Lentivirus-delivered Short Hairpin RNA Research Articles

Keratin 15 protects against cigarette smoke-induced epithelial mesenchymal transformation by MMP-9

BackgroundChronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease, is a leading cause of morbidity and mortality worldwide. Prolonged cigarette smoking (CS) that causes irreversible airway remodeling and significantly reduces lung function is a major risk factor for COPD. Keratin15+ (Krt15+) cells with the potential of self-renewal and differentiation properties have been implicated in the maintenance, proliferation, and differentiation of airway basal cells; however, the role of Krt15 in COPD is not clear.MethodsKrt15 knockout (Krt15−/−) and wild-type (WT) mice of C57BL/6 background were exposed to CS for six months to establish COPD models. Krt15-CrePGR;Rosa26-LSL-tdTomato mice were used to trace the fate of the Krt15+ cells. Hematoxylin and eosin (H&E) and Masson stainings were performed to assess histopathology and fibrosis, respectively. Furthermore, lentivirus-delivered short hairpin RNA (shRNA) was used to knock down KRT15 in human bronchial epithelial (HBE) cells stimulated with cigarette smoke extract (CSE). The protein expression was assessed using western blot, immunohistochemistry, and enzyme-linked immunosorbent assay.ResultsKrt15−/− CS mice developed severe inflammatory cell infiltration, airway remodeling, and emphysema. Moreover, Krt15 knockout aggravated CS-induced secretion of matrix metalloproteinase-9 (MMP-9) and epithelial–mesenchymal transformation (EMT), which was reversed by SB-3CT, an MMP-9 inhibitor. Consistent with this finding, KRT15 knockdown promoted MMP-9 expression and EMT progression in vitro. Furthermore, Krt15+ cells gradually increased in the bronchial epithelial cells and were transformed into alveolar type II (AT2) cells.ConclusionKrt15 regulates the EMT process by promoting MMP-9 expression and protects the lung tissue from CS-induced injury, inflammatory infiltration, and apoptosis. Furthermore, Krt15+ cells transformed into AT2 cells to protect alveoli. These results suggest Krt15 as a potential therapeutic target for COPD.

Inhibition of Spinal Interleukin-33 Attenuates Peripheral Inflammation and Hyperalgesia in Experimental Arthritis.

Accumulating evidence indicates that the continuous and intense nociceptive from inflamed tissue may increase the excitability of spinal dorsal horn neurons, which can signal back and modulate peripheral inflammation. Previous studies have demonstrated that spinal interleukin (IL)-33 contributes to the hyperexcitability of spinal dorsal horn neurons. This study was undertaken to investigate whether spinal IL-33 can also influence a peripheral inflammatory response in a rat model of arthritis. Lentivirus-delivered short hairpin RNA targeting IL-33 (LV-shIL-33) was constructed for gene silencing. Rats with adjuvant-induced arthritis (AIA) were injected intrathecally with LV-shIL-33 3days before the complete Freund's adjuvant (CFA) injection. During an observation period of 21days, pain-related behavior and inflammation were assessed. In addition, the expression of spinal proinflammatory cytokines and the activation of spinal extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways were evaluated on 9days after CFA treatment. The existence of tissue injury or inflammation in rats with AIA resulted in the upregulation of spinal IL-33, which is predominantly expressed in neurons, astrocytes, and oligodendrocytes. Intrathecal administration of LV-shIL-33 significantly alleviated hyperalgesia, paw swelling, and joint destruction, and attenuated the expression of proinflammatory cytokines [IL-6, IL-1β, and tumor necrosis factor-α (TNF-α)], as well as the activation of ERK and NF-κB/p65 in the spinal cord. Our data suggest that spinal IL-33 contributes to the development of both peripheral inflammation and hyperalgesia. Thus, interference with IL-33 at the spinal level might represent a novel therapeutic target for painful inflammatory disorders.

Knockdown of ISOC1 suppresses cell proliferation in pancreatic cancer in vitro.

