genes of MZ1, and lentiviral transfection was employed to establish stably-expressing/knockdown cell lines. RESULTS: MZ1 has been observed to induce the degradation of Bromodomain Containing 4 (BRD4), Bromodomain Containing 3 (BRD3), and Bromodomain Containing 2 (BRD2) in B-ALL cell strains, leading to inhibited cell growth and induction of cell apoptosis and cycle arrest in vitro. These findings suggest that MZ1 exhibits cytotoxic effects on two distinct molecular subtypes of B-ALL, namely 697 (TCF3/PBX1) and RS4;11 (MLL-AF4) B-ALL cell lines. Additionally, RNA-sequencing analysis revealed that MZ1 significantly downregulated the expression of Cyclin D3 (CCND3) gene in B-ALL cell lines, which in turn promoted cell apoptosis, blocked cell cycle, and caused cell proliferation inhibition. CONCLUSION: Our results suggest that MZ1 has potential anti-B-ALL effects and might be a novel therapeutic target.

IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation.

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced.

D-Type Cyclins in Development and Disease.

D-type cyclins encode G1/S cell cycle checkpoint proteins, which play a crucial role in defining cell cycle exit and progression. Precise control of cell cycle exit is vital during embryonic development, with defects in the pathways regulating intracellular D-type cyclins resulting in abnormal initiation of stem cell differentiation in a variety of different organ systems. Furthermore, stabilisation of D-type cyclins is observed in a wide range of disorders characterized by cellular over-proliferation, including cancers and overgrowth disorders. In this review, we will summarize and compare the roles played by each D-type cyclin during development and provide examples of how their intracellular dysregulation can be an underlying cause of disease.

Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

Targeting diacylglycerol kinase α impairs lung tumorigenesis by inhibiting cyclin D3.

BACKGROUND: Diacylglycerol kinase α (DGKA) is the first member discovered from the diacylglycerol kinase family, and it has been linked to the progression of various types of tumors. However, it is unclear whether DGKA is linked to the development of lung cancer. METHODS: We investigated the levels of DGKA in the lung cancer tissues. Cell growth assay, colony formation assay and EdU assay were used to examine the effects of DGKA-targeted siRNAs/shRNAs/drugs on the proliferation of lung cancer cells in vitro. Xenograft mouse model was used to investigate the role of DGKA inhibitor ritanserin on the proliferation of lung cancer cells in vivo. The downstream target of DGKA in lung tumorigenesis was identified by RNA sequencing. RESULTS: DGKA is upregulated in the lung cancer cells. Functional assays and xenograft mouse model indicated that the proliferation ability of lung cancer cells was impaired after inhibiting DGKA. And cyclin D3(CCND3) is the downstream target of DGKA promoting lung cancer. CONCLUSIONS: Our study demonstrated that DGKA promotes lung tumorigenesis by regulating the CCND3 expression and hence it can be considered as a potential molecular biomarker to evaluate the prognosis of lung cancer patients. What's more, we also demonstrated the efficacy of ritanserin as a promising new medication for treating lung cancer.

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance.

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

A rapidly growing cutaneous malignant glomus tumor with a CCND3 mutation.

Glomus tumors are rare mesenchymal neoplasms composed of cells resembling the glomus body. They are most frequently seen in subungual regions but have been reported to arise in almost every anatomic location. Malignant glomus tumors, also called glomangiosarcomas, of cutaneous origin are exceedingly rare with only 47 reported cases. The genetic alterations that lead to the development of cutaneous malignant glomus tumors are not well understood. Small studies report glomus tumors with mutations in glomulin (GLMN), NF1, BRAF, NOTCH, PDGFRB, KRAS, and SMARCB1. These mutations have mostly been studied in deep or visceral glomus tumors. We report a case of a cutaneous malignant glomus tumor with a CCND3 point mutation identified on next generation sequencing, without any of the previously described genetic mutations. CCND3 mutations that cause cyclin D3 amplification may prove to be targets for CDK4/6 inhibitors in the treatment of malignant glomus tumors.

[LncRNA RPL22P1-201 affects prostate cancer cell proliferation, cell cycle, and sensitivity to docetaxel by regulating miR-216b-5p expression].

