基于单细胞转录组的视网膜母细胞瘤进展特征分析

视网膜母细胞瘤（retinoblastoma,RB）是婴幼儿最常见的眼内恶性肿瘤。在RB进展过程中的关键致病因素目前尚不十分清楚。因此,识别与RB进展密切相关的基因能为病情诊断及基因治疗提供重要信息。然而,肿瘤组织具有很强的细胞异质性,不同病理状态下的细胞,其功能及基因表达都可能呈现显著的差异。本研究从公共基因表达数据库（gene expression omnibus,GEO）下载了1例4个月肿瘤患者和1例2年患者的肿瘤及癌旁组织的单细胞转录组测序数据,从单细胞水平解析不同患病时长的RB肿瘤转录图谱,鉴定与RB进展有潜在关联的细胞亚群及基因集。结果显示,肿瘤组织与癌旁组织在单细胞转录图谱上具有整体的一致性,但视锥前体G1期细胞群、G2期细胞群以及小胶质细胞群在肿瘤与癌旁组织中的分布比例存在明显差异。进一步分析了这3种细胞群在RB肿瘤进展过程中的作用。研究发现,在RB肿瘤的早期阶段,视锥前体细胞在G1期异常增殖,随着RB肿瘤的进展,视锥前体G2期细胞比例显著增加。同时,RB进展过程的小胶质细胞群差异分析结果显示,主要参与免疫应答的关键基因包括RPL23、B2M、HLA家族基因。本研究可为RB发病机制及进展研究提供更多新视角和数据资源。     

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.     

Putative roles of retinoblastoma protein in apoptosis

Cell numbers are regulated by a balance between processes of proliferation and apoptosis (programmed cell death). Proper regulation in a cell requires an accurate co-ordination between these two processes. Indeed, it has recently been found that dysregulation of cell cycle progression is essential for the initiation of apoptosis. Retinoblastoma protein (RB) is an important tumour suppressor and a cell cycle regulator. Most recent studies suggest that RB also plays a regulatory role in the process of apoptosis. During the onset of apoptosis, the hyperphosphorylated form of RB (p120/hyper) is converted to a hypophosphorylated form (p115/hypo), which is mediated by a specific protein-serine/ threonine phosphatase activity. The p115/hypo/RB may play an active role in the regulation of apoptosis. Accompanied by the endonucleosomal fragmentation of DNA, the newly formed p115/hypo/RB is immediately cleaved by a protease that has properties of the interleukin-1beta-converting enzyme family. By contrast, the unphosphorylated form of RB (p110/unphos) remains uncleaved during apoptosis. Further studies suggest that p110/unphos/RB functions as an inhibitor of apoptosis. Therefore, a balance between RB phosphatases and kinases and consequent RB phosphorylation status may be important for the determination of cellular fate.     

The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.     

Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53

p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.     

Phosphoinositide 3-kinases p110alpha and p110beta regulate cell cycle entry, exhibiting distinct activation kinetics in G1 phase

Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G(0)/G(1) transition) and again in late G(1). The two ubiquitous PI3K isoforms (p110alpha and p110beta) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110alpha was activated first in G(0)/G(1), followed by a minor p110beta activity peak. In late G(1), p110alpha activation preceded that of p110beta, which showed the maximum activity at this time. p110beta activation required Ras activity, whereas p110alpha was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110alpha and -beta activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110alpha in blocking early G(1) events. We show that inhibition of either p110alpha or p110beta reduced cell cycle entry. These results reveal that PI3Kalpha and -beta present distinct activation requirements and kinetics in G(1) phase, with a selective action of PI3Kalpha at the G(0)/G(1) phase transition. Nevertheless, PI3Kalpha and -beta both regulate S-phase entry.     

Primary and compensatory roles for RB family members at cell cycle gene promoters that are deacetylated and downregulated in doxorubicin-induced senescence of breast cancer cells

When treated with DNA-damaging chemotherapy agents, many cancer cells, in vivo and in vitro, undergo a terminal growth arrest and acquire a senescence-like phenotype. We investigated the molecular basis for this in breast cancer cells following a 2-hour treatment with 1 muM doxorubicin. Treated cells arrested in G1 and G2 phases of the cell cycle, with concomitant reductions in S-phase and G2-M regulatory genes. p53 and p21 protein levels increased within hours after treatment and were maintained for 5 to 6 days but were reduced 8 days posttreatment, though the cells remained growth arrested. Levels of p130 rose after drug treatment, and it was the primary RB family member recruited to the S-phase promoters cyclin A and PCNA and G2-M promoters cyclin B and cdc2, remaining present for the entire 8-day time period. In contrast, p107 protein and promoter occupancy levels declined sharply after drug treatment. RB was recruited to only the PCNA promoter. In MCF-7 cells with p130 knockdown, p107 compensated for p130 loss at all cell cycle gene promoters examined, allowing cells to retain the growth arrest phenotype. Cells with p130 and p107 knockdown similarly arrested, while cells with knockdown of all three family members failed to downregulate cyclin A and cyclin B. These results demonstrate a mechanistic role for p130 and compensatory roles for p107 and RB in the long-term senescence-like growth arrest response of breast cancer cells to DNA damage.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system

The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression.     

Ceramide mimics tumour necrosis factor-alpha in the induction of cell cycle arrest in endothelial cells. Induction of the tumour suppressor p53 with decrease in retinoblastoma/protein levels.

Tumour necrosis factor (TNF)-alpha induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-alpha signalling. We tested the ability of C8-ceramide to reproduce the TNF-alpha-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-alpha (10 ng.mL-1) and C8-ceramide (20 microM) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-alpha and C8-ceramide induced increased expression of p53. Treatment with TNF-alpha or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-alpha prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway.     

Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation

The growth-suppressive function of the retinoblastoma gene product, RB, has been ascribed to the underphosphorylated RB form that prevails during G1 phase in the cell cycle. We show that addition of the paracrine growth inhibitor transforming growth factor beta 1 (TGF-beta 1) to Mv1Lu lung epithelial cells in mid to late G1 prevents phosphorylation of RB scheduled for this cell cycle stage and arrests cells in late G1. Expression of SV40 T antigen, a transforming protein that binds underphosphorylated RB, does not block the effect of TGF-beta 1 on RB phosphorylation but greatly reduces the growth-inhibitory response to TGF-beta 1. TGF-beta 1 and RB appear to function in a common growth-inhibitory pathway in which TGF-beta 1 acts to retain RB in the underphosphorylated, growth-suppressive state.     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.