Hairpin RNA Expression Vector Research Articles

Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA

BackgroundClassical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA.ResultsFirst, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection.ConclusionThe IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid β, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid β deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Carbon-based Nanomaterials for delivery of small RNA molecules: a focus on potential cancer treatment applications.

Nucleic acid-mediated therapy holds immense potential in the treatment of recalcitrant human diseases such as cancer. This is underscored by advances in understanding the mechanisms of gene regulation. In particular, the endogenous protective mechanism of gene silencing known as RNA interference (RNAi) has been extensively exploited. We review here the developments from 2011 to 2021, in the use of nanographene oxide, carbon nanotubes, fullerenes, carbon nanohorns, carbon nanodots and nanodiamonds for the delivery of therapeutic small RNA molecules. Appropriately designed effector molecules such as small interfering RNA (siRNA), can, in theory, silence the expression of any disease-causing gene. Alternatively, siRNA can be generated in vivo through the introduction of plasmid-based short hairpin RNA (shRNA) expression vectors. Other small RNAs such as micro RNA (miRNA) also function in post-transcriptional gene regulation and are aberrantly expressed under disease conditions. The miRNA-based therapy involves either restoration of miRNA function through the introduction of miRNA mimics; or the inhibition of miRNA function by delivering anti-miRNA oligomers. However, the large size, hydrophilicity, negative charge and nuclease-sensitivity of nucleic acids necessitate an appropriate carrier for their introduction as medicine into cells. While numerous organic and inorganic materials have been investigated for this purpose, the perfect carrier agent remains elusive. In recent years, carbon-based nanomaterials have received widespread attention in biotechnology due to their tunable surface characteristics, mechanical, electrical, optical and chemical properties.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation.

Conditional expression of short hairpin RNAs with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell–cell communication in vivo benefits from simultaneous use of 2 independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-short hairpin RNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based short hairpin RNA expression vector to achieve short hairpin RNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

CD9 knockdown suppresses cell proliferation, adhesion, migration and invasion, while promoting apoptosis and the efficacy of chemotherapeutic drugs and imatinib in Ph+ ALL SUP‑B15 cells.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is regarded as a prognostically unfavorable subgroup, as this ALL subgroup has an increased risk of relapse/refractory disease. CD9, which belongs to the tetraspanin membrane proteins, is implicated in several pathological processes, including tumor progression. However, the role of CD9 in the pathogenesis of Ph+ ALL and the potential benefit of applying CD9-targeted RNA interference strategies for treatment of Ph+ ALL require further investigation. The aim of the present study was to determine the effects of CD9 on leukemic cell progression and the efficacy of therapeutic agents in Ph+ ALL cells, in addition to assessing the in vitro anti-leukemia activity of CD9-targeted RNA interference in Ph+ ALL cells. In the present study, a lentiviral short hairpin RNA (shRNA) expression vector targeting CD9 gene in Ph+ ALL SUP-B15 cells was constructed. The present results demonstrated that treatment of SUP-B15 cells with lentiviral-mediated shRNA against CD9 decreased CD9 mRNA and protein expression compared with the shControl cells transduced with a blank vector. In addition, CD9 knockdown could suppress cell proliferation, adhesion, migration and invasion, and promote apoptosis and the efficacy of chemotherapeutic drugs (such as vincristine, daunorubicin, cyclophosphamide and dexamethasone) and the tyrosine kinase inhibitor imatinib in SUP-B15 cells. Furthermore, CD9 knockdown suppressed cell proliferation and promoted apoptosis in SUP-B15 cells via a p53-dependent pathway. These findings suggested that gene silencing of CD9 using a shRNA-expressing lentivirus vector may provide a promising treatment for Ph+ ALL.

Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo

Background and Aim: Our previous study demonstrated that SLC22A18 downregulation through promoter methylation and Sequence binding protein 1 (SATB1) upregulation are associated with the development and progression of glioma. This study aimed to examine the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. Materials and Methods: Here, a combined gene therapy to upregulate SLC22A18 and downregulate SATB1 in malignant glioma was evaluated both in vitro and in vivo. Glioma U251 cells overexpressing SLC22A18 and underexpressing SATB1 were generated to investigate the effects of these changes on cell survival, as measured by the methyl thiazol tetrazolium assay, and on cell invasion, as measured by cell invasion and migration assays. In addition, analysis of the cell cycle was performed using flow cytometry in vitro. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). Results: The upregulation of SLC22A18 and downregulation of SATB1 significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of human glioma U251 cells. Furthermore, we revealed that U251 cells were arrested at the G0/G1 phase. Similar data for apoptotic glioma cell death were obtained in tumor cells from an in vivo glioma xenograft model after transfection. At the same total dose, the therapeutic effect was markedly better in the glioma xenografts transfected with both SLC22A18 gene and SATB1 short hairpin RNA (shRNA) expression vectors compared with tumors transfected with either agent alone. The levels of SLC22A18 and SATB1 protein expression were respectively increased and decreased in the glioma cells.Conclusion: These results demonstrate that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibits the growth of human malignant glioma cells both in vitro and in glioma xenografts in vivo, suggesting a promising novel strategy for glioma therapy that warrants further study.

Downregulation of lncRNA AWPPH inhibits colon cancer cell proliferation by downregulating GLUT-1.

Long non-coding RNA (lncRNA) associated with poor prognosis of hepatocellular carcinoma (AWPPH) serves pivotal roles in bladder cancer and liver cancer; however, to the best of our knowledge, its functionality in colon cancer has not been characterized. The present study aimed to investigate the involvement of lncRNA AWPPH in colon cancer. Serum levels of lncRNA AWPPH and glucose transporter 1 (GLUT-1) in patients with early stage colon cancer and healthy controls were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA. Correlation between lncRNA AWPPH and GLUT-1 expression was analyzed by Pearson's correlation coefficient. χ2 test was performed to investigate the associations between serum levels of lncRNA AWPPH and clinical data of patients with colon cancer. lncRNA AWPPH short hairpin RNA and GLUT-1 expression vectors were transfected into colon cancer cells, and the effects on lncRNA AWPPH, GLUT-1 and cell proliferation were detected by RT-qPCR, western blotting and Cell Counting Kit-8 assay. It was observed that serum levels of lncRNA AWPPH and GLUT-1 were significantly higher in patients with colon cancer patients compared with healthy controls. Serum levels of AWPPH and GLUT-1 were significantly positively correlated in patients with colon cancer. Serum levels of lncRNA AWPPH were associated with the tumor size. Furthermore, AWPPH-silencing significantly inhibited GLUT-1 expression and inhibited cancer cell proliferation. GLUT-1 overexpression promoted cancer cell proliferation and attenuated the inhibitory effects of AWPPH-silencing on cancer cell proliferation. However, GLUT-1 overexpression failed to significantly affect the expression of AWPPH. Therefore, it can be concluded that a downregulation of lncRNA AWPPH may inhibit colon cancer cell proliferation by downregulating GLUT-1.

Expression of exogenous dsRNA by Lecanicillium attenuatum enhances its virulence to Dialeurodes citri.

Dialeurodes citri is an important pest in citrus-producing areas of the world. Lecanicillium attenuatum parasitizes D. citri and kills it, suggesting a potential approach for the biological control of pests. However, the low virulence of the fungus and its slow rate of killing have limited its commercial competitiveness. The objective reason for these disadvantages is immunological rejection by the host. Our strategy was to use fungi to express the double-stranded RNA (dsRNA) of the host immune genes. The fungal hyphae release siRNA at the time of infection, thus interfering with the expression of immune genes in the host and facilitating fungal invasion. We selected prophenoloxidase (DcPPO), prophenoloxidase-activating factor (DcPPO-AF), and lysozyme (DcLZM) as target genes to construct intron-splicing hairpin RNA expression vectors and to successfully obtain transgenic fungi. Two days after infection, the immune genes of D. citri showed varying degrees of silencing compared with those in the positive control group. The median lethal concentration (LC50 ; spores mL-1 ) values of La::GFP, La::DcPPO, La::DcPPO-AF, and La::DcLZM were 9.63×104 , 2.66×104 , 1.21×105 , and 3.31×104 , respectively. The 50% lethal time (LT50 ) values of these fungi were 5.15, 3.60, 5.34, and 4.04 days, respectively. The virulence of La::DcPPO and La::DcLZM increased 3.62- and 2.91-fold, respectively, and their LT50 decreased by 30.10% and 21.55%, respectively. The results indicate that this method, which uses tens of thousands of hyphae to inject dsRNA to improve the virulence of transgenic fungi, can play a greater role in the prevention and control of pests in the future. © 2018 Society of Chemical Industry.