Pancreatic cancer is a deadly disease that is frequently associated with mortality at the time of diagnosis due to rapid metastasis, which makes it unsuitable for operative surgery, and resistant to chemotherapy and radiation therapy. Isochorismatase domain-containing protein 1 (ISOC1) has putative isochorismatase activity, and is positively regulated by estrogen in human breast cancer. However, its role in pancreatic cancer has yet to be fully elucidated. Analysis from datasets downloaded from The Cancer Genome Atlas and Genotype-Tissue Expression databases indicated that the ISOC1 mRNA expression level was increased in pancreatic cancer tissues, compared with normal pancreatic tissues. In the present study, it was determined that the human pancreatic cancer cell lines SW 1990, PANC-1 and AsPC-1 had increased expression levels of ISOC1 mRNA, compared with human pancreatic ductal epithelial cells. Additionally, two of the pancreatic cancer cell lines, SW 1990 and PANC-1, transfected with lentivirus-delivered short hairpin RNA, to knockdown the expression of ISOC1, were established. Cell counting and MTT assays indicated that knockdown of ISOC1 decreased the ability of cell growth and proliferation in pancreatic cancer cells. Furthermore, Annexin V staining and caspase-3/7 activity assays demonstrated that inhibition of ISOC1 promoted cell apoptosis via elevation of the expression of caspase-3/7. Furthermore, inhibition of ISOC1 impaired the cell migration and invasive capability of the cells. In conclusion, ISOC1 exerts a role in pancreatic cancer cell growth and apoptosis, and may have a role in pancreatic cancer tumorigenesis.

Knockdown of CCNO decreases the tumorigenicity of gastric cancer by inducing apoptosis.

PurposeRecently, Cyclin O (CCNO) has been reported to be a novel protein of the cyclin family. However, the clinical significance and functional roles of CCNO in human cancer, including gastric cancer (GC), remain largely unexplored. In this study, we investigated the clinical and functional roles of CCNO in GC.MethodsWe analyzed CCNO expression patterns in GC patients. To investigate the role of CCNO in malignancy of GC, we used lentivirus-delivered short hairpin RNA to knockdown CCNO expression in GC cell lines. Then multiparametric high-content screening and MTT incorporation assay were used to assess the cell proliferation capability. Cell apoptosis was detected by flow cytometry and Caspase 3/7 assays. Furthermore, the effect of CCNO on tumorigenicity of GC was also determined in vivo. Finally, microarray analysis was performed to elucidate the molecular mechanisms by which shCCNO inhibited the malignancy of GC cells.ResultsThe analysis from The Cancer Genome Atlas database revealed elevated CCNO mRNA expression in GC tissue than in the adjacent normal tissue. Immunohistochemical studies also showed that stronger cytoplasmic staining of CCNO was detected in GC tissues. Downregulation of CCNO in GC cells efficiently, through infection with the lentivirus-mediated specific short hairpin RNA, could significantly induce cell apoptosis and inhibit the proliferative properties both in vitro and in vivo. Microarray analysis further revealed 652 upregulated genes and 527 downregulated genes in the shCCNO group compared with control, and indicated that CCNO knockdown could inhibit the malignancy of GC cells through inducing genome-wide gene expression changes.ConclusionOur work is the first to reveal that elevated CCNO expression is closely associated with human GC development and that CCNO knockdown could efficiently inhibit the malignant properties of GC cells by inducing cell apoptosis. Therefore, CCNO could be used as a potential biomarker for prognosis or even as a therapeutic target in human GC.

Decoy receptor 3 promotes cell adhesion and enhances endometriosis development.

