An Optimized Telomerase-Specific Lentivirus for Optical Imaging of Tumors

hTERT is the catalytic subunit of the telomerase complex. Elevated expression of hTERT is associated with the expansion and metastasis of gastric tumor. In this study, we aimed to identify novel tumor suppressor miRNAs that restrain hTERT expression. We began our screen for hTERT-targeting miRNAs with a miRNA microarray. miRNA candidates were further filtered by bioinformatic analysis, general expression pattern in different cell lines, gain-of-function effects on hTERT protein and the potential of these effects to suppress hTERT 3′ untranslated region (3′UTR) luciferase activity. The clinical relevance of two miRNAs (miR-1207-5p and miR-1266) was evaluated by real-time RT-PCR. The effects of these miRNAs on cell growth, cell cycle and invasion of gastric cancer cells were measured with CCK-8, flow cytometry and transwell assays. Finally, the ability of these miRNAs to suppress the transplanted tumors was also investigated. Fourteen miRNAs were identified using a combination of bioinformatics and miRNA microarray analysis. Of these fourteen miRNAs, nine were expressed at significantly lower levels in hTERT-positive cell lines compared with hTERT-negative cell lines and five could downregulate hTERT protein expression. Only miR-1207-5p and miR-1266 interacted with the 3′ UTR of hTERT and the expression levels of these two miRNAs were significantly decreased in gastric cancer tissues. These two miRNAs also inhibited gastric tumor growth in vitro and in vivo. Altogether, miR-1207-5p and miR-1266 were determined to be hTERT suppressors in gastric cancer, and the delivery of these two miRNAs represents a novel therapeutic strategy for gastric cancer treatment.

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“…The signals were detected with the ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ, USA), as described previously. 38 …”

Section: Methodsmentioning

confidence: 99%

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

et al. 2014

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“…Subconfluent MG-63 cells were infected with GFP-lentivirus in the presence of 8 ìg/mL polybrene (Sigma, St. Louis, Mo). At 24 h post-infection, media were removed and replaced with fresh growth media containing puromycin (1 ìg/mL) to select for infected cells[10], [11].…”

Section: Methodsmentioning

confidence: 99%

Manipulation Therapy Prior to Diagnosis Induced Primary Osteosarcoma Metastasis—From Clinical to Basic Research

Wang

Chen

et al. 2014

PLoS ONE

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Osteosarcoma (OS) patients who suffer manipulation therapy (MT) prior to diagnosis resulted in poor prognosis with increasing metastasis or recurrence rate. The aim of the study is to establish an in vivo model to identify the effects of MT on OS. The enrolled 235 OS patients were followed up in this study. In vivo nude mice model with tibia injection of GFP-labeled human OS cells were randomly allocated into MT(+) that with repeated massage on tumor site twice a week and no treatment as MT(−) group. The five-year survival, metastasis and recurrence rates were recorded in clinical subjects. X-ray plainfilm, micro-PET/CT scan, histopathology, serum metalloproteinase 2 (MMP2), metalloproteinase 9 (MMP9) level and human kinase domain insert receptor (KDR) pattern were assayed in mice model. The results showed that patient with MT decreased 5-year survival and higher recurrence or metastasis rate. Compatible with clinical findings, the decreased body weight (30.5±0.65 g) and an increased tumor volume (8.3±1.18 mm3) in MT(+) mice were observed. The increasing signal intensity over lymph node region of hind limb by micro-PET/CT and the tumor cells were detected in lung and bilateral lymph nodes only in MT(+) group. MMP2 (214±9.8 ng/ml) and MMP9 (25.5±1.81 ng/ml) were higher in MT(+) group than in MT(−) group (165±7.8 ng/ml and 16.9±1.40 ng/ml, individually) as well as KDR expression. Taking clinical observations and in vivo evidence together, MT treatment leads to poor prognosis of primary osteosarcoma; physicians should pay more attention on patients who seek MT before diagnosis.

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“…If the PDCD5 gene was used to treat a tumor, it would be preferable to select a tumor-specific promoter or enhancer, such as hTERT, to insert into the upstream region of the gene. This would be important for virus amplification, so that gene transcription can be well controlled (28,29). Finally, specific receptors might be able to be used to increase the targeting capacity of the viral envelope.…”

Section: Discussionmentioning

confidence: 99%

Construction and characterization of a PDCD5 recombinant lentivirus vector and its expression in tumor cells

et al. 2012

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Abstract. The function and mechanism of the programmed cell death 5 (PDCD5) gene is not completely understood, so it is necessary to build a stable and efficient PDCD5 recombinant lentiviral expression vector. The coding region of the PDCD5 gene was PCR amplified from pCMV-SPORT6. Next, the PDCD5 fragment and the pGC-FU vector were digested with the restriction enzyme AgeI and ligated by in-Fusion technology to build the pGC-FU-PDCD5 plasmid. Competent E. coli DH5α cells were transformed and positive clones were identified by PCR. The PCR products were digested and sequenced. After sequencing, positive clones were selected to grow and propagate. The pGC-FU-PDCD5 plasmid DNA was purified and mixed with the pHelper1.0 and pHelper2.0 packaging plasmids. They were co-transfected into 293T cells. The viral titer was measured by real-time PCR. The expression of GFP and PDCD5 was detected by both Western blotting and fluorescence microscopy. Additionally, the A498, ACHN, HCT116 and LoVo tumor cell lines were transfected with the virus supernatant, and PDCD5 expression was detected in these cells. The constructed pGC-FU-PDCD5 plasmid contained the correct PDCD5 gene sequence. Strong green fluorescence in both the cytoplasm and cell membrane was observed following transfection with purified pGC-FU-PDCD5 into 293T cells. The transfection rate was greater than 95%, and the expression of the PDCD5-GFP fusion protein was confirmed by Western blotting. The titer of the recombinant PDCD5 lentiviral condensed supernatant was measured to be 2x10 9 Tu/ml. The transfection efficiencies of A498, ACHN and HCT116 cells were greater than 90%. However, transfection efficiency of LoVo cells was lower but still greater than 70%. The PDCD5-GFP fusion protein was stable in these transfected tumor cells, as detected by Western blotting. In conclusion, a PDCD5 recombinant lentiviral vector was successfully constructed, and high expression of the plasmid was observed in tumor cells.

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An Optimized Telomerase-Specific Lentivirus for Optical Imaging of Tumors

Cited by 17 publications

References 35 publications

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

Manipulation Therapy Prior to Diagnosis Induced Primary Osteosarcoma Metastasis—From Clinical to Basic Research

Construction and characterization of a PDCD5 recombinant lentivirus vector and its expression in tumor cells

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