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Dynamic changing of miR-7 expression pattern affected by culture age of Chinese hamster ovary cells

Zedan, Wahed (2014) MOBM19 20141
Degree Projects in Molecular Biology
Abstract
MiRNA molecules have the ability to regulate hundreds of gene expressions and play a central role in a wide range of biological processes. MiR-7 may be a potential tool and promising target to improve and optimize the growth characteristic and antibody productivity in Chinese Hamster Ovary (CHO) cells which represent the most commonly used mammalian cell line for therapeutic recombinant protein production. The molecular mechanisms of gene regulation that control many physiological processes of CHO cells, such cell growth, apoptosis and secretion, are not fully understood. Samples are taken out during the logarithmic phase in most studies of miRNA’s expression profile, while here we try to examine the expression pattern of miR-7 in a... (More)
MiRNA molecules have the ability to regulate hundreds of gene expressions and play a central role in a wide range of biological processes. MiR-7 may be a potential tool and promising target to improve and optimize the growth characteristic and antibody productivity in Chinese Hamster Ovary (CHO) cells which represent the most commonly used mammalian cell line for therapeutic recombinant protein production. The molecular mechanisms of gene regulation that control many physiological processes of CHO cells, such cell growth, apoptosis and secretion, are not fully understood. Samples are taken out during the logarithmic phase in most studies of miRNA’s expression profile, while here we try to examine the expression pattern of miR-7 in a different producer of GS-CHO cell line during the whole time of batch culture. In our study the presented data revealed that miR-7 expression levels were gradually decreased over culture time. In other words, miR-7 was upregulated during lag and exponential phases and was then down-regulated in stationary and decline phases, with no relation between those expressions and a different producer of CHO cell lines. A negative correlation between cumulative viable cell time and miR-7 expression
level was counted and the results were p = –0.97, p = –0.95 and p = –0.96 for CL 47, CL 160 and CL 164 respectively, while a positive correlation between glucose consumption and miR- 7 expression level was counted and the results were p = 0.86, p = 0.90 and p = 0.92 for CL 47, CL 160 and CL 164 respectively. Positive correlations were shown between the miR-7 expression level and the specific growth rate – the results were p = 0.75, p = 0.86 and p = 0.92 for CL 47, CL 160 and CL 164, respectively. After the variable factors affecting miR-7 were minimized, the differences of miR-7 expression between different cell densities for each cell line were non-significant (P-value > 0.05) and miR-7 expression levels were approximately constant. Based on our findings, we suggest that the changes in internal and external factors such as a cumulative viable cell time, growth rate and glucose consumption level during different phases of batch culture have a large impact on dynamically altering miR7 expression levels.

Glucose consumption level has a central role in increasing and decreasing the expression of miR-7. Therefore, we suggest studying the internal cellular mechanisms including miR-7 expressions that may be affected by glucose depletion and glucose abundance in growth media of CHO cells. Our data highlighted the importance of cellular environment and culture age on the expression pattern of miR-7. (Less)
Popular Abstract
Dynamic changes in miR-7 expression pattern in Chinese hamster ovary cells

MicroRNAs are non-coding, highly conserved, short strand RNA of 18 to 24 nucleotides in length which have the ability to regulate hundreds of gene expressions and play a central role in a wide range of biological processes. MiR-7 has been identified as a promising target to improve and optimize the growth characteristic and antibody productivity in Chinese Hamster Ovary (CHO) cells which represent the most commonly used mammalian cell line for therapeutic recombinant protein production. The molecular mechanisms of gene regulation that control many physiological processes of CHO cells, such cell growth, apoptosis and secretion, are not fully understood. Samples... (More)
Dynamic changes in miR-7 expression pattern in Chinese hamster ovary cells

MicroRNAs are non-coding, highly conserved, short strand RNA of 18 to 24 nucleotides in length which have the ability to regulate hundreds of gene expressions and play a central role in a wide range of biological processes. MiR-7 has been identified as a promising target to improve and optimize the growth characteristic and antibody productivity in Chinese Hamster Ovary (CHO) cells which represent the most commonly used mammalian cell line for therapeutic recombinant protein production. The molecular mechanisms of gene regulation that control many physiological processes of CHO cells, such cell growth, apoptosis and secretion, are not fully understood. Samples are taken out during the logarithmic phase in most studies of miRNA’s expression profile. Here, we examine the expression pattern of miR-7 in a group of GS-CHO clonal cell lines with varying levels of productivity over the entire course of batch culture. In addition to study the relationship of miR-7 with cellular metabolic activity during batch cultivation.

