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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 the host gene encoding miR-143 and -145. Real-time RT-PCR analysis was performed on human normal tissues and cancer cell lines (A) and human colon adenocarcinoma samples (B) by using host gene-specific primers. Ten pairs of colon samples, including 10 human colon adenocarcinoma tissue samples and 10 matched normal colon tissue samples, were obtained from Osaka Medical College Hospital (Takatsuki, Osaka, Japan) and Fujita Health University Hospital (Toyoake, Aichi, Japan) with patients' informed consent. Collection and distribution of the samples were approved by each of the appropriate institutional review boards. The category of colon samples was confirmed by pathological analysis. Total RNA was extracted from the colon samples by using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. Expression levels of the host gene in each sample (LOC3 and LOC10, refer to Fig. 2) were detected by using a Superscript III reverse transcription kit (Invitrogen, Carlsbad, USA) and SYBR Premix Ex Taq (Takara, Otsu, Japan) and normalized with GAPDH. Ruled lines present the medians of samples. Data were analyzed by using Student's t-test. A value of p < 0.05 was considered significant.
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Figure 4: Real-time RT-PCR analysis of the host gene encoding miR-143 and -145. Real-time RT-PCR analysis was performed on human normal tissues and cancer cell lines (A) and human colon adenocarcinoma samples (B) by using host gene-specific primers. Ten pairs of colon samples, including 10 human colon adenocarcinoma tissue samples and 10 matched normal colon tissue samples, were obtained from Osaka Medical College Hospital (Takatsuki, Osaka, Japan) and Fujita Health University Hospital (Toyoake, Aichi, Japan) with patients' informed consent. Collection and distribution of the samples were approved by each of the appropriate institutional review boards. The category of colon samples was confirmed by pathological analysis. Total RNA was extracted from the colon samples by using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. Expression levels of the host gene in each sample (LOC3 and LOC10, refer to Fig. 2) were detected by using a Superscript III reverse transcription kit (Invitrogen, Carlsbad, USA) and SYBR Premix Ex Taq (Takara, Otsu, Japan) and normalized with GAPDH. Ruled lines present the medians of samples. Data were analyzed by using Student's t-test. A value of p < 0.05 was considered significant.

Mentions: Most miRNAs located within protein-coding or non-coding genes are transcriptionally linked to the expression of their host genes [27]. In order to investigate the coordinated expression of the host gene identified in this study with mature miR-143 and -145, we performed real-time RT-PCR analysis by using the host gene-specific primer set shown in Fig. 2. In human normal tissues, the host gene was highly expressed, as were both miRNAs; but in the corresponding cancer cell lines, the signal was hardly detected (Fig. 4A), though the host gene and both miRNAs were highly expressed in normal human cell lines (Additional files 1 - Figure S1 & S2). Also in human cancer tissues, the host gene was down-regulated compared with its expression in normal human tissues (Figs. 3, 4B and additional file 1 - Figure S3). As a result, the down-regulation of host gene NCR143/145 expression caused low expression of both mature miRNAs in human cancer cell lines. Thus, the aberrant transcription of NCR143/145 could contribute to the low expression of miR-143 and -145.


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 the host gene encoding miR-143 and -145. Real-time RT-PCR analysis was performed on human normal tissues and cancer cell lines (A) and human colon adenocarcinoma samples (B) by using host gene-specific primers. Ten pairs of colon samples, including 10 human colon adenocarcinoma tissue samples and 10 matched normal colon tissue samples, were obtained from Osaka Medical College Hospital (Takatsuki, Osaka, Japan) and Fujita Health University Hospital (Toyoake, Aichi, Japan) with patients' informed consent. Collection and distribution of the samples were approved by each of the appropriate institutional review boards. The category of colon samples was confirmed by pathological analysis. Total RNA was extracted from the colon samples by using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. Expression levels of the host gene in each sample (LOC3 and LOC10, refer to Fig. 2) were detected by using a Superscript III reverse transcription kit (Invitrogen, Carlsbad, USA) and SYBR Premix Ex Taq (Takara, Otsu, Japan) and normalized with GAPDH. Ruled lines present the medians of samples. Data were analyzed by using Student's t-test. A value of p < 0.05 was considered significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Real-time RT-PCR analysis of the host gene encoding miR-143 and -145. Real-time RT-PCR analysis was performed on human normal tissues and cancer cell lines (A) and human colon adenocarcinoma samples (B) by using host gene-specific primers. Ten pairs of colon samples, including 10 human colon adenocarcinoma tissue samples and 10 matched normal colon tissue samples, were obtained from Osaka Medical College Hospital (Takatsuki, Osaka, Japan) and Fujita Health University Hospital (Toyoake, Aichi, Japan) with patients' informed consent. Collection and distribution of the samples were approved by each of the appropriate institutional review boards. The category of colon samples was confirmed by pathological analysis. Total RNA was extracted from the colon samples by using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. Expression levels of the host gene in each sample (LOC3 and LOC10, refer to Fig. 2) were detected by using a Superscript III reverse transcription kit (Invitrogen, Carlsbad, USA) and SYBR Premix Ex Taq (Takara, Otsu, Japan) and normalized with GAPDH. Ruled lines present the medians of samples. Data were analyzed by using Student's t-test. A value of p < 0.05 was considered significant.
Mentions: Most miRNAs located within protein-coding or non-coding genes are transcriptionally linked to the expression of their host genes [27]. In order to investigate the coordinated expression of the host gene identified in this study with mature miR-143 and -145, we performed real-time RT-PCR analysis by using the host gene-specific primer set shown in Fig. 2. In human normal tissues, the host gene was highly expressed, as were both miRNAs; but in the corresponding cancer cell lines, the signal was hardly detected (Fig. 4A), though the host gene and both miRNAs were highly expressed in normal human cell lines (Additional files 1 - Figure S1 & S2). Also in human cancer tissues, the host gene was down-regulated compared with its expression in normal human tissues (Figs. 3, 4B and additional file 1 - Figure S3). As a result, the down-regulation of host gene NCR143/145 expression caused low expression of both mature miRNAs in human cancer cell lines. Thus, the aberrant transcription of NCR143/145 could contribute to the low expression of miR-143 and -145.

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