Passage Overexpression of the miR-17~92 gene cluster occurs in many human cancers involving c-Myc, a gene coding for a transcription factor that regulates expression of proteins such as p21, an inhibitor of cell cycle progression. miR-17~92 encodes six distinct microRNAs (miRNAs) that can be divided into four families based on sequence identity at positions 2-7 (Table 1).Table 1 Sequence Identities of miRNA Families Encoded by the mir-17∼92 Gene Cluster Experiment 1Two independent, diffuse B-cell lymphoma cell lines were used to study the functional relationship between miR-17~92 transcripts and c-Myc in cancer pathogenesis. The first cell line was derived from Eμ-Myc mice (Eμ), which overexpress a c-Myc transgene under the control of a B-cell-specific enhancer that induces early development of B-cell lymphoma. The second cell line was derived from Eμ-Myc mice carrying a miR-17∼92 knockout allele (Δ/Δ). As shown in Figure 1, researchers measured the growth of both cell lines, as well as that of Δ/Δ cells infected with a retrovirus expressing the entire miR-17∼92 cluster (Δ/Δ + miR-17∼92). Figure 1 Growth curves of Eμ cells, Δ/Δ cells, and Δ/Δ + miR-17∼92 cells (error bars = standard deviation)Experiment 2To assess the relative contribution of each miRNA to the oncogenic ability of the miR-17∼92 cluster, Δ/Δ cell lines were transduced with an empty vector or with miR-17∼92 mutant allele constructs that were engineered to lack expression of miRNA from at least one of the four families. In each of the constructs, the family that was knocked out of the vector is indicated by the Δ symbol. After verifying that the transduced cells correctly expressed the desired miRNA, the fraction of apoptotic cells in each line was measured (Figure 2). Figure 2 Percentage of apoptotic cells in Eμ cells and Δ/Δ cells transduced with the indicated miR-17~92 construct (error bars = standard deviation) Adapted from Mu P, Han YC, Betel D, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 2009;23(24):2806-11. -If changes in p21 expression contribute to the development of B-cell lymphoma in Eμ-Myc mice, what is the expected result of real-time PCR analysis of c-Myc and p21 expression in wild-type (wt) and Eμ-Myc (Eμ) mice? (Note: mRNA levels are standardized to the expression of ubiquitin (Ub), which is not regulated by c-Myc.)

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Quizplus · Accepted Answer

11ecba2d_0f60_0c35_a3bf_69777e024092_MD0008_00 Standard polymerase chain reaction (PCR) measures DNA amplification after all the thermal cycles are complete, whereas real-time PCR quantifies product amplification as the reaction progresses in real time. The researchers assessed expression of c-Myc and p21 in wild-type (normal) and Eμ-Myc (Eμ) mice using real-time PCR. According to the passage, c-Myc overexpression leads to early cancer development (ie, B-cell lymphoma) in Eμ-Myc mice. Therefore, c-Myc is most likely an oncogene, a mutated or overexpressed gene that induces uncontrolled cell growth by promoting cell cycle progression or inhibition of apoptosis. As a result, c-Myc levels would be higher in Eμ-Myc (Eμ) mice than in wild-type mice to promote cancerous cell growth. Based on the passage, regulation of p21 expression by c-Myc also contributes to B-cell lymphoma development in Eμ-Myc mice. In addition, the passage states that p21 inhibits cell cycle progression, a key feature of a tumor suppressor gene. Tumor suppressor genes regulate DNA repair by repressing or pausing the cell cycle to ensure that only normal cells proceed to the division stage and induce programmed cell death if repair fails. As a result, p21 levels would be lower in Eμ-Myc mice compared to wild-type mice because p21 was likely inactivated by loss of function mutations and lost the ability to prevent abnormal growth/division of cancerous cells. (Choice B) p21 expression levels are correct in this graph; however, c-Myc expression should be increased (not decreased) in Eμ-Myc mice compared to wild-type mice. (Choice C) Compared to wild-type mice, c-Myc should be expressed more abundantly and p21 should be expressed less abundantly in Eμ-Myc mice. (Choice D) Although c-Myc expression levels are correct in this graph, p21 levels should be lower (not higher) in Eμ-Myc mice compared to wild-type mice. Educational objective: In the setting of cancer, oncogenes are mutated or expressed at abnormally high levels and contribute to cancer development by promoting cell growth/proliferation or suppressing apoptosis. In contrast, tumor suppressor genes, which induce programmed cell death, are inhibited in cancerous cells.

Passage Overexpression of the MiR-17~92 Gene Cluster Occurs in Many Human