The cyclin D1-dependent kinase associates with the pre-replication complex and modulates RB.MCM7 binding

The capacity of the cyclin D-dependent kinase to promote G(1) progression through modulation of RB.E2F is well documented. We now demonstrate that the cyclin D1/CDK4 kinase binds to components of the MCM complex. MCM7 and MCM3 were identified as cyclin D1-binding proteins. Catalytically active cyclin D1/CDK4 complexes were incorporated into chromatin-bound protein complexes with the same kinetics as MCM7 and MCM3, where they associated specifically with MCM7. Although the cyclin D1-dependent kinase did not phosphorylate MCM7, active cyclin D1/CDK4, but not cyclin E/CDK2, did catalyze the dissociation of an RB.MCM7 complex. Finally, expression of an active D1/CDK4 kinase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data suggest that D1/CDK4 complexes play a direct role in altering an inhibitory RB.MCM7 complex possibly allowing for setting of the origin in preparation for DNA replication.     

MiR‐106b inhibition suppresses inflammatory bone destruction of wear debris‐induced periprosthetic osteolysis in rats

Aseptic loosening caused by periprosthetic osteolysis (PPO) is the main reason for the primary artificial joint replacement. Inhibition of inflammatory osteolysis has become the main target of drug therapy for prosthesis loosening. MiR‐106b is a newly discovered miRNA that plays an important role in tumour biology, inflammation and the regulation of bone mass. In this study, we analysed the in vivo effect of miR‐106b on wear debris‐induced PPO. A rat implant loosening model was established. The rats were then administrated a lentivirus‐mediated miR‐106b inhibitor, miR‐106b mimics or an equivalent volume of PBS by tail vein injection. The expression levels of miR‐106b were analysed by real‐time PCR. Morphological changes in the distal femurs were assessed via micro‐CT and histopathological analysis, and cytokine expression levels were examined via immunohistochemical staining and ELISA. The results showed that treatment with the miR‐106b inhibitor markedly suppressed the expression of miR‐106b in distal femur and alleviated titanium particle‐induced osteolysis and bone loss. Moreover, the miR‐106b inhibitor decreased TRAP‐positive cell numbers and suppressed osteoclast formation, in addition to promoting the activity of osteoblasts and increasing bone formation. MiR‐106b inhibition also significantly regulated macrophage polarization and decreased the inflammatory response as compared to the control group. Furthermore, miR‐106b inhibition blocked the activation of the PTEN/PI3K/AKT and NF‐κB signalling pathways. Our findings indicated that miR‐106b inhibition suppresses wear particles‐induced osteolysis and bone destruction and thus may serve as a potential therapy for PPO and aseptic loosening.     

Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

Pathological cardiac hypertrophy (CH) is a key factor leading to heart failure and ultimately sudden death. Long non‐coding RNAs (lncRNAs) are emerging as a new player in gene regulation relevant to a wide spectrum of human disease including cardiac disorders. Here, we characterize the role of a specific lncRNA named cardiac hypertrophy‐associated regulator (CHAR) in CH and delineate the underlying signalling pathway. CHAR was found markedly down‐regulated in both in vivo mouse model of cardiac hypertrophy induced by pressure overload and in vitro cellular model of cardiomyocyte hypertrophy induced by angiotensin II (AngII) insult. CHAR down‐regulation alone was sufficient to induce hypertrophic phenotypes in healthy mice and neonatal rat ventricular cells (NRVCs). Overexpression of CHAR reduced the hypertrophic responses. CHAR was found to act as a competitive endogenous RNA (ceRNA) to down‐regulate miR‐20b that we established as a pro‐hypertrophic miRNA. We experimentally established phosphatase and tensin homolog (PTEN), an anti‐hypertrophic signalling molecule, as a target gene for miR‐20b. We found that miR‐20b induced CH by directly repressing PTEN expression and indirectly increasing AKT activity. Moreover, CHAR overexpression mitigated the repression of PTEN and activation of AKT by miR‐20b, and as such, it abrogated the deleterious effects of miR‐20b on CH. Collectively, this study characterized a new lncRNA CHAR and unravelled a new pro‐hypertrophic signalling pathway: lncRNA‐CHAR/miR‐20b/PTEN/AKT. The findings therefore should improve our understanding of the cellular functionality and pathophysiological role of lncRNAs in the heart.     

