Synergistic action of the microRNA-17 polycistron and Myc in aggressive cancer development - PubMed (original) (raw)

Comparative Study

Synergistic action of the microRNA-17 polycistron and Myc in aggressive cancer development

Hiroyuki Tagawa et al. Cancer Sci. 2007 Sep.

Erratum in

Abstract

The c13orf25/miR-17 cluster, which is responsible for 13q31-q32 amplification in malignant lymphoma, contains the microRNA-17-18-19-20-92 polycistron. A previous study demonstrated that this polycistron could modulate tumor formation following transplantation of microRNA 17-19b into Eu-myc mice. Another study reported that Myc can upregulate the miR-17 cluster by binding directly upstream of the miR-17 locus. These findings suggest that Myc and the miR-17 cluster synergistically contribute to cancer development. In the study presented here, we observed recurrent 13q31-32 amplification in MYC-rearranged lymphomas (11 of 47 cases). Quantitative real-time polymerase chain reaction analysis of c13orf25 for MYC-rearranged lymphomas demonstrated that cases with 13q31-32 amplification showed significantly higher expression of c13orf25 than cases without such amplification, although cases without 13q31-32 amplification still showed slight upregulation of c13orf25. To investigate the relationship between Myc and the miR-17 polycistron in tumorigenesis, we engineered rat fibroblasts (Rat-1) that constitutively express the miR-17 polycistron (miR), Myc, or both miR and Myc. The highest level of miR expression was detected in Rat-1 transfected with both miR and Myc, whereas Myc transfectant cells alone also showed slight upregulation of miR. Furthermore, we demonstrated that nude mice injected with Rat-1 transfected with both miR and Myc presented more accelerated tumor growth than those injected with Myc transfectant cells. These results suggest that miR is stably upregulated in the presence of constitutive expression of Myc, and that the deregulation of miR and Myc synergistically contribute to aggressive cancer development, probably by repressing tumor suppressor genes.

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Figures

Figure 1

Figure 1

13q31‐32 amplification in _MYC_‐rearranged malignant lymphomas. (a) Frequencies of genomic gains of 13q in a series of samples of 26 sporadic Burkitt's lymphomas (sBL) and 21 diffuse large B‐cell lymphomas with MYC rearrangement. Bold bars indicate bacterial artificial chromosome (BAC) clones that showed recurrent genomic amplifications (n ≥ 2; log2 ratio ≥ +1.0). The arrow indicates the location of BAC RP11‐121J17, which includes a miR‐17 cluster. (b) Individual genome profiles of three cases of sBL with 13q amplification. Arrows indicate the location of BAC RP11‐121J17. Genomic gain: log2 ratio > 0.2; genomic loss: log2 ratio < –0.2.

Figure 2

Figure 2

Overexpression of the miR‐17 cluster in lymphoma with or without MYC rearrangement. (a) Real‐time quantitative polymerase chain reaction of the miR‐17 cluster in lymphomas with (n = 14) and without (n = 27) MYC rearrangement. Sporadic Burkitt's lymphoma (sBL) = 14 cases, diffuse large B‐cell lymphoma (DLBCL) = 12 cases, and adult T cell lymphoma (ATLL) = 15 cases. (b) Northern blot analysis of miR‐19a and ‐20 in sBL samples with (n = 5) and without (n = 5) 13q amplification. Average c13orf25 expression: sBL with 13q amplification = 21.94, sBL without 13q amplification = 2.23, DLBCL with 13q amplification = 6.99, DLBCL without 13q amplification = 0.74, ATLL = 0.27.

Figure 3

Figure 3

Overexpression of the miR‐17 cluster in the presence of constitutive expression of Myc. (a) Schematic illustrations of c13orf25 and the miR‐17 polycistron, and of the PMX vector constructs PMX‐Myc (puromycin resistant), PMX‐miR (neomycin resistant) and PMX‐miR‐Myc (puromycin resistant). (b) The expression of miR‐20 of rat fibroblast (Rat‐1) cells transfected with Mycpuro, miR‐17‐19bneo, miR‐17‐19bneo + Mycpuro, miR‐17‐92neo, miR‐17‐92neo + Mycpuro, and vector control (mock). The quantitative expression for miR‐20 was monitored using a densitometer. (c) Expression of MYC mRNA of Rat‐1 transfectants. (d) Expression of miR‐19a, miR17‐5p, miR‐17‐3p and miR‐18 in Rat‐1 cells transfected with miRpuro + Mycpuro, Mycpuro and mock. Exposure times are indicated below the boxes. (e) Expression of mature miR‐17‐5p, miR‐19a, miR‐17‐3p and miR‐18 of indicated B cell lymphoma cell lines. (f) Comparison of mature miR‐20 expression in Rat‐1 transfectants, B cell lymphoma cell lines with 13q amplification (Karpas 1718 and REC1),( 7, 8 ) and the HeLa cell line, which was derived from an epithelial cancer cell line without 13q amplification. The figure shows standard deviations for three independent experiments. (g) Rat‐1 expressing Mycpuro were subsequently transduced with miR‐17‐19bneo. ‘Mycpuro + miRneo’ represents a Rat‐1 cell transduced with both Mycpuro and miR‐17‐19bneo by dual drug selection as shown in the schematic illustration next to the photograph.

