microRNA-16-5p suppresses cell proliferation and angiogenesis in colorectal cancer by negatively regulating forkhead box K1 to block the PI3K/Akt/mTOR pathway

MicroRNAs (miRNAs/miRs) have aroused increasing attention in colorectal cancer (CRC) therapy. This study is designed for a detailed analysis of the roles of miR-16-5p and forkhead box K1 (FOXK1) in cell angiogenesis and proliferation during CRC in addition to their underlying mechanisms. CRC tissues and colon cancer cell lines (SW620 and HCT8) were investigated. qRT-PCR and Western blot were utilized to evaluate miR-16-5p and FOXK1 expression. Following gain- and loss-of-function assays on miR-16-5p or FOXK1, the effects of miR-16-5p and FOXK1 were assessed on cell angiogenesis and proliferation in CRC cells. A dual-luciferase reporter assay was employed to evaluate the binding relationship of miR-16-5p and FOXK1. Western blot was used to determine the effects of miR-16-5p and FOXK1 on key molecules of the PI3K/Akt/mTOR pathway. Highly expressed FOXK1 and lowly expressed miR-16-5p were observed in CRC cells and tissues. miR-16-5p overexpression or FOXK1 knockdown reduced CRC cell proliferation and angiogenesis of human umbilical vein endothelial cells co-cultured with the supernatant of CRC cells, whereas miR-16-5p silencing or FOXK1 upregulation caused opposite trends. Additionally, miR-16-5p negatively modulated FOXK1 expression. The blockade of the PI3K/Akt/mTOR pathway was triggered by miR-16-5p overexpression or FOXK1 silencing. In conclusion, miR-16-5p hampers cell angiogenesis and proliferation during CRC by targeting FOXK1 to block the PI3K/Akt/mTOR pathway.Key words: microRNA-16-5p, forkhead box K1, PI3K/Akt/mTOR pathway, colorectal cancer, proliferation, angiogenesis     

Angiogenesis and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle

Prolactin (PRL) is a pituitary hormone involved in a broad spectrum of physiological processes, including lactation, development, and immune function. To further investigate the in vivo roles of PRL, rat PRL cDNA, fused to the cytomegalovirus promoter, was introduced into mouse muscle by direct injection. Prolactin mRNA and protein were detected in the muscle following injection. As a result the number of white blood cells (WBC) increased. When injection was combined with adrenalectomy there was an even greater increase. The augmentation of WBCs persisted for at least 20 days after injection of the rPRL plasmid either on its own and after injection combined with adrenalectomy. The increase in WBCs was accompanied in both cases by an increase in blood cell DNA content. We also observed an increase in heart volume, particularly of the left ventricle. Evidence of marked angiogenesis was found in the testis of rPRL- injected mice. New blood vessels were first found at 8 weeks of age and fully developed blood vessels with complex branching patterns were found after 11 weeks. When PRL fused with EGFP was introduced into mice by intramuscular injection, the EGFP localized to areas of the testis that corresponded to the sites of new blood vessel formation. PRL inhibited this binding. Taken together, our data reveal that intramuscularly expressed PRL augments WBC numbers and induces formation of new blood vessels in the testis, suggesting important roles for PRL in hematopoiesis and angiogenesis. They also indicate that direct intramuscular injection of naked DNA can be used effectively to study the function of secreted proteins, including endocrine signaling molecules.     

Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle

Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.     

The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1(23), that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1(23) effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-1(23) was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-1(23) treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-1(23) blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-1(23) stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-1(23) anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.     

Sperm motility is dependent on a unique isoform of the Na,K-ATPase

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, alpha and beta, and is responsible for translocating Na(+) out of the cell and K(+) into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the alpha and beta subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The alpha4 isoform is the most recently identified Na, K-ATPase alpha isoform, and its expression has been found only in testis. Here we show that expression of the alpha4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the alpha4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.     

Angiogenesis modulators expression in culture cell lines positives for HPV-16 oncoproteins

Altered angiogenesis response is observed in patients with cervical cancer. In this study we examined whether Human Papilloma Virus (HPV) positive epithelial cells are able to produce angiogenic modulators. When added to human umbilical vein endothelial cells (HUVEC) the media conditioned by HPV-16 positive cells was able to induce proliferation, whereas a contrary effect was observed for media derived from non-tumorigenic keratinocytes. The analyses of angiogenesis modulator's mRNA levels result in a decrease of the antiangiogenic factors TSP-1 and 2 in HPV-16 positive cells. In contrast the expression of the pro-angiogenic molecules: bFGF, IL-8, TGF-beta, TNFalpha, and VEGF were higher in these cells as compared to control keratinocytes. Furthermore the pattern of VEGF isoforms observed in the cells positive for the viral genome point to a preferential induction of the VEGF(189) isoform. We therefore conclude that cervical cancer cells expressing HPV-16 genome are able to contribute to the pro-angiogenic response that might support tumor growth and invasion of the surrounding tissues.     

Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis

In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998     

Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms.

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2alpha or CK2alpha') together with the regulatory CK2beta subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2alpha or CK2alpha' (i. e. GK2alpha (K68M) or CK2alpha'(K69M)) together with CK2beta. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2alpha or CK2alpha' resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2alpha differed significantly from the effects of induced expression of kinase-inactive CK2alpha'. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha'(K69M), but not following induction of CK2alpha(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions.     

Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.     

哺乳动物雷帕霉素靶蛋白（mTOR）在不同发育阶段SD大鼠睾丸中的表达及作用

精子发生是男性生殖中的主要过程,精原细胞的不断分裂增殖又保证了精子发生的顺利进行。随着年龄的不断增长,男性精子的数量、质量出现下降趋势。mTOR信号转导通路在细胞增殖分化中发挥着中心调控作用,因此,mTOR信号通路可能在精子发生过程中有着重要的地位。为了探明mTOR信号通路与精子发生的关系,首先,通过SD大鼠睾丸组织切片的免疫组化,发现mTOR是在生精小管的精原细胞胞浆中表达;其次,采用FQ-PCR检测mTOR mRNA在SD大鼠睾丸中的表达。结果显示,80周龄组mTOR的转录与8周龄组相比差异显著。最后利用Western blot检测出mTOR蛋白的表达及其对下游靶蛋白P70S6K的磷酸化效率均随年龄的增长逐渐下降。同时,在用雷帕霉素处理8周龄SD大鼠中,发现精子数量减少,P70S6K磷酸化效率降低并伴随生精小管萎缩和空泡化。通过这些结果,可以看出mTOR信号转导通路可能在精子发生中发挥着重要作用。     

FLIP is expressed in mouse testis and protects germ cells from apoptosis

Apoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIP(L)) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIP(L) expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIP(L) expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIP(L) expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIP(L) expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIP(L), are sensitive to Fas-mediated apoptosis.These data indicate for the first time that c-FLIP(L) might control germ cell apoptosis and caspase activity in the adult testis.     

Bevacizumab 联合吉西他滨对荷肝癌裸鼠移植瘤生长的抑制作用

目的:探讨抗血管生成药物Bevacizumab联合吉西他滨对人肝癌裸鼠皮下移植瘤生长的抑制作用。方法:构建人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为空白对照组、Bevacizumab组、吉西他滨组和联合用药组。观察用药前后肿瘤体积,绘制肿瘤生长曲线;应用免疫组化检测肿瘤微血管密度(MVD);Western Blot检测Bcl-2蛋白的表达。结果:Bevacizumab和吉西他滨单药均能抑制肿瘤生长,两药联合疗效明显增强(P=0.000)。与对照组和吉西他滨组相比,Bevacizumab组和联合用药组能明显抑制肿瘤血管生成,MVD值均明显降低,以联合用药组最为明显(P均0.000)。Bevacizumab和吉西他滨单药均能下调Bcl-2的表达,两药联合下调作用明显增强。结论:Bevacizumab联合吉西他滨能增强对人肝癌裸鼠移植瘤的生长及微血管生成的抑制作用,其机制可能与调控Bcl-2的表达有关。     

Epigallocatechin gallate suppresses hepatic cholesterol synthesis by targeting SREBP-2 through SIRT1/FOXO1 signaling pathway

To investigate the mechanism of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and human adipose-derived stem cells (hADSCs) in the treatment of Achilles tendinitis. Biomechanical indices of stiffness, stress, and maximum load-to-failure were detected by biomechanical test. mRNA and protein levels of miR-363, p-PI3K/AKT, tendon-related genes Collagen I, Scleraxis (Scx), and Tenascin C (TNC) were measured by qRT-PCR and western blot. The proliferation of hADSCs was accessed by MTT assay. Biomechanical indices of stiffness, stress, and maximum load-to-failure, and mRNA and protein levels of tendon-related genes could be improved by rhPDGF-BB or hADSCs alone, and could be further improved by rhPDGF-BB + hADSCs. rhPDGF-BB and hADSCs downregulated the expression of miR-363 and upregulated the levels of p-PI3K/Akt, and rhPDGF-BB + hADSCs further strengthened these effects. In addition, rhPDGF-BB promoted the proliferation of hADSCs in vitro and upregulated the expression of tendon-related genes. miR-363 mimic downregulated the levels of p-PI3K/Akt, miR-363 inhibitor upregulated the levels of p-PI3K/Akt, and miR-363 mimic and PI3K/Akt pathway inhibitor LY294002 reversed the positive effect of rhPDGF-BB on the proliferation of hADSCs, which suggested that rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway. Biomechanical indices and tendon-related genes could be improved by rhPDGF-BB and hADSCs. Moreover, rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway, indicating that rhPDGF-BB combined with ADSCs could treat Achilles tendinitis via miR-363/PI3K/Akt pathway.