SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.     

Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer

The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).     

Proteasome-dependent degradation of human CDC25B phosphatase

The CDC25 dual specificity phosphatase is a universal cell cycle regulator. The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery. CDC25 phosphatase both dephosphorylates and activates CDKs. Three human CDC25s have been identified. CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK1-cyclin B. The exact function of CDC25B however remains elusive. We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B. Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a mitotic starter triggering the activation of an auto-amplification loop before being degraded.     

MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells

Highly selective therapy for hepatocellular carcinoma (HCC) remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4%) and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011(let7T), by introducing eight copies of let-7 target sites (let7T) into the 3' untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels) of the SG7011(let7T) was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011(let7T) was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68) expressing high level of let-7 (>300 folds), whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5) with low level of let-7. Consequently, the cytotoxicity of SG7011(let7T) to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011(let7T)in vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011(let7T) may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Background

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.

Methodology/Principal

Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.

Conclusions/Significance

These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer.     

Extracellular vesicles-encapsulated let-7i shed from bone mesenchymal stem cells suppress lung cancer via KDM3A/DCLK1/FXYD3 axis

Accumulating evidence has suggested that extracellular vesicles (EVs) play a crucial role in lung cancer treatment. Thus, we aimed to investigate the modulatory role of bone marrow mesenchymal stem cell (BMSC)-EV-derived let-7i and their molecular mechanism in lung cancer progression. Microarray-based analysis was applied to predict lung cancer-related miRNAs and their downstream genes. RT-qPCR and Western blot analyses were conducted to determine Let-7i, lysine demethylase 3A (KDM3A), doublecortin-like kinase 1 (DCLK1) and FXYD domain-containing ion transport regulator 3 (FXYD3) expressions, after which dual-luciferase reporter gene assay and ChIP assay were used to identify the relationship among them. After loss- and gain-of-function assays, the effects of let-7i, KDM3A, DCLK1 and FXYD3 on the biological characteristics of lung cancer cells were assessed. Finally, tumour growth in nude mice was assessed by xenograft tumours in nude mice. Bioinformatics analysis screened out the let-7i and its downstream gene, that is KDM3A. The findings showed the presence of a high expression of KDM3A and DCLK1 and reduced expression of let-7i and FXYD3 in lung cancer. KDM3A elevated DCLK1 by removing the methylation of H3K9me2. Moreover, DCLK1 suppressed the FXYD3 expression. BMSC-EV-derived let-7i resulted in the down-regulation of KDM3A expression and reversed its promoting role in lung cancer development. Consistently, in vivo experiments in nude mice also confirmed that tumour growth was suppressed by the BMSC-EV-derived let-7i. In conclusion, our findings demonstrated that the BMSC-EV-derived let-7i possesses an inhibitory role in lung cancer progression through the KDM3A/DCLK1/FXYD3 axis, suggesting a new molecular target for lung cancer treatment.     

CDC25B Phosphorylation by Aurora A Occurs at the G2/M Transition and is Inhibited by DNA Damage

CDC25B is one of the three human dual-specificity phosphatases involved in the activation ofcyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsiblefor the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-Akinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/Mtransition. Here we have investigated this phosphorylation event during the cell cycle, and inresponse to activation of the G2 DNA damage checkpoint. We show that accumulation of theS353-phosphorylated form of CDC25B at the centrosome correlates with the relocalisation ofcyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation ofthe G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated andconsequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-Aresults in a bypass of the checkpoint that partially overcome by a S353A mutant of CDC25B.Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These resultsstrongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is animportant step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclinB1 activation.     

Gankyrin: a new oncoprotein and regulator of pRb and p53

Gankyrin is a new oncoprotein with potent cell cycle and apoptotic properties that is overexpressed early in hepatocarcinogenesis and in hepatocellular carcinomas. Gankyrin regulates the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and enhances the ubiquitylation of p53 by the RING ubiquitin ligase MDM2. Purified preparations of the 26S proteasome contain gankyrin, which specifically interacts with the S6b (Rpt3) ATPase of the 19S regulator. In conclusion, gankyrin is a small versatile cell cycle regulator that illustrates the essential interplay between the ubiquitin proteasome system and gene expression in the cell. Here, we discuss the activities of gankyrin and present a model for its function in the regulation of pRb and p53.     

高原鼢鼠肝脏组织细胞周期相关基因的进化和表达

高原鼢鼠Myospalax baileyi是一种世居青藏高原的地下鼠,对严重的低氧环境有很强的适应性。低氧诱导细胞周期G1、G2期阻滞。为了探讨高原鼢鼠适应低氧环境的分子机制,应用生物信息学方法对p53下游细胞周期基因p21、CyclinD1、CyclinE、CDK6、CDK2、14-3-3-σ、Gadd45α、B99和CyclinB1的序列和编码的氨基酸序列进行了进化分析,并以SD大鼠Rattus norvegicus为对照,研究了这些基因在不同海拔(3300 m、2260 m)条件下的表达模式。结果表明:(1)高原鼢鼠细胞周期相关基因的序列与以色列鼹鼠Nannospalax galili同源性最高,达到90%以上;p21、CyclinD1、CyclinE和CyclinB1编码蛋白与以色列鼹鼠存在明显的趋同进化位点;SIFT评估发现,p21和CyclinB1氨基酸序列分别在第27号位点和第105号位点的变异对细胞周期调控功能有显著影响;(2)与低海拔条件相比,在高海拔条件下,高原鼢鼠肝脏组织中与G1期相关的基因p21表达水平显著上升,p21下游基因CyclinD1、CyclinE、CDK6和CDK2表达水平显著下降,而在SD大鼠中没有显著变化;与G2期相关的基因Gadd45α、B99、14-3-3-δ和CyclinB1在高原鼢鼠和SD大鼠中随海拔变化不发生明显变化。在不同海拔条件下,高原鼢鼠肝脏组织中的上述细胞周期相关基因的表达水平均极显著高于SD大鼠(P<0.01)。以上结果提示,高原鼢鼠经过长期的低氧适应,通过上调p21基因的表达抑制下游CyclinD1、CyclinE、CDK6和CDK2基因的表达,导致细胞周期G1期阻滞,从而提供充足的时间进行DNA修复,保证了DNA复制的准确性;同时高原鼢鼠肝脏组织中细胞周期的调控不仅与细胞周期基因的表达水平有关,而且可能与细胞周期因子p21的第27号位点和CyclinB1的第105号位点的变异有关。     

Induction of apoptosis in human leukemia cells by the CDK1 inhibitor CGP74514A

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 microM CGP74514A induced mitochondrial damage (i.e., loss of Delta psi(m)) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 microM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor lETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bel- 2, a loop-deleted mutant Bcl-2, and Bcl-x(L). CGP74514A treatment (5 microM; 18 hr) resulted in increased p21(CIP1) expression, p27(KIP1) degradation, diminished E2F1 expression, and dephosphorylation of p34(CDC2). It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.