Endometriosis is a multifactorial inflammatory disease with persistent activation of the nuclear factor-κB (NF-κB) signalling pathway. Aberrant adhesion of endometrium is the essential step in the progression of endometriosis, but the molecular mechanism of ectopic growth of endometrium is still unclear. Decoy receptor 3 (DcR3)/TNFRSF6B, a pleiotropic immunomodulator regulated by oestrogen, is able to activate focal adhesion kinase to promote cell adhesion. We found that DcR3 is upregulated in human ectopic endometrial cells via activation of the Akt-NF-κB signalling pathway, and its expression level correlates positively with that of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and homing cell adhesion molecule (HCAM; CD44). In a multivariate regression model, DcR3 expression level was the most significant parameter associated with endometriosis severity. Knockdown of DcR3 not only downregulated the expression of ICAM-1 and HCAM, but also reduced cell adhesion and migration. In vivo investigation further showed that DcR3 promoted the growth and spread of endometrium, whereas knockdown of DcR3 by lentivirus-delivered short hairpin RNA inhibited ectopic adhesion of endometrium and abrogated endometriosis progression. These observations are in support of DcR3 playing a critical role in the pathogenesis of endometriosis, and the inhibition of DcR3 expression being a promising approach for the treatment of endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Combined Knockdown of D-dopachrome Tautomerase and Migration Inhibitory Factor Inhibits the Proliferation, Migration, and Invasion in Human Cervical Cancer

D-dopachrome tautomerase (D-DT) is a homologue of macrophage migration inhibitory factor (MIF) with similar functions. However, the possible biological roles of D-DT in cervical cancer remain unknown so far. D-dopachrome tautomerase was assessed by immunohistochemistry in 83 cervical cancer and 31 normal cervix tissues. The stable knockdown of D-DT and MIF by lentivirus-delivered short hairpin RNA was established, and tumor growth was examined in vitro and in vivo. The effects of D-DT and MIF on the migration and invasion were further detected by wound healing assay and transwell assay. Western blot was used to explore the mechanism of D-DT and MIF in cervical cancer pathogenesis. We found that D-DT was significantly high in cervical cancer, which correlated with lymph node metastasis. The knockdown of D-DT and MIF, individually and additively, inhibited the proliferation, migration, and invasion in HeLa and SiHa cells and restrained the growth of xenograft tumor. The ablation of D-DT and MIF rescued the expression of E-cadherin and inhibited the expression of PCNA, cyclin D1, gankyrin, Sam68, and vimentin, as well as phospho-Akt and phospho-glycogen synthase kinase 3-β. The inhibition of D-DT and MIF in combination may represent a potential therapeutic strategy for cervical cancer.

Glucocorticoid-induced leucine zipper overexpression inhibits lipopolysaccharide-induced retinal inflammation in rats

Glucocorticoid-induced leucine zipper (GILZ) mediates several effects of glucocorticoids and has important anti-inflammatory properties. Here, we explored the role of GILZ in inhibiting retinal inflammation. Endotoxin-induced uveitis (EIU) was established in rats by intravitreal injection of lipopolysaccharide (LPS). GILZ levels decreased in the EIU retina after LPS injection. Retinal GILZ was downregulated by recombinant lentivirus-delivered short-hairpin RNA targeting GILZ (shRNA-GILZ-rLV) and upregulated by recombinant lentivirus-mediated GILZ overexpression (Oe-GILZ-rLV). GILZ silencing attenuated the anti-inflammatory effects of intravitreal injection of triamcinolone acetonide (TA) in the EIU retina, as demonstrated by increased retinal interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1and intercellular cell adhesion molecule-1 expression at 18 h after TA injection. A Bio-Plex cytokine assay and western blotting demonstrated that GILZ overexpression inhibited the effects of LPS, downregulating retinal IL-1β, MCP-1, MIP-1α, and IL-17 and inhibiting LPS-induced activation of the retinal toll-like receptor 4–myeloid differentiation factor 88 signaling pathway. At 48 and 72 h after LPS injection, the clinical score of inflammation was significantly lower in Oe-GILZ-rLV–transfected eyes than in blank-rLV–transfected eyes. Histological examination showed a 67.85% reduction of infiltrating inflammatory cells in the anterior chamber and a 58.97% reduction in vitreous cavity of Oe-GILZ-rLV transfected eyes at 48 h after LPS injection. Taken together, our results suggest that GILZ is a novel therapeutic target for the treatment of retinal inflammatory diseases.