The roles and mechanism of chemokine receptor type 5 in colorectal cancer proliferation

Objective To examine the expression of chemokine receptor type 5 (CXCR5) in colorectal cancer (CRC) and to explore its relationship with clinicopathological features and cell proliferation in CRC. Methods Immunohistochemical staining method was used to detect the protein expression of CXCR5 in 132 paired specimens of CRC, adjacent and 62 samples of adenoma. Analysing the relationship between CXCR5 with clinicopathological features in CRC. RNA interference (RNAi) technique was applied to construct four CXCR5 short hairpin RNA (shRNA) expression vectors. Lipofectamine 2000 was used to transfer plasmids into colon cancer cell lines LoVo and HCT116. Real-time PCR and Western blotting were used to observe the inhibitory effect of RNAi on CXCR5 expression. Lentivirus production and transduction were used to construct CXCR5 shRNA stably expressed CRC cell lines. CXCR5 overexpressed plasmid was used to construct CXCR5 stably expressed CRC cell lines. cell counting kit-8 (CCK-8) assay and colony formation assay were used to detect the influence of specific CXCR5 on CRC cell. Tumor formation in nude mouse was performed to detect the effects of CXCR5 on the proliferation ability of CRC cells in vivo. FCM were used to detect the cell cycle. Results CXCR5 protein was expressed in 46.97% (62/132) of CRC tissues and 24.19% (15/62) of adenoma tissues, respectively. The protein expression of CXCR5 was negative in adjacent tissues. The protein expression level of CXCR5 in CRC tissues was significantly higher than ademona tissues (P=0.020). The expression of CXCR5 was correlated with relapse, TNM stage, lymph node metastasis and distant metastasis (P=0.004, 0.025, 0.002, 0.008). CCK-8 and colony formation results showed that the CXCR5 shRNA inhibited the proliferation of CRC significantly, whereas overexpression of CXCR5 promoted CRC proliferation in vitro. Tumor formation results in nude mouse showed that CXCR5 silencing inhibited the proliferation and metastasis ability of CRC cells, whereas overexpression of CXCR5 promoted proliferation of CRC cells in vivo. CXCR5 may influence CRC cells proliferation through cell cycle in vitro. Conclusion CXCR5 may play a crucial role in carcinogenesis, development of CRC. CXCR5 could promote proliferation of colon cancer cell both in vitro and in vivo, and may influence CRC development through cell cycle. Key words: Colorectal cancer; Chemokine receptor type 5; Proliferation

Down-regulation of CD19 expression inhibits proliferation, adhesion, migration and invasion and promotes apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells

The survival rate of childhood acute lymphoblastic leukemia (ALL) has increased while that of Philadelphia-positive (Ph+) ALL remains low. CD19 is a B-cell specific molecule related to the survival and proliferation of normal B cells. However, there is little information available on the effects of CD19 on the biological behavior of Ph+ ALL cells. In this study, we explored a lentiviral vector-mediated short hairpin RNA (shRNA) expression vector to stably reduce CD19 expression in Ph+ ALL cell line SUP-B15 cells and investigated the effects of CD19 downregulation on cell proliferation, apoptosis, drug sensitivity, cell adhesion, cell migration and cell invasion in vitro. CD19 mRNA and protein expression levels were inhibited significantly by CD19 shRNA. Down-regulation of CD19 could inhibit cell proliferation, adhesion, migration and invasion, and increase cell apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells. Moreover, we found that down-regulation of CD19 expression inhibits cell proliferation and induces apoptosis in SUP-B15 cells in a p53-dependent manner. Taken together, our results suggest that lentiviral vector-mediated RNA interference of CD19 gene may be a promising strategy in the treatment of Ph+ ALL.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy

AimsThis investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. Main methodsTo construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. Key findingsThe silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%–25%) under normoxia, whereas it was significantly increased to approximately 50%–60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. SignificanceThis novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo.