During this project, six different CHO cell lines which have different levels of cB72.3 IgG4 monoclonal antibody productivity were cultivated for one week batch culture. The concentration of monoclonal antibody was measured using a sandwich enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction RTqPCR was Performed to detect and quantify the expression level of miRNA-7 during the batch culture of cell lines. In our study the presented data revealed that miR-7 expression levels were gradually decreased over culture time. In other words, miR-7 was up-regulated during lag and exponential phases and was then down-regulated in stationary and decline phases, with no relation between those expressions and a different producer of CHO cell lines. A negative correlation between cumulative viable cell time and miR- 7 expression level was found, while MiR-7 expression levels were positively related to each of specific growth rate and glucose concentration level during culture time. After the variable factors affecting miR-7 were minimized, the differences of miR-7 expression between different cell densities for each cell line were non-significant and miR-7 expression levels were approximately constant. Our findings suggest that changes in parameters such as a cumulative viable cell time, growth rate, and in particular glucose during different phases of batch culture have a major impact on miR7 expression. Therefore, we suggest studying the internal cellular mechanisms including miR-7 expressions that may be affected by glucose depletion and glucose abundance in growth media of CHO cells. Our data highlighted the importance of cellular environment and culture age on the expression pattern of miR-7.

Advisor’s name: Professor Mohamed Al-Rubeai (University College Dublin, Ireland)
Master’s degree Project in Molecular Genetics, 30 Credits, 2014
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Zedan, Wahed
supervisor
organization
course
MOBM19 20141
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
4698925
date added to LUP
2014-10-20 10:10:46
date last changed
2014-10-21 14:00:03
@misc{4698925,
  abstract     = {MiRNA molecules have the ability to regulate hundreds of gene expressions and play a central role in a wide range of biological processes. MiR-7 may be a potential tool and promising target to improve and optimize the growth characteristic and antibody productivity in Chinese Hamster Ovary (CHO) cells which represent the most commonly used mammalian cell line for therapeutic recombinant protein production. The molecular mechanisms of gene regulation that control many physiological processes of CHO cells, such cell growth, apoptosis and secretion, are not fully understood. Samples are taken out during the logarithmic phase in most studies of miRNA’s expression profile, while here we try to examine the expression pattern of miR-7 in a different producer of GS-CHO cell line during the whole time of batch culture. In our study the presented data revealed that miR-7 expression levels were gradually decreased over culture time. In other words, miR-7 was upregulated during lag and exponential phases and was then down-regulated in stationary and decline phases, with no relation between those expressions and a different producer of CHO cell lines. A negative correlation between cumulative viable cell time and miR-7 expression
level was counted and the results were p = –0.97, p = –0.95 and p = –0.96 for CL 47, CL 160 and CL 164 respectively, while a positive correlation between glucose consumption and miR- 7 expression level was counted and the results were p = 0.86, p = 0.90 and p = 0.92 for CL 47, CL 160 and CL 164 respectively. Positive correlations were shown between the miR-7 expression level and the specific growth rate – the results were p = 0.75, p = 0.86 and p = 0.92 for CL 47, CL 160 and CL 164, respectively. After the variable factors affecting miR-7 were minimized, the differences of miR-7 expression between different cell densities for each cell line were non-significant (P-value > 0.05) and miR-7 expression levels were approximately constant. Based on our findings, we suggest that the changes in internal and external factors such as a cumulative viable cell time, growth rate and glucose consumption level during different phases of batch culture have a large impact on dynamically altering miR7 expression levels.

Glucose consumption level has a central role in increasing and decreasing the expression of miR-7. Therefore, we suggest studying the internal cellular mechanisms including miR-7 expressions that may be affected by glucose depletion and glucose abundance in growth media of CHO cells. Our data highlighted the importance of cellular environment and culture age on the expression pattern of miR-7.},
  author       = {Zedan, Wahed},
  language     = {eng},
  note         = {Student Paper},
  title        = {Dynamic changing of miR-7 expression pattern affected by culture age of Chinese hamster ovary cells},
  year         = {2014},
}