Effects of lentivirus-delivered short hairpin RNA targeting human papillomavirus 16 E7 gene on the expression of DNA methyltransferases in SiHa cells

Objective To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) targeting human papillomavirus 16 (HPV16) E7 gene on the expression of 4 kinds of DNA methyl-transferases (DNMTs) , including DNMT1, DNMT3A, DNMT3B and DNMT3L, in HPV16-positive cervical cancer cell line SiHa. Methods The recombinant plasmid containing HPV16 E7 gene-targeting shRNA was constructed firstly. Then, the BLOCK-iTTM lentiviral RNAi expression system kit was used to package the lentiviral vector, which was transfected into 293T cells. The lentivirus-containing supernatants were collected at 48 and 72 hours after transfection. The SiHa cells were divided into 3 groups to be cultured with lentiviral supernatant containing HPV16 E7 gene-targeting shRNA recombinant plasmids mixed with complete medium at a ratio of 1∶1 (shRNA group) , lentiviral supernatant containing empty plasmids mixed with complete medium at a ratio of 1∶1 (negative control group) , and complete medium alone (blank control group) , respectively. Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure mRNA expression of HPV16 E7 and 4 kinds of DNMTs in the above 3 groups at 0, 48, 96 hours after infection, and Western blot analysis to determine protein expression of the 4 DNMTs at 48, 96 hours after infection. Results There were no significant differences in the mRNA expression of HPV16 E7 and the 4 DNMTs among the shRNA group, negative control group and blank control group at 0 hour after infection (all P > 0.05) . At 48, 96 hours after infection, the mRNA expression of HPV16 E7 and the 4 DNMTs decreased significantly in the shRNA group compared with the negative control group and blank control group (all P 0.05) . Additionally, E7, DNMT1, DNMT3A, DNMT3B and DNMT3L gene-silencing efficiencies in the shRNA group were 71.13%, 50.53%, 13.72%, 46.27% and 17.92% at 48 hours, and 83.50%, 74.2%, 47.8%, 64.7% and 48.9% at 96 hours after infection, respectively. Western blot analysis showed that the protein expression of the 4 DNMTs significantly decreased in the shRNA group compared with the negative control group and blank control group at 48, 96 hours after infection (all P < 0.01) . Moreover, the protein expression of DNMT1, DNMT3A, DNMT3B and DNMT3L in the shRNA group gradually decreased over time, and was inhibited by 84%, 37.2%, 59.8% and 49.3% at 48 hours respectively, and by 73.1%, 68.7%, 55.5% and 65.5% at 96 hours after infection respectively. Conclusion Targeted silencing of E7 gene in HPV16-positive SiHa cells can interfere with the mRNA and protein expression of DNMT1, DNMT3A, DNMT3B and DNMT3L. Key words: Human papillomavirus 16; Papillomavirus E7 proteins; RNA, small interfering; Methyl-transferases; Cell line, tumor

Retraction notice to: Myosin VI contributes to malignant proliferation of human glioma cells.

[This retracts the article on p. 139 in vol. 20, PMID: 26937209.].

Myosin VI contributes to malignant proliferation of human glioma cells.

Previously characterized as a backward motor, myosin VI (MYO6), which belongs to myosin family, moves toward the minus end of the actin track, a direction opposite to all other known myosin members. Recent researches have illuminated the role of MYO6 in human cancers, particularly in prostate cancer. However, the role of MYO6 in glioma has not yet been determined. In this study, to explore the role of MYO6 in human glioma, lentivirus-delivered short hairpin RNA (shRNA) targeting MYO6 was designed to stably down-regulate its endogenous expression in glioblastoma cells U251. Knockdown of MYO6 signifi cantly inhibited viability and proliferation of U251 cells in vitro. Moreover, the cell cycle of U251 cells was arrested at G0/G1 phase with the absence of MYO6, which could contribute to the suppression of cell proliferation. In conclusion, we firstly identified the crucial involvement of MYO6 in human glioma. The inhibition of MYO6 by shRNA might be a potential therapeutic method in human glioma.

