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Identification of non-coding RNAs embracing microRNA-143/145 cluster.

Iio A, Nakagawa Y, Hirata I, Naoe T, Akao Y - Mol. Cancer (2010)

Bottom Line: However, the mechanism of this down-regulation has not been investigated in detail.Then we identified the host gene encoding both miRNAs.The transcripts of this gene were approximately 11, 7.5, and 5.5 kb long; and the expression of these transcripts was coordinated with that of its resident miRNAs and down-regulated in the cancer cell lines tested as well as in colorectal cancer tissue samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Oncology, Gifu International Institute of Biotechnology, 1-1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, Japan. aiio@giib.or.jp

ABSTRACT
In a variety of cancers, altered patterns of microRNA (miRNA) expression are reported and may affect the cell cycle and cell survival. Recent studies suggest that the expression level of miRNAs that act as tumor suppressors is frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription and disturbances in miRNA processing. miR-143 and -145, which are located approximately 1.3 kb from each other at chromosome 5q33, are highly expressed in several tissues, but down-regulated in most cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that both miRNAs were expressed well under the same control program in human tissues, but were down-regulated equally in the most of the cancer cell lines tested. Then we identified the host gene encoding both miRNAs. The transcripts of this gene were approximately 11, 7.5, and 5.5 kb long; and the expression of these transcripts was coordinated with that of its resident miRNAs and down-regulated in the cancer cell lines tested as well as in colorectal cancer tissue samples. These data demonstrate that the host gene can function as a primary miRNA transcript and suggest that the down-regulation of host gene expression caused the low-expression of its encoded microRNAs-143 and -145 in human cancer cell lines and in cancer tissues.

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Real-time RT-PCR analysis of mature miR-143 and -145 expression in human normal tissues. Total RNAs from human normal tissues were purchased from Biochain (Hayward, USA) and Takara (Otsu, Japan). Expression of both miRNAs in each sample was detected by using a TaqMan microRNA reverse transcription kit and TaqMan microRNA Assay Kit (Applied Biosystems, Foster City, USA) and normalized with RNU6B. Calculation of the Ct value was done by using the second derivative maximum method, and relative quantification was analyzed by the comparative Ct (ΔCt) method. All reactions were run in triplicate. Relative miR-143 (A) and -145(B) expression levels (value of 2-ΔCt(miR-RNU6B)) are indicated on the left axis, with error bars indicating the standard deviation for these analyses.
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Figure 1: Real-time RT-PCR analysis of mature miR-143 and -145 expression in human normal tissues. Total RNAs from human normal tissues were purchased from Biochain (Hayward, USA) and Takara (Otsu, Japan). Expression of both miRNAs in each sample was detected by using a TaqMan microRNA reverse transcription kit and TaqMan microRNA Assay Kit (Applied Biosystems, Foster City, USA) and normalized with RNU6B. Calculation of the Ct value was done by using the second derivative maximum method, and relative quantification was analyzed by the comparative Ct (ΔCt) method. All reactions were run in triplicate. Relative miR-143 (A) and -145(B) expression levels (value of 2-ΔCt(miR-RNU6B)) are indicated on the left axis, with error bars indicating the standard deviation for these analyses.

Mentions: We examined the expression levels of mature miR-143 and -145 in human normal tissues by performing TaqMan microRNA assays (Fig. 1). In human normal tissues, miR-143 and -145 showed good expression in stomach, intestine, cervix, uterus, colon, and prostate (Fig. 1A, B). Whereas, in cancer cell lines, they were expressed at an extremely low level compared with that in human normal cell lines (Additional file 1 - Figure S1). Compared with their expression in corresponding normal tissues, the expression levels of both miRNAs were obviously down-regulated in all cancer cell lines and cancer tissue samples tested, just as many groups had previously reported [6-18]. Such a similar expression pattern of them indicates that the expression of both miRNAs may be regulated by a similar mechanism. Additionally, the DNA loci of both miRNAs are very close to each other, within 1.3 kb, which led us to speculate that both precursors may originate from the same primary transcript. Genomic PCR spanning this region demonstrates the fragment in most of the cancer cell lines tested [6,7,9]. Therefore, we decided to isolate the gene that carried both miRNAs in a cluster.


