A Novel Quality Control Methodology on Screening out for Human Osteosarcoma Cell Adaptor Based on the Cell-SELEX Technique

Abstract Background: We have successfully developed a novel molecular probe for recognition of human osteosarcoma cell using the cell-SELEX method. The study aims to establish an accurate, time-saving quality-monitoring method in screening for tumour cell adaptors in order to shorten the screening process and ensure the accurate preparation of the adaptor. Methods: Two kinds of human osteosarcoma cells (U2-OS, HOS) were selected as the forward screening target cells and human fibrosarcoma cells (HT-1080) as the reverse screening cells to screen the adaptors from the candidate oligonucleotide library. In each round of preparation of the library, PCR was optimised by using quantitative template concentration instead of percentage volume. Each round of forward screening was conducted with reverse screening; Fluorescence spectroscopy and flow cytometry were used to monitor and compare the aptamer libraries. Results: During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the adaptor groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion: The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell adaptor. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.

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Development and Validation of a Novel Quality Control Methodology on Screening Out for Human Osteosarcoma Cell Aptamers

10.21203/rs.3.rs-116152/v1 ◽

2020 ◽

Author(s):

Hua Wang ◽

Ji Liang ◽

Yan Ma ◽

Lei Zhou ◽

Yufei Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

Quality Control ◽

Fluorescence Spectroscopy ◽

Quality Monitoring ◽

Target Cells ◽

Osteosarcoma Cell ◽

Time Saving ◽

Screening Process ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

Abstract Background: We have successfully established an aptamer library for recognition of human osteosarcoma cell using the cell-SELEX method. However, we found that the quality monitoring become a key, resulting in success or failure in the screening process. The study aims to establish an accurate, time-saving quality-monitoring method in screening for tumour cell aptamers in order to shorten the screening process and ensure the accurate preparation of the aptamer. Methods: Two kinds of human osteosarcoma cells (U2-OS, HOS) were selected as the forward screening target cells and human fibrosarcoma cells (HT-1080) as the reverse screening cells to screen the aptamers from the candidate oligonucleotide library. In each round of preparation of the library, PCR was optimised by using quantitative template concentration instead of percentage volume. Each round of forward screening was conducted with reverse screening; Fluorescence spectroscopy and flow cytometry were used to monitor and compare the aptamer libraries. Results: During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the aptamer groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion: The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell aptamers. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.

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Development and validation of a novel quality control methodology on screening out for human osteosarcoma cell aptamers

10.21203/rs.3.rs-175435/v1 ◽

2021 ◽

Author(s):

Hua Wang ◽

Ji Liang ◽

Yan Ma ◽

Lei Zhou ◽

Yufei Zhang ◽

...

Keyword(s):

Quality Control ◽

Fluorescence Spectroscopy ◽

Tumour Cell ◽

Rapid Screening ◽

Target Cells ◽

Osteosarcoma Cell ◽

Time Saving ◽

Monitoring Method ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

Abstract Objectives To establish an accurate, time-saving quality-monitoring method in screening for tumour cell aptamers in order to shorten the screening process and ensure the accurate preparation of the aptamer. Results During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the aptamer groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell aptamers. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.

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Anti-Metastatic and Anti-Angiogenic Effects of Curcumin Analog DK1 on Human Osteosarcoma Cells In Vitro

Pharmaceuticals ◽

10.3390/ph14060532 ◽

2021 ◽

Vol 14 (6) ◽

pp. 532

Author(s):

Muhammad Nazirul Mubin Aziz ◽

Nurul Fattin Che Rahim ◽

Yazmin Hussin ◽

Swee Keong Yeap ◽

Mas Jaffri Masarudin ◽

...

Keyword(s):

Cell Lines ◽

Safety Profile ◽

Quantitative Polymerase Chain Reaction ◽

Tube Formation ◽

Osteosarcoma Cell ◽

Curcumin Analog ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.

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Establishment and characterization of human osteosarcoma cell lines with different pulmonary metastatic potentials

Cytotechnology ◽

10.1007/s10616-009-9239-3 ◽

2009 ◽

Vol 61 (1-2) ◽

pp. 37-44 ◽

Cited By ~ 25

Author(s):

Xiang Chen ◽

Tong-Tao Yang ◽

Wei Wang ◽

Hong-Hui Sun ◽

Bao-An Ma ◽

...

Keyword(s):

Cell Lines ◽

Osteosarcoma Cell ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

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Deep Learning Approach to Human Osteosarcoma Cell Detection and Classification

Cryptology and Network Security - Lecture Notes in Computer Science ◽

10.1007/978-3-319-98678-4_36 ◽

2018 ◽

pp. 353-361

Author(s):

Mario D’Acunto ◽

Massimo Martinelli ◽

Davide Moroni

Keyword(s):

Deep Learning ◽

Cell Detection ◽

Learning Approach ◽

Osteosarcoma Cell ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

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Rhotekin 2 silencing inhibits proliferation and induces apoptosis in human osteosarcoma cells

Bioscience Reports ◽

10.1042/bsr20181384 ◽

2018 ◽

Vol 38 (6) ◽

Author(s):

Xiong Wang ◽

Lei Zhang ◽

Wenji Wang ◽

Yuchen Wang ◽

Ye Chen ◽

...

Keyword(s):

Cell Lines ◽

Phase Cell ◽

Osteosarcoma Cell ◽

Cyclin Dependent Kinase ◽

Potential Therapeutic Target ◽

Human Osteosarcoma ◽

Cycle Arrest ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell ◽

Induced Apoptosis

Human osteosarcoma is the most frequent primary malignant of bone, and often occurs in adolescents. However, molecular mechanism of this disease remains unclear. In the present study, we found that the level of Rhotekin 2 (RTKN2) was up-regulated in osteosarcoma tissues and cell lines. In addition, silencing of RTKN2 of human osteosarcoma cell lines U2OS, inhibited proliferation, and induced G1 phase cell cycle arrest via reducing the level of the cyclin-dependent kinase 2 (CDK2). Furthermore, RTKN2 knockdown in the U2OS cells induced apoptosis by increasing the level of Bax and decreasing the level of Bcl2. These results suggested that RTKN2 is involved in the progression of human osteosarcoma, and may be a potential therapeutic target.

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