Reduced USP39 expression inhibits malignant proliferation of medullary thyroid carcinoma in vitro.

BackgroundMedullary thyroid carcinoma (MTC) constitutes approximately 5 % of all thyroid cancers and carries a worse prognosis than other differentiated thyroid cancers. Targeted therapies are being investigated for systemic treatment of MTC. Ubiquitin-specific peptidase 39 (USP39) functions in pre-mRNA splicing as a component of the U4/U6-U5 tri-snRNP and also participates in spindle checkpoint and cytokinesis. In this study, we aimed to evaluate the potential role in MTC.MethodsWe used lentivirus-delivered short hairpin RNA (shRNA) to silence USP39 expression in one MTC cell line TT. USP39 expression was detected by qPCR and Western blot. For functional analysis, MTT assay was performed to evaluate the proliferation activity, and FACS was used to assess the cell distribution in the cell cycle. Moreover, the expressions of cell cycle-related proteins were examined by Western blot.ResultsBoth two shRNA sequences against USP39 could efficiently reduce its expression in TT cells. Knockdown of USP39 significantly decreased cell proliferation and caused cell cycle arrest at G2/M phase. Moreover, G2/M phase-associated proteins, Cyclin B1 and CDK1, were obviously down-regulated in TT cells after USP39 silencing.ConclusionsTherefore, knockdown of USP39 is likely to provide a novel alternative to targeted therapy of MTC and deserves further investigation.

Efficient inhibition of human glioma development by RNA interference-mediated silencing of PAK5.

Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment.

Silencing of Aurora kinase A by RNA interference inhibits tumor growth in human osteosarcoma cells by inducing apoptosis and G2/M cell cycle arrest

The overexpression of Aurora kinase A (AURKA), a member of serine/threonine kinase family, has been observed in various types of human cancers. However, the role of AURKA in osteosarcoma (OS), the most common type of primary malignancy arising from bone, has not been clarified. We used AURKA-specific lentivirus-delivered short hairpin RNA (shRNA) to significantly and sustainably silence the endogenous AURKA expression in human OS cells SAOS-2 and U2OS. We found that AURKA downregulation in OS cells prominently decreased colony formation ability in vitro and tumorigenesis ability in vivo. We further evaluated the effect of AURKA silence on cell viability by MTT assay, cell apoptosis and cell cycle by flow cytometer detection. The results showed that AURKA silence inhibited cell viability by inducing cell apoptosis and G2/M cell cycle arrest in OS cells. Taken together, our findings indicate that AURKA plays a crucial role on OS growth by inhibiting cell apoptosis and propelling cell cycle. Inhibition of AURKA by lentivirus-delivered specific shRNA showed the therapeutic potential in treatment of osteosarcoma.

Lentivirus-delivered short hairpin RNA targeting SNAIL inhibits HepG2 cell growth

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, and the highest incidence rates are reported in East Asia. We previously showed that SNAIL is upregulated in HCC tissues. In the present study, we aimed to investigate RNA interference-mediated targeting of SNAIL on the growth of HepG2 cells. We constructed three RNA interference plasmids targeting the SNAIL gene and selected the most efficient shRNA expression cassette. After the lentivirus (LV)-SNAIL small interfering (si)RNA vector was transfected into the HepG2 cell line, cell proliferation was measured using the MTT assay. E-cadherin mRNA and protein expression levels were examined by quantitative PCR and western blotting, respectively. We successfully constructed an LV-SNAIL siRNA lentiviral vector and demonstrated that it suppressed the expression of the SNAIL gene in HepG2 cells. RNA interference of SNAIL by the LV-SNAIL siRNA construct significantly inhibited the growth of HepG2 cells, in addition to significantly increasing E-cadherin mRNA and protein expression. Our findings strongly suggest that SNAIL and E-cadherin play a significant role in HCC progression, and exhibit a negative correlation. Furthermore, the expression of E-cadherin may be responsible for the reduced proliferation and survival of HepG2 cells. Thus, the SNAIL signaling pathway may provide a novel therapeutic target for the treatment of HCC.