miR-106b-93-25簇对子宫内膜癌细胞的调控作用研究

目的:检测mi R-106b-93-25基因簇对子宫内膜癌细胞增殖及凋亡的影响,并探讨其机制。方法:q RT-PCR检测临床子宫内膜癌标本及癌旁正常组织中mi R-106b、mi R-93和mi R-25及其宿主基因MCM7的表达情况。将micro RNA及其拮抗剂转染ECC-1细胞后,MTT实验检测ECC-1细胞增殖情况,流式细胞术检测ECC-1细胞周期及细胞凋亡情况。荧光素酶报告系统验证mi R-106b和mi R-25分别直接调控p21和Bim。结果:临床标本子宫内膜癌组织与癌旁正常组织相比mi R-106b-93-25簇及其宿主基因MCM7的表达明显增高。mi R-106b-93-25簇能够促进ECC-1细胞增殖,减少凋亡。转染mi R-106b和mi R-93的细胞出现明显的S期阻滞,过表达mi R-25的细胞凋亡明显减少。mi R-106b-93-25簇通过抑制靶基因p21和Bim的表达,引起促增殖、抗凋亡作用。结论:mi R-106b-93-25簇能够促进子宫内膜癌细胞增殖,抑制凋亡,并使细胞发生S期阻滞。mi R-106b-93-25簇在子宫内膜癌的发生与发展中具有重要的作用。     

Shikonin suppresses progression and epithelial–mesenchymal transition in hepatocellular carcinoma (HCC) cells by modulating miR‐106b/SMAD7/TGF‐β signaling pathway

Shikonin is a natural naphthoquinone component with antioxidant and anti‐tumor function and has been used for hepatocellular carcinoma (HCC) treatment. According to the previous study, many herbs can regulate cancer cell progression by targeting specific microRNA (miRNA) (Liu, 2016). However, the underlying pathological mechanism of shikonin in HCC therapy is still unclear. The detection of cell growth and death rate were performed by hemacytometry and trypan blue staining, respectively. The expression of miR‐106b and SMAD7 messenger RNA (mRNA) in HCC cells was evaluated by quantitative real‐time polymerase chain reaction. Cell proliferation, apoptosis, and migration ability were measured by cell counting kit‐8 (CCK‐8), flow cytometry, and transwell assay. The expression of proteins E‐cadherin, N‐cadherin, vimentin, SMAD7, TGF‐β1, p‐SMAD3, SMAD3, and GAPDH was examined by western blot. The interaction between SMAD7 and miR‐106b was assessed by luciferase reporter system. Shikonin inhibited Huh7 and HepG2 cell growth in a dose‐dependent manner while induced cell death in a time‐dependent manner. In addition, the expression of miR‐106b was reduced after shikonin treatment. Moreover, miR‐106b attenuated the suppressive effects of shikonin on HCC cell migration and epithelial–mesenchymal transition (EMT). SMAD7 was predicted as a target of miR‐106b and the prediction was confirmed by luciferase reporter system. Additionally, we observed that SMAD7 reversed the promotive effects of miR‐106b on HCC cell progression and EMT. The subsequent western blot assay revealed that shikonin could modulate SMAD7/TGF‐β signaling pathway by targeting miR‐106b. In conclusion, Shikonin suppresses cell progression and EMT and accelerates cell death of HCC cells via modulating miR‐106b/SMAD7/TGF‐β signaling pathway, suggesting shikonin could be an effective agent for HCC treatment.     

Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.     

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Background

Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines.

Results

The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other.

Conclusion

Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.     

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKT^Ser473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.Subject terms: Cancer, Chronic lymphocytic leukaemia     

Distinct action of the retinoblastoma pathway on the DNA replication machinery defines specific roles for cyclin-dependent kinase complexes in prereplication complex assembly and S-phase progression

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G1, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.