OBJECTIVE: Exploring the effects and mechanisms of long non coding RNA (lncRNA) RPL22P1-201 on prostate cancer cell proliferation, cell cycle, and docetaxel sensitivity by regulating miR-216b-5p expression. METHODS: The Cancer LncRNA Census database was used to analyze the differential expression of RPL22P1-201 between prostate cancer tissue and normal tissue. Real time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of RPL22P1-201 in prostate cancer cell lines (DU-145, C4-2B, PC3, 22Rv1, LNCaP) and normal prostate epithelial cells (RWPE-1). PC3 cells were divided into si-RPL22P1-201 group (transfected with RPL22P1-201 interference sequence) and si-NC group (transfected with si-NC sequence). Colony formation assay was used to detect the proliferation ability of PC3 cells. Flow cytometry was used to detect the PC3 cell cycle. The CCK-8 method was used to detect the proliferation of PC3 cells in each group after treatment with docetaxel. The dual luciferase reporter gene experiment verifies the binding of RPL22P1-201 to the target gene. qRT-PCR was used to detect the expression level of miR-216b-5p. Western blot was used to detect the expression levels of TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 proteins. RESULTS: The expression level of RPL22P1-201 in prostate cancer tissue was higher than that in normal tissue (P<0.01). The expression level of RPL22P1-201 in prostate cancer cell lines was higher than that in normal prostate epithelial cells (P<0.01). The number of colonies in the si-NC group and si-RPL22P1-201 group was (256.1 ± 28.79) and (78.77 ± 14.52), respectively. The difference was statistically significant (P<0.01). The G0/G1 cell rates in the si-NC group and si-RPL22P1-201 group were (43.18 ± 4.56)% and (68.85 ± 3.40)%, respectively. The S cell rates were (36.84 ± 2.28)% and (24.27 ± 2.74)%, respectively. The G2/M cell rates were (19.98 ± 2.69)% and (6.88 ± 1.57)%, respectively, and the differences were statistically significant (all P<0.05). The cell survival rate of the si-RPL22P1-201 group under the action of docetaxel was lower than that of the si-NC group (all P<0.05). RPL22P1-201 can pair and bind with miR-216b-5p (P<0.01). Compared with the si-NC group, the si-RPL22P1-201 group showed a decrease in miR-216b-5p expression in PC3 cells (P<0.01), and a decrease in TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 protein expression. CONCLUSIONS: RPL22P1-201 is highly expressed in prostate cancer, and silencing RPL22P1-201 inhibits prostate cancer PC3 cell proliferation and cell cycle by increasing miR-216b-5p expression, and enhances PC3 cell sensitivity to docetaxel.

Age- and cell cycle-related expression patterns of transcription factors and cell cycle regulators in Müller glia.

Mammalian Müller glia express transcription factors and cell cycle regulators essential for the function of retinal progenitors, indicating the latent neurogenic capacity; however, the role of these regulators remains unclear. To gain insights into the role of these regulators in Müller glia, we analyzed expression of transcription factors (Pax6, Vsx2 and Nfia) and cell cycle regulators (cyclin D1 and D3) in rodent Müller glia, focusing on their age- and cell cycle-related expression patterns. Expression of Pax6, Vsx2, Nfia and cyclin D3, but not cyclin D1, increased in Müller glia during development. Photoreceptor injury induced cell cycle-associated increase of Vsx2 and cyclin D1, but not Pax6, Nfia, and cyclin D3. In dissociated cultures, cell cycle-associated increase of Pax6 and Vsx2 was observed in Müller glia from P10 mice but not from P21 mice. Nfia levels were highly correlated with EdU incorporation suggesting their activation during S phase progression. Cyclin D1 and D3 were transiently upregulated in G1 phase but downregulated after S phase entry. Our findings revealed previously unknown links between cell cycle progression and regulator protein expression, which likely affect the cell fate decision of proliferating Müller glia.

Non-canonical miRNA-RNA base-pairing impedes tumor suppressor activity of miR-16.

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as &lt;i&gt;cyclin D3&lt;/i&gt;, is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient's OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease.

BMP and activin membrane-bound inhibitor regulate connective tissue growth factor controlling mesothelioma cell proliferation.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive mesothelial cell cancer type linked mainly to asbestos inhalation. MM characterizes by rapid progression and resistance to standard therapeutic modalities such as surgery, chemotherapy, and radiotherapy. Our previous studies have suggested that tumor cell-derived connective tissue growth factor (CTGF) regulates the proliferation of MM cells as well as the tumor growth in mouse xenograft models. METHODS: In this study, we knock downed the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and CTGF in MM cells and investigated the relationship between both and their impact on the cell cycle and cell proliferation. RESULTS: The knockdown of CTGF or BAMBI reduced MM cell proliferation. In contrast to CTGF knockdown which decreased BAMBI, knockdown of BAMBI increased CTGF levels. Knockdown of either BAMBI or CTGF reduced expression of the cell cycle regulators; cyclin D3, cyclin-dependent kinase (CDK)2, and CDK4. Further, in silico analysis revealed that higher BAMBI expression was associated with shorter overall survival rates among MM patients. CONCLUSIONS: Our findings suggest that BAMBI is regulated by CTGF promoting mesothelioma growth by driving cell cycle progression. Therefore, the crosstalk between BAMBI and CTGF may be an effective therapeutic target for MM treatment.