Knockdown of Checkpoint Kinase 1 Is Associated with the Increased Radiosensitivity of Glioblastoma Stem-Like Cells

Glioblastoma multiforme is an aggressive brain tumor with a poor prognosis. The glioblastoma stem-like cells (GSCs) represent a rare fraction of human glioblastoma cells with the capacity for multi-lineage differentiation, self-renewal and exact recapitulation of the original tumor. Interestingly, GSCs are more radioresistant compared with other tumor cells. In addition, the remarkable radioresistance of GSCs has been known to promote radiotherapy failure and therefore is associated with a significantly higher risk of a local tumor recurrence. Moreover, the hyperactive cell cycle checkpoint kinase (Chk) 1 and 2 play a pivotal role in the DNA damage response including radiation and chemical therapy. Based on aforementioned, we hypothesized that knockdown of Chk1 or Chk2 might confer radiosensitivity on GSCs and thereby increases the efficiency of radiotherapy. In this study, we knocked down the expression of Chk1 or Chk2 in human GSCs using lentivirus-delivered short hairpin RNA (shRNA) to examine its effect on the radiosensitivity. After radiation, the apoptosis rate and the cell cycle of GSCs were measured with Flow Cytometry. Compared with control GSCs (apoptosis, 7.82 ± 0.38%; G2/M arrest, 60.20 ± 1.28%), Chk1 knockdown in GSCs increased the apoptosis rate (37.87 ± 0.32%) and decreased the degree of the G2/M arrest (22.37 ± 2.01%). In contrast, the radiosensitivity was not enhanced by Chk2 knockdown in GSCs. These results suggest that depletion of Chk1 may improve the radio-sensitivity of GSCs via inducing cell apoptosis. In summary, the therapy targeting Chk1 gene in the GSCs may be a novel way to treat glioblastoma.

Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.

Bub1 and CENP-F Can Contribute to Kaposi's Sarcoma-Associated Herpesvirus Genome Persistence by Targeting LANA to Kinetochores

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for segregation of viral episomes to progeny nuclei and allows for maintenance of the viral genome in newly divided daughter cells. LANA binds to KSHV terminal repeat (TR) DNA and simultaneously associates with chromatin-bound cellular proteins. This process tethers the viral episomes to host chromosomes. However, the mechanism of tethering is complex and involves multiple protein-protein interactions. Our previous proteomics studies which showed the association of LANA with centromeric protein F (CENP-F) prompted us to further study whether LANA targets centromeric proteins for persistence of KSHV episomes during cell division. Here we show that LANA colocalized with CENP-F as speckles, some of which are paired at centromeric regions of a subset of chromosomes in KSHV-infected JSC-1 cells. We also confirm that both the amino and carboxy termini of LANA can bind to CENP-F. Moreover, LANA associated with another kinetochore protein, Bub1 (budding uninhibited by benzimidazole 1), which is known to form a complex with CENP-F. Importantly, we demonstrated the dynamic association of LANA and Bub1/CENP-F and the colocalization between Bub1, LANA, and the KSHV episome tethered to the host chromosome using fluorescence in situ hybridization (FISH). Knockdown of Bub1 expression by lentivirus-delivered short hairpin RNA (shRNA) dramatically reduced the number of KSHV genome copies, whereas no dramatic effect was seen with CENP-F knockdown. Therefore, the interaction between LANA and the kinetochore proteins CENP-F and Bub1 is important for KSHV genome tethering and its segregation to new daughter cells, with Bub1 potentially playing a more critical role in the long-term persistence of the viral genome in the infected cell.

