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Published on: June 2, 2016
Identification of genes involved in liver cancer cell growth using an antisense library of phage genomic DNA
Yun Han Lee1, Young Ho Kim, Jong Gu Park
1WelGENE Inc., Daegu, Korea.
Purpose:
Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage.
Materials And Methods:
A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1,200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2.
Results:
When examined for growth inhibition after antisense transfection, 153 out of 1,200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays.
Conclusion:
These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery.
Insights
Researchers identified genes influencing liver cancer cell growth using a novel large circular (LC-) antisense DNA library. This method screened 1,200 clones, revealing 58 unique genes with growth inhibitory effects, aiding drug discovery.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Liver cancer is a significant global health concern.
- Identifying genes that drive cancer cell proliferation is crucial for developing effective treatments.
- Current methods for gene discovery can be time-consuming and lack high-throughput capabilities.
Purpose of the Study:
- To develop and utilize a novel large circular (LC-) genomic DNA antisense library for identifying genes involved in liver cancer cell growth.
- To screen this library for molecules that inhibit HepG2 liver cancer cell proliferation.
- To validate the identified genes through further cell-based assays.
Main Methods:
- Construction of a subtracted cDNA library using suppression subtractive hybridization (SSH) and unidirectional cloning into an M13 phagemid vector.
- Preparation of LC-antisense molecules from 1,200 random cDNA clones using M13 bacteriophage life cycle.
- Transfection of HepG2 cells with LC-antisense molecules and assessment of growth inhibition.
Main Results:
- Out of 1,200 LC-antisense molecules tested, 153 demonstrated growth inhibitory effects on HepG2 cells.
- Sequence analysis of these 153 clones led to the identification of 58 unique genes.
- Further cell-based assays corroborated these findings.
Conclusions:
- The developed LC-antisense library is a valuable tool for high-throughput screening to discover genes implicated in specific biological functions, such as liver cancer cell growth.
- This system shows promise as an effective target validation platform for gene-based drug discovery in oncology.