Figure 3

Figure 3

Overexpression of the miR‐17 cluster in the presence of constitutive expression of Myc. (a) Schematic illustrations of c13orf25 and the miR‐17 polycistron, and of the PMX vector constructs PMX‐Myc (puromycin resistant), PMX‐miR (neomycin resistant) and PMX‐miR‐Myc (puromycin resistant). (b) The expression of miR‐20 of rat fibroblast (Rat‐1) cells transfected with Mycpuro, miR‐17‐19bneo, miR‐17‐19bneo + Mycpuro, miR‐17‐92neo, miR‐17‐92neo + Mycpuro, and vector control (mock). The quantitative expression for miR‐20 was monitored using a densitometer. (c) Expression of MYC mRNA of Rat‐1 transfectants. (d) Expression of miR‐19a, miR17‐5p, miR‐17‐3p and miR‐18 in Rat‐1 cells transfected with miRpuro + Mycpuro, Mycpuro and mock. Exposure times are indicated below the boxes. (e) Expression of mature miR‐17‐5p, miR‐19a, miR‐17‐3p and miR‐18 of indicated B cell lymphoma cell lines. (f) Comparison of mature miR‐20 expression in Rat‐1 transfectants, B cell lymphoma cell lines with 13q amplification (Karpas 1718 and REC1),( 7, 8 ) and the HeLa cell line, which was derived from an epithelial cancer cell line without 13q amplification. The figure shows standard deviations for three independent experiments. (g) Rat‐1 expressing Mycpuro were subsequently transduced with miR‐17‐19bneo. ‘Mycpuro + miRneo’ represents a Rat‐1 cell transduced with both Mycpuro and miR‐17‐19bneo by dual drug selection as shown in the schematic illustration next to the photograph.

Figure 4

Figure 4

Tumorigenic assays. (a) Growth assay. Rat fibroblast (Rat‐1) cells containing the indicated vectors were drug selected and then subjected to a growth assay. Standard deviations for three independent transductions are shown. (b) Colony assay. Rat‐1 cells containing Myc, the miR‐17 polycistron (miR), miRpuro + Mycpuro and Mycpuro + miRneo were plated in soft agar and counted after 2 weeks. ‘miRpuro + Mycpuro’ represents a Rat‐1 cell transfected with both miR‐17‐19b and Myc by simultaneous construct as shown in Fig. 3a. ‘Mycpuro + miRneo’ represents a Rat‐1 cell transfected with both Myc and miR‐17‐19b by dual transfection as shown in Fig. 3g. Standard deviations for three independent experiments are shown. (c) Nude mice assay. A total of 2 × 106 Rat‐1 cells expressing Myc (n = 8), miR‐17‐19b (n = 8), miR + Myc (n = 8) and mock (n = 6) were injected subcutaneously. The ‘miRpuro + Mycpuro’ transfectant was used in this experiment. Averages and standard deviations are shown. The tumor size was monitored twice a week until days 35–42.

Figure 5

Figure 5

Transforming growth factor‐β receptor type II (TβRII) as a target of the miR‐17 cluster. (a) Western blot analysis of TβRII for the indicated rat fibroblast (Rat‐1) transfectants. α‐Tublin was use as a control. (b) Northern blot analysis of TβRII (rat) for the indicated Rat‐1 (rat) transfectants. Ethidium bromide (EtBr) was used as a control. (c) Luciferase assay. pGL3‐TβRIImut, pGL3‐inserted mutated TβRII 3′ untranslated region (UTR); pGL3‐TβRII, pGL3‐inserted wild‐type TβRII 3′ UTR; PGL3‐17 or ‐20, negative control containing a miR‐20 sequence. PGL3‐17c and pGL3‐20c are positive controls containing their complementary sequence inserted into the pGL3 _Xba_I site. The firefly luciferase vector was modified from the pGL3 Control Vector (Promega), such that TβRII 3′ UTR was inserted into the _Xba_I site immediately downstream of the stop codon. Similarly, miR‐17‐5p (pGL‐17), miR‐20 (pGL3‐20) and their complementary sequences (miR‐17c, miR‐20c) were inserted immediately downstream of the stop codon of the luciferase gene (_Xba_I site).

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