IGF-I Activation of the AKT Pathway Is Impaired in Visceral But Not Subcutaneous Preadipocytes from Obese Subjects

Obesity morbidity is associated with excess visceral adiposity, whereas sc adipose tissue is much less metabolically hazardous. Human abdominal sc preadipocytes have greater capacity for proliferation, differentiation, and survival than omental preadipocytes. IGF-I is a critical mediator of preadipocyte proliferation, differentiation, and survival through multiple signaling pathways. We investigated IGF-I action in primary cultures of human preadipocytes isolated from sc and omental adipose tissue of obese subjects. IGF-I-stimulated DNA synthesis was significantly lower in omental compared with sc preadipocytes. IGF-I phosphorylation of the IGF-I receptor and the ERK pathway was comparable in sc and omental cells. However, omental preadipocytes had decreased insulin receptor substrate (IRS)-1 protein associated with increased IRS-1-serine(636/639) phosphorylation and degradation. IGF-I-stimulated phosphorylation of AKT on serine(473) but not threonine(308) was decreased in omental cells, and activation of downstream targets, including S6Kinase, glycogen synthase kinase-3, and Forkhead box O1 was also impaired. CyclinD1 abundance was decreased in omental cells due to increased degradation. Over-expression of IRS-1 by lentivirus in omental preadipocytes increased IGF-I-stimulated AKT-serine(473) phosphorylation. The mammalian target of rapamycin (mTOR)-Rictor complex regulates phosphorylation of AKT-serine(473) in 3T3-L1 adipocytes, but knockdown of Rictor by lentivirus-delivered short hairpin RNA in sc preadipocytes did not affect AKT-serine(473) phosphorylation by IGF-I. These data reveal an intrinsic defect in IGF-I activation of the AKT pathway in omental preadipocytes from obese subjects that involves IRS-1 but probably not mTOR-Rictor complex. We conclude that impaired cell cycle regulation by AKT contributes to the distinct growth phenotype of preadipocytes in visceral fat of obese subjects.

Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up-regulation of eotaxin, which, together with interleukin (IL)-5, is important for differentiation, chemo-attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus-delivered short hairpin RNA (shRNA) to suppress the expression of IL-5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL-5 shRNAs on airway inflammation in an ovalbumin (OVA)-induced murine model of asthma. Lentivirus-delivered shRNAs were used to suppress the expression of eotaxin and/or IL-5 in local tissue in an OVA-induced murine asthma model. Intra-tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper-responsiveness, cellular infiltration of lung tissues, and eotaxin and IL-5 levels in bronchio-alveolar lavage fluid. Administration of lentiviruses expressing IL-5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model. Local delivery of lentiviruses expressing IL-5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia.

Insulin‐like growth factor binding protein‐5 enhances the migration and differentiation of gingival epithelial cells

The objective was to define the roles of insulin-like growth factor binding protein-5 (IGFBP-5) in gingival epithelial cells (GEC). Human IGFBP-5 is expressed in many cell types and has diverse biological functions. It stimulates the growth of bone cells and is associated with the impedance of gingival fibroblast apoptosis. In gingival epithelium, IGFBP-5 is expressed in the cells of the differentiated stratum spinosum layer. Recombinant IGFBP-5 protein treatment and knockdown of IGFBP-5 expression using a lentivirus-delivered short hairpin RNA was carried out in human GEC. Proliferation, apoptosis, anoikis, migration, differentiation and gene expression in GEC were analyzed and molecular images were obtained. The IGFBP-5 had no effect on proliferation, but it slightly suppressed apoptosis and anoikis of GEC. It also induced GEC migration and upregulated the expression of involucrin, transglutaminase-1, keratin and focal adhesion kinase. The IGFBP-5 induced migration partly via an insulin-like growth factor-independent mechanism. The knockdown of IGFBP-5 downregulated the expression of involucrin, transglutaminase-1 and focal adhesion kinase. Expression of IGFBP-5 in GEC is associated with anti-apoptosis, migration and differentiation of GEC. These phenotypic effects may be associated with focal adhesion kinase and are advantageous for re-epithelization of GEC and the maintenance of gingival health.