ena-STUDY-Isfahan University of Medical Sciences-13-03-2018-10:15:10:922-17
Bioinformatics prediction and experimental validation of two novel miRNAs within SNHG21 and CDH4 luci in breast cancer tumorogenic and metastatic cell lines
Introduction: microRNAs (miRNAs) have critical roles in tumorigenesis and cancer metastasis. Despite the importance of these small molecules, considerable number of miRNAs have not been identified or experimentally validated yet. There are different approaches for miRNA discovery and next generation sequencing (NGS) is one of the newest and most sensitive approaches. In the current study, we employed bioinformatics tools to re-analyze NGS data obtained from a study by Ryu et. al. (PMID: 21346806) and then tried to experimentally validate two novel candidate miRNAs: miR-B6 and miR-B43Materials and methods: Several online softwares were employed for bioinformatics confirmation.Candidate pri-miRNAs sequences was then cloned in an expression vector and transfected into the AGS cells (a gastric cancer cell line). After over-expression, cells were harvested, RNAs were isolated and cDNAs were synthesized. Real-time PCR by specific primers was applied for detection of the mature form of the candidate miRNA.Results: Bioinformatics verification showed that the sequence of candidate miR-B43 is located in CDH4 gene which has an important role in cell-cell adhesion. Also miR-B6 is located within human SNHG21 gene which encodes for a long non-coding RNA. miR-B6 sequence is a highly conserved one in different species. Nearly all examined softwares predict both of miRNA as a real microRNAs. Transfection of the candidate pri-miRNAs sequences yielded in a detection of the mature form of miR-B6 and miR-B43 in AGS cells.of Conclusions: Our preliminary data shows that both candidate pri-miRNAs can be processed in cells yielding a novel mature miRNAs. Further experiments need to be performed to elucidate the functional roles of these two novel miRNAs in cancer cells.
Introduction: microRNAs (miRNAs) have critical roles in tumorigenesis and cancer metastasis. Despite the importance of these small molecules, considerable number of miRNAs have not been identified or experimentally validated yet. There are different approaches for miRNA discovery and next generation sequencing (NGS) is one of the newest and most sensitive approaches. In the current study, we employed bioinformatics tools to re-analyze NGS data obtained from a study by Ryu et. al. (PMID: 21346806) and then tried to experimentally validate two novel candidate miRNAs: miR-B6 and miR-B43Materials and methods: Several online softwares were employed for bioinformatics confirmation.Candidate pri-miRNAs sequences was then cloned in an expression vector and transfected into the AGS cells (a gastric cancer cell line). After over-expression, cells were harvested, RNAs were isolated and cDNAs were synthesized. Real-time PCR by specific primers was applied for detection of the mature form of the candidate miRNA.Results: Bioinformatics verification showed that the sequence of candidate miR-B43 is located in CDH4 gene which has an important role in cell-cell adhesion. Also miR-B6 is located within human SNHG21 gene which encodes for a long non-coding RNA. miR-B6 sequence is a highly conserved one in different species. Nearly all examined softwares predict both of miRNA as a real microRNAs. Transfection of the candidate pri-miRNAs sequences yielded in a detection of the mature form of miR-B6 and miR-B43 in AGS cells.of Conclusions: Our preliminary data shows that both candidate pri-miRNAs can be processed in cells yielding a novel mature miRNAs. Further experiments need to be performed to elucidate the functional roles of these two novel miRNAs in cancer cells.