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Year : 2015  |  Volume : 52  |  Issue : 1  |  Page : 22--25

miR-17-92 host gene, uderexpressed in gastric cancer and its expression was negatively correlated with the metastasis

F Bahari1, M Emadi-Baygi2, P Nikpour3,  
1 Department of Genetics, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran
2 Department of Genetics, Faculty of Basic Sciences, Shahrekord University; Research Institute of Biotechnology, Shahrekord University, Shahrekord, Iran
3 Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences; Applied Physiology Research Center, Isfahan University of Medical Sciences; Child Growth and Development Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Correspondence Address:
P Nikpour
Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences; Applied Physiology Research Center, Isfahan University of Medical Sciences; Child Growth and Development Research Center, Isfahan University of Medical Sciences, Isfahan
Iran

Abstract

Background: This study aimed to evaluate the expression of miR-17-92 host gene (MIR17HG), in gastric cancer and paired normal adjacent tissues for the 1st time. Methods: Using quantitative real-time-polymerase chain reaction, the MIR17HG expression was assessed in 30 paired tumoral and nontumoral gastric tissue samples. Results: Our results showed that this transcript was significantly underexpressed in gastric tumors compared with normal ones. Furthermore, there was an association between the expression levels of MIR17HG and gastric cancer grades and stages. Moreover, the expression level of MIR17HG was conversely associated with the size of tumoral specimens in early stages (stages I and II). We also observed an association between the presence of metastasis and lower expression of MIR17HG. Conclusion: Our results suggest that MIR17HG gene expression is dysregulated in gastric cancer in which it may indicate a tumor suppressive function of this miRNA cluster host gene in gastric cancer.



How to cite this article:
Bahari F, Emadi-Baygi M, Nikpour P. miR-17-92 host gene, uderexpressed in gastric cancer and its expression was negatively correlated with the metastasis.Indian J Cancer 2015;52:22-25


How to cite this URL:
Bahari F, Emadi-Baygi M, Nikpour P. miR-17-92 host gene, uderexpressed in gastric cancer and its expression was negatively correlated with the metastasis. Indian J Cancer [serial online] 2015 [cited 2019 Oct 15 ];52:22-25
Available from: http://www.indianjcancer.com/text.asp?2015/52/1/22/175605


Full Text

 Introduction



Gastric cancer is the second cause of cancer-related death and is the fourth most common malignancy in the world.[1] In Iranian men and women, this type of cancer is ranked the first and third common cancer, respectively.[2] There are two histological subtypes of gastric carcinoma, the intestinal and the diffuse type, both of which are associated with Helicobacterpylori infection.[3] Study on biological properties of tumors is helpful to find clinical behavior of tumors; that, in turn, help us to develop biomarkers for various purposes.

One of the best characterized miRNA clusters is the human miR-17-92 cluster, also known as oncomiR-1. Located at chromosome 13q31, miR-17-92 cluster, resides in the third intron of miR-17-92 host gene (MIR17HG). Transcription of this gene produces a single polycistronic transcript that yields at least six mature miRNAs with evolutionary conserved sequences. Elevated expression of miR-17-92 has been observed in a variety of hematological malignancies and solid tumors. Despite over-expression of the miR-17-92cluster in different tumor types, other studies indicate that DNA region encoding the miR-17-92 cluster is deleted in some cancers.[4]

Due to the overexpression of miR-17-92 cluster components in gastric cancer [5] and the lack of evidence in evaluating the expression of the host gene of the cluster, we aimed to evaluate the expression of MIR17HG in gastric cancer. To this end, we evaluated MIR17HG expression in 30 paired tumoral and non-tumoral gastric tissue samples using quantitative real-time-polymerase chain reaction (qRT-PCR). We further analyzed the correlation between the MIR17HG gene expression with clinicopathological features of gastric tumors such as TNM staging, tumor grades, perineural invasion, distant metastasis, age of patients and tumor size.

 Materials and Methods



Subjects

As previously described,[6] a total of 60 specimens of gastric cancerous and nonconcerous tissues were assessed in the current study. All tissue samples were provided by the Iran National Tumor Bank which is funded by Cancer Institute of Tehran University of Medical Sciences, for Cancer Research. The patients' written informed consents were obtained by Iran National Tumor Bank.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from all cancerous and nonconcerous tissues using Qiazol reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The quality of RNA was verified by electrophoresis. The quantity and quality of RNA were determined by ultraviolet spectroscopy at 260 and 280 nm, respectively. cDNA was synthesized using random hexamer primers and an M-MLV reverse transcriptase (Fermentas, Vilnius, Lithuania) as described previously.[6]

Quantitative real-time polymerase chain reaction

The expression level of MIR17HG was determined by qRT-PCR. The TATA box binding protein (TBP) gene was used as an internal control.[7] The primers for the target gene, MIR17HG, were as follows: 5' -CCTCCGGTCGTAGTAAAGC-3' and 5'-GTTAGGTCCACGTGTATGACAG-3' with an amplicon size of 110 bps. Relative gene expression was calculated from the crossing points by the 2-ΔΔCT method. qRT-PCR was performed using the SYBR premix Ex Taq, Tli RNaseH Plus (Takara, Japan) and run on the Rotor-gene 6000 (Qiagen, Hilden, Germany). The cycling conditions for PCR included an initial denaturation step at 95°C for 10 min, followed by 35 amplification cycles consisting of denaturation at 95°C for 40 s, annealing at 55°C and 58.5°C, for TBP and MIR17HG genes respectively, for 40 s and an extension at 72°C for 40 s. A few PCR products were loaded into an agarose gel, electrophoresed, stained with green viewer, and then visualized under the ultraviolet light.

Statistical analysis

Statistical analyses were performed using SPSS version 18.0 (SPSS Inc, Chicago, IL, USA). Differences between groups were analyzed using a paired t-test or one-way ANOVA with post-hoc multiple comparisons. All data are expressed as means ± standard error of mean. P < 0.05 was considered