This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Summary:
The scientific article, "MicroRNA Let-7a Down-regulates MYC and Reverts MYC-induced Growth in Burkitt Lymphoma Cells", published by Sampson et al. tested the relationship between the MYC oncogene and an inhibiting compound, 10058-F4. To test this relationship, the authors used microRNAs (miRNA) to silence the MYC gene. The 10058-F4 compound was used because it was previously shown to inhibit MYC-MAX, a transcription factor. As well as a transcription factor inhibitor, 10058-F4 has been noted to decay MYC mRNA in Burkitt Lymphoma tumor cells.
The first step in this experiment was to determine the potential roles that miRNA served in regulating the decay of Burkitt Lymphoma MYC mRNAs. These mRNAs were evaluated through real-time TaqMan relative quantitation. The regulator mRNAs were then subjected to microRNA multianalyte suspension array (mirMASA) by xMAP platform. The authors also used a time-dependent study to measure concentrations of miRNAs in accordance with levels of the MYC-MAX transcription factor and levels of MYC mRNA. The last study used to evaluate the capacity of the miRNAs was through transfection analyses of pre-let-7a miRNA.
The authors were then interested in the levels of the MYC mRNA and cell growth in cells treated with the MYC inhibitor 10058-F4. A special cell line was used to do so and was called Namalwa along with two other Burkitt lymphoma cell lines. 10058-F4 was administered in increasing concentrations (60, 90, 120 μmol/L) to logarithmically growing cultures and measured MYC mRNA with real-time RT-PCR. They found that the MYC mRNA levels were down-regulated in a dose-dependent and time-related manner.
As levels of MYC mRNA were found to be down-regulated, interest was focused on whether there was an association of the compound-induced declines of mRNA levels with the MYC-MAX transcription factor. This was performed through immunoprecipitations and immunoblots. After the exposure of the cells to the 10058-F4 inhibitor, MAX protein levels were found not to change. A decrease in the MYC-MAX transcription factor however, was noted. The inhibitor was also shown to decrease levels of the MYC protein. This result was obtained using monoclonal antibodies to MYC protein.
The authors also performed experiments using overexpression of miRNAs. The overexpression of the miRNAs showed a time-dependent increase in miRNA amounts using locked nucleic acid-based technology. Using this methodology, increases were found in let-7a, let-7b and mir-98 miRNAs. The authors also tested for the expression of mir-17-5p due to previous studies that have showed MYC induction of the miRNA. In contrast to the three previously mentioned miRNAs, mir-17-5p levels were reduced in inhibited MYC cells. The most significant changes of all four of these miRNAs were observed within 24 hours of the lines being treated with 10058-F4. A washout of 10058-F4 proved that the changes in let-7a, let-7b, mir-98, and MYC mRNA were drug inducible and also capable of being reversed.
The last experiment dealt with the transfection of rat lines with a pre-let-7a molecule. In both an immortalized fibroblast line (homozygous knockout of MYC) , the levels of let-7a increased and a decrease in MYC mRNA.
The siRNA MYC-transfected, 10058-F4-treated, and pre-let-7a-transfected fibroblasts showed an increase in let-7a and less MYC mRNA compared to the 1a-c Myc-transformed cells. The knockout rat also showed decrease mir-17-5p in comparison to the 1a-c Myc-transformed line. Plotting the ratios of let-7a and mir-17-5p against Myc mRNA levels there was a positive, linear correlation (r =. 99) between the two.
From the results of all the experiments, it was found that the levels of let-7a and mir-17-5p fluctuated in opposite directions with let-7a increasing and mir-17-5p decreasing without the presence of MYC mRNA. MYC was also noted as being down regulated. With this relationship between the miRNA and the MYC mRNA, the authors proposed an autoloop as a means of regulation.
Article Reaction:
The article summarized above was published in an easy and logical manner. First, the authors demonstrated that, indeed, the MYC inhibitor affected the levels of MYC mRNA. This finding allowed for subsequent experiments that showed a decrease in MYC-MAX complex levels affected the MYC mRNA level and that these changes lead to a change in the expression of the genes targeted by the complex. Now that the authors had identified the target genes of the MYC-MAX complex, they tested the target genes through the method of post-transcriptional silencing called miRNA. Essentially, when these miRNAs are not working in a functionally proper manner the results are the formation of the phenotypes associated with Burkitt lymphoma.
References:
1Sampson et al. (2007) MicroRNA Let-7a Down-regulates MYC and Reverts MYC-induced Growth in Burkitt Lymphoma Cells. Cancer Research. 67(20): 9762-9770 Retrieved from: http://cancerres.aacrjournals.org/cgi/content/full/67/20/9762?maxtoshow=&HITS=&hits=&RESULTFORMAT=&fulltext=MYC+and+Burkitt+Lymphoma&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT
Contact Info: Deeter Neumann, [email protected], May 14, 2009