Construction of COX-2 short hairpin RNA expression vector and its inhibitory effect on hepatic fibrosis

ABSTRACTThe aim of this study was to construct recombinant adenovirus vector carrying a short hairpin RNA (shRNA) that can exclusively target cyclooxygenase-2 (COX-2) gene expression and observe its inhibitory effect on hepatic fibrosis. We designed and synthesized three oligonucleotide sequences, and cloned those into a shuttle vector, pYr-1.1-hU6-EGFP, after annealing. The restriction-enzyme digestion and sequencing analyses confirmed that the constructed recombinant eukaryotic expression vector was correct. A recombination reaction using LR Clonase was performed for the pYr-1.1-hU6-EGFP COX-2shRNA and the adenovirus vector pAd/BL-DEST to form Ad-COX-2shRNA. The adenoviruses containing the recombinant plasmids were transfected into hepatic stellate cells (HSCs). The transfection efficiency of the three COX-2 shRNAs exceeded 70%. Reverse transcription PCR (RT-PCR) and western blot confirmed that the target gene expression was decreased at the level of mRNA and protein, and the interference effect of COX-2 shRNA-1 was better than that of the other two COX-2 shRNAs. COX-2 shRNA-1 recombinant adenovirus vectors (1 × 109 PFU/mL) were injected via the tail vein into rats fed a high-fat diet with a 40% carbon tetrachloride peanut oil lavage, which induced liver fibrosis. Rats were euthanized at the end of the 12th week, and their liver was removed. Liver expression of COX-2 mRNA and protein was detected by RT-PCR and immunohistochemistry, respectively. RT-PCR and immunohistochemistry showed that COX-2 shRNA-1 inhibited COX-2 expression in liver tissue. Hematoxylin/eosin and Masson staining showed that COX-2 shRNA-1 ameliorated the severity of liver fibrosis. The COX-2 shRNA eukaryotic expression vectors were constructed successfully and COX-2 shRNA-1 ameliorated liver fibrosis.

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells.

Apoptosis is considered to serve an important role in the pathogenesis of rheumatoid arthritis. The aim of the present study was to construct Bcl-2-short hairpin (sh)RNA expression vectors and transfect them into human synovial sarcoma SW982 cells, in order to screen for an effective interference sequence and analyze the effects of this interference on the expression levels of Bcl-2 and other molecules associated with the mitochondrial apoptosis pathway. Three different shRNAs (Bcl-2-sh1, 2 and 3) were designed according to the human Bcl-2 mRNA target sequence and were transformed into competent DH5α Escherichiacoli cells following the construction of an expression vector, which was then transfected into SW982 cells. SW982 cells were grouped into a control group (transfected with a negative control shRNA), and Bcl-2-sh1, Bcl-2-sh2 and Bcl-2-sh3 groups (transfected with Bcl-2-sh1, 2 and 3, respectively). The expression levels of Bcl-2 mRNA were detected using reverse transcription-quantitative PCR (RT-qPCR). Bcl-2-sh1 was identified as the most effective shRNA sequence for interference, and was used for subsequent experiments. The mRNA and protein expression levels of Bcl-2, Bax, CytC and Caspase-3 were detected in SW982 cells by RT-qPCR and western blotting at various time-points (48 and 72h) following transfection with Bcl-2-sh1, in order to observe the effectiveness of this interference. Compared with the control group, the expression levels of Bcl-2 were decreased, while those of Bax, CytC and Caspase-3 were increased in Bcl-2-sh1-transfected cells (P<0.01). The interference effect was greater at 48h than at 72h. In summary, an effective shRNA sequence (Bcl-2-sh1) targeting the Bcl-2 gene was identified from three candidates, and was demonstrated to significantly interfere with the expression of Bcl-2, Bax, CytC and Caspase-3 when transfected into SW982 cells. The interference effect of Bcl-2-sh1 was more pronounced at 48h than at 72h post-transfection.

Wip1 is associated with tumorigenity and metastasis through MMP-2 in human intrahepatic cholangiocarcinoma.