Identification of non-coding RNAs embracing microRNA-143/145 cluster.

Iio A, Nakagawa Y, Hirata I, Naoe T, Akao Y - Mol. Cancer (2010)

Real-time RT-PCR analysis of mature miR-143 and -145 expression in human normal tissues. Total RNAs from human normal tissues were purchased from Biochain (Hayward, USA) and Takara (Otsu, Japan). Expression of both miRNAs in each sample was detected by using a TaqMan microRNA reverse transcription kit and TaqMan microRNA Assay Kit (Applied Biosystems, Foster City, USA) and normalized with RNU6B. Calculation of the Ct value was done by using the second derivative maximum method, and relative quantification was analyzed by the comparative Ct (ΔCt) method. All reactions were run in triplicate. Relative miR-143 (A) and -145(B) expression levels (value of 2-ΔCt(miR-RNU6B)) are indicated on the left axis, with error bars indicating the standard deviation for these analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903500&req=5

Figure 1: Real-time RT-PCR analysis of mature miR-143 and -145 expression in human normal tissues. Total RNAs from human normal tissues were purchased from Biochain (Hayward, USA) and Takara (Otsu, Japan). Expression of both miRNAs in each sample was detected by using a TaqMan microRNA reverse transcription kit and TaqMan microRNA Assay Kit (Applied Biosystems, Foster City, USA) and normalized with RNU6B. Calculation of the Ct value was done by using the second derivative maximum method, and relative quantification was analyzed by the comparative Ct (ΔCt) method. All reactions were run in triplicate. Relative miR-143 (A) and -145(B) expression levels (value of 2-ΔCt(miR-RNU6B)) are indicated on the left axis, with error bars indicating the standard deviation for these analyses.
Mentions: We examined the expression levels of mature miR-143 and -145 in human normal tissues by performing TaqMan microRNA assays (Fig. 1). In human normal tissues, miR-143 and -145 showed good expression in stomach, intestine, cervix, uterus, colon, and prostate (Fig. 1A, B). Whereas, in cancer cell lines, they were expressed at an extremely low level compared with that in human normal cell lines (Additional file 1 - Figure S1). Compared with their expression in corresponding normal tissues, the expression levels of both miRNAs were obviously down-regulated in all cancer cell lines and cancer tissue samples tested, just as many groups had previously reported [6-18]. Such a similar expression pattern of them indicates that the expression of both miRNAs may be regulated by a similar mechanism. Additionally, the DNA loci of both miRNAs are very close to each other, within 1.3 kb, which led us to speculate that both precursors may originate from the same primary transcript. Genomic PCR spanning this region demonstrates the fragment in most of the cancer cell lines tested [6,7,9]. Therefore, we decided to isolate the gene that carried both miRNAs in a cluster.

Bottom Line: However, the mechanism of this down-regulation has not been investigated in detail.Then we identified the host gene encoding both miRNAs.The transcripts of this gene were approximately 11, 7.5, and 5.5 kb long; and the expression of these transcripts was coordinated with that of its resident miRNAs and down-regulated in the cancer cell lines tested as well as in colorectal cancer tissue samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Oncology, Gifu International Institute of Biotechnology, 1-1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, Japan. aiio@giib.or.jp

ABSTRACT
In a variety of cancers, altered patterns of microRNA (miRNA) expression are reported and may affect the cell cycle and cell survival. Recent studies suggest that the expression level of miRNAs that act as tumor suppressors is frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription and disturbances in miRNA processing. miR-143 and -145, which are located approximately 1.3 kb from each other at chromosome 5q33, are highly expressed in several tissues, but down-regulated in most cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that both miRNAs were expressed well under the same control program in human tissues, but were down-regulated equally in the most of the cancer cell lines tested. Then we identified the host gene encoding both miRNAs. The transcripts of this gene were approximately 11, 7.5, and 5.5 kb long; and the expression of these transcripts was coordinated with that of its resident miRNAs and down-regulated in the cancer cell lines tested as well as in colorectal cancer tissue samples. These data demonstrate that the host gene can function as a primary miRNA transcript and suggest that the down-regulation of host gene expression caused the low-expression of its encoded microRNAs-143 and -145 in human cancer cell lines and in cancer tissues.

Show MeSH
Related in: MedlinePlus