Wip1 has been shown to correlate with the metastasis/invasion of several tumors. This study was designed to investigate the clinical significance and biological function of Wip1 in intrahepatic cholangiocarcinoma (ICC). The expression of Wip1 was investigated in sixty human ICC biopsy samples by immunohistochemistry. Transient and stable knockdown of Wip1 in two human ICC cells (ICC-9810 and SSP25) were established using short hairpin RNA expression vector. Immunohistochemistry revealed that Wip1 was up-regulated in human ICC tissues (47/60, 78.3%). High levels of Wip1 in human ICC correlated with metastasis to the lymph metastasis (P=0.022). Genetic depletion of Wip1 in ICC cells resulted in significantly inhibited proliferation and invasion compared with controls. Most importantly, Wip1 down-regulation impaired tumor migration capacity of ICC cells in vivo. Subsequent investigations revealed that matrix metalloproteinase-2 (MMP-2) is an important target of Wip1. Consistently, in human ICC tissues, Wip1 level was positively correlated with MMP-2 expression. Taken together, our founding indicates that Wip1 may be a crucial regulator in the tumorigenicity and invasion of human ICC, Wip1 exerts its pro-invasion function at least in part through the MMP-2 signaling pathway, suggesting Wip1 as a potential therapeutic target for ICC.

Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non‑small cell lung cancer cells.

Lung cancer is reported to be a major public health issue worldwide and the overall prognosis of patients remains poor. The expression levels of Livin and Survivin, of the inhibitors of apoptosis (IAP) family, are associated with prognostic significance in the majority of solid tumors. Therefore, in the presents study, short hairpin (sh)RNA expression vectors inhibiting the Livin and Survivin genes were constructed to examine the effects of the transfection of Livin shRNA and/or Survivin shRNA on the biological functions of tumor cells. The transfection efficiency was measured using fluorescence reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The cell growth inhibition ratio was measured using a CCK assay. Cell apoptosis following transfection and in tumor tissues were measured using a TUNEL assay, and a cancer xenograft model was used to investigate the effect of Livin shRNA and/or Survivin shRNA on tumor growth. The results indicated that the mRNA and protein expression levels were suppressed following the transfection of Livin and Survivin shRNA into tumor cells (P<0.05, compared with control group). The growth of tumor cells invivo and invitro was significantly inhibited following transfection with Livin and Survivin shRNA, compared with that in the other groups (P<0.05). Taken together, the transfection of cells with Livin and Survivin inhibited tumor growth invivo and invitro, with the co‑transfection of Livin and Survivin shRNA showing increased efficiency, compared with transfection of either the Livin vector or Survivin vector alone. The combined inhibition of Livin and Survivin may be a promising multitargeted gene therapeutic strategy in cancer treatment.

High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis.

Purpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter could lead to mitochondrial dysfunction and RGC death.Methods: Primary cultured rat RGCs were exposed to 30 mm Hg of hydrostatic pressure (HP) for 12, 24, 48, 72, 96 and 120 h. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and polymerase gamma (POLG) expressions were also analyzed. The RGCs were then infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting POLG (POLG-shRNA), and mtDNA alterations as well as mitochondrial function, including complex I/III activities and ATP production were subsequently studied at appropriate times. Finally, RGC apoptosis and the mitochondrial-apoptosis pathway-related protein cleaved caspase-3 were detected using a Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and western blotting, respectively.Results: mtDNA damage was observed as early as 48 h after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching >40% and 4.8 ± 0.3-fold, respectively, compared with the control values. Twelve hours after HP, the expressions of OGG1, MYH and POLG mRNA in the RGCs were obviously increased 5.02 ± 0.6-fold (p < 0.01), 4.3 ± 0.2-fold (p < 0.05), and 0.8 ± 0.09-fold (p < 0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 h after HP (p < 0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p < 0.01), while complex I/III activities gradually decreased (p < 0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p < 0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations.Conclusions: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death.

Effect of silencing key proteins in telomerase mechanism and alternative lengthening of telomeres mechanism in laryngeal cancer cells

PurposeTo explore the influences of telomerase and alternative lengthening of telomeres mechanism on telomere length and laryngeal squamous cell carcinoma in vitro and in vivo. Materials and methodsShort hairpin RNA expression vectors targeting the messenger TERT, TRF2, RAD51 and NBS1 were constructed. The mRNA and protein expression of targeted genes in human laryngeal squamous carcinoma cell line HEp-2 was evaluated by reverse transcription polymerase chain reaction and Western blotting separately. The length of telomere was analyzed by fluorescent in-situ hybridization. Cell viability was examined by cell counting Kit-8. Effects on tumor growth were also investigated in vivo. ResultsThe transfection of multiple short hairpin RNAs expression plasmid significantly inhibited the mRNA and protein expression of related genes. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes resulted in the shortening of telomere length in HEp-2 cell. However, silence of alternative lengthening of telomeres mechanism related genes could shorten the telomere length but had no significant difference. Both simultaneously and separately blocking telomerase mechanism and alternative lengthening of telomeres mechanism resulted in reduction of tumor cell viability. Silence of alternative lengthening of telomeres mechanism and telomerase mechanism related genes inhibited the tumor growth in vivo. ConclusionsThe inhibition of telomere related gene may be a promising strategy for the treatment of laryngeal squamous cell carcinoma.

Dickkopf-1 expression is associated with tumorigenity and lymphatic metastasis in human hilar cholangiocarcinoma.

Dickkopf-1 (DKK1) is involved in tumorigenesis and the invasion of several tumors. However, its biological function in human hilar cholangiocarcinoma (HCCA) has not yet been documented. This study was designed to investigate the clinical significance and biological function of DKK1 in HCCA. The expression of DKK1 was investigated in thirty-seven human HCCA biopsy samples by immunohistochemistry. To further explore the biological effects of DKK1 in HCCA, transient and stable knockdown of DKK1 in two human HCCA cells (QBC939 and FRH0201) were established using small interfering or short hairpin RNA expression vector. In the present study, immunohistochemistry revealed that DKK1 was up-regulated in human HCCA tissues (24/37, 64.9%). High levels of DKK1 in human HCCA correlated with metastasis to the hilar lymph nodes (P=0.038). Genetic depletion of DKK1 in HCCA cells resulted in significantly inhibited proliferation, colony formation and migration compared with controls. Most importantly, DKK1 down-regulation impaired tumor formation capacity of HCCA cells in vivo. Subsequent investigations revealed that β-catenin is an important target of DKK1 and DKK1 exerts its pro-invasion function at least in part through the β-catenin/ matrix metalloproteinase-7 (MMP-7) signaling pathway. Consistently, in human HCCA tissues, DKK1 level was positively correlated with β-catenin and MMP-7 expression, as well as tumor hilar lymphatic metastasis. Taken together, our findings indicate that DKK1 may be a crucial regulator in the tumorigenicity and invasion of human HCCA, DKK1 exerts its pro-invasion function at least in part through the β-catenin/ MMP-7 signaling pathway, suggesting DKK1 as a potential therapeutic target for HCCA.

Abstract 5173: Role of Gli2 in mediating the progression of prostate cancer to CRPC

Abstract Background: Prostate cancer is a leading cause of cancer-related death in the US men, largely due to the development of castration resistant prostate cancer (CRPC) in the patients treated with androgen deprivation therapy. There is increasing evidence indicating that Hedgehog pathway is involved in the malignancy of prostate cancer, which includes the finding of two prostate tumors with loss-of-function mutations in the SuFu gene, correlation of tumor grade with high expression of Sonic hedgehog, Gli2 and/or hedgehog target genes including Ptch1 and HIP, and increased Gli2 expression in therapy-resistant tumor cells from patients. In addition, treatment of mice with hedgehog inhibitors significantly attenuated the growth of prostate tumor xenografts. Some recent studies also implicated a crosstalk between Hedgehog/Gli signaling and androgen signaling pathways in prostate cancer. However, the role of Gli2 in CRPC progression has not been investigated. Methods: A human Gli2 specific small hairpin RNA (shRNA) expression vector was constructed using Tet-PLKO-puro lentivector, which was delivered into the androgen-dependent human prostate cancer LNCaP cells. RT-PCR, Western Blot and Luciferase Reporter Assay were performed to confirm doxycycline-inducible Gli2 knockdown. The effect of Gli2 knockdown on anchorage-dependent or -independent growth was assessed with MTT and Soft Agar Assays respectively. Cells were cultured in androgen depleted medium or treated with the anti-androgen, Casodex, to mimic androgen deprivation condition. Mouse xenograft models were used to assess the role of Gli2 in the development of CRPC in vivo. Results: Androgen deprivation promoted the expression of Hedgehog signaling components including Gli2 in the LNCaP cells. Gli2 knockdown inhibited anchorage-dependent and -independent growth of LNCaP cells. Casodex complemented the inhibition of Gli2 knockdown on the growth of LNCaP cells. Gli2 knockdown prevented the outgrowth of androgen-independent cells from LNCaP cells cultured in androgen depleted medium. Induced-knockdown of Gli2 by doxycycline inhibited the growth of castration-resistant tumors in vivo in mice. Conclusions: Our study demonstrated the important role of Gli2 in the development of CRPC. Therefore, it may have the potential to serve as a novel therapeutic target for the prevention of CRPC. Citation Format: Lu Xia, Tai Qin, Luzhe Sun. Role of Gli2 in mediating the progression of prostate cancer to CRPC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5173.

Abstract 4577: Fibroblast growth factor receptor-4 (FGFR-4) as a novel therapeutic target for pancreatic cancer

Abstract The fibroblast growth factor receptor (FGFR) family plays crucial roles in development, tissue repair, and malignant tumors. FGFR-1, -2, and -3 each exist in two isoforms, IIIb and IIIc, due to alternative splicing of the extracellular domain, whereas FGFR-4 does not have these isoforms. FGFR-4 is reportedly over-expressed in various cancers such as breast, prostate, hepatocellular, ovarian, gastric, colorectal, and pancreatic cancers, wherein it contributes to tumor progression. Recent studies have shown that a decrease in FGFR-4 levels suppresses the aggressiveness of gastric, colorectal, and ovarian cancers. We found that pancreatic cancer cell lines expressed similar levels of the IIIb and IIIc isoforms of FGFR-1 to -3, but showed different FGFR-4 levels. These findings suggest that the cancer cells surviving after treatment with recently developed anti-FGFR drugs, which target FGFR-1 to -3, tend to express high levels of FGFR-4. Moreover, a single-nucleotide polymorphism (SNP) in exon 9 of the gene encoding FGFR-4 that results in the substitution of glycine with arginine at codon 388 (388 Gly/Arg) in the transmembrane domain is associated with poor outcomes of high-grade soft tissue sarcoma, prostate, lung, head and neck carcinoma, and advanced and treatment-resistant breast cancer. It has been reported that 40-50% of Caucasians carry at least one copy of the 388 Gly/Arg SNP of FGFR-4. In the present study, we examined the expression and roles of the 388 Gly/Arg SNP in pancreatic cancer and tried to clarify whether FGFR-4 may be a novel therapeutic target for cancer treatment. In human pancreatic tissues, FGFR-4 was weakly localized in the normal exocrine and endocrine pancreas, and was strongly expressed in 67 of 136 pancreatic ductal adenocarcinoma cases (49%). FGFR-4 expression positively correlated with larger primary tumors and more advanced stages of pancreatic cancer. FGFR-4 mRNA was expressed in 5 pancreatic cancer cell lines at various levels, and the mutation in codon 388 was detected in 3 of the cell lines, including PK-1 cells. A short hairpin RNA expression vector targeting FGFR-4 was stably transfected to these mutant-expressing PK-1 cells (388 Gly/Arg), which then showed lower growth rates and cell migration and invasion abilities compared to sham vector-transfected cells. DNA microarray analysis showed that decreased expression of FGFR-4 in PK-1 cells altered the expression levels of molecules related to cellular movement, cellular development, and cell-to-cell signaling and interactions. FGF-19—one of the major ligands for FGFR-4—was expressed in all 5 of the pancreatic cancer cell lines tested. These findings suggest that inhibition of the expression of FGFR-4 harboring the 388 Gly/Arg SNP may be a novel therapeutic strategy for pancreatic cancer, especially after treatment with the newly developed anti-FGFR-1 to -3-targeted drugs. Citation Format: Toshiyuki Ishiwata, Hisashi Yoshimura, Yoko Matsuda, Shunji Ishiwata. Fibroblast growth factor receptor-4 (FGFR-4) as a novel therapeutic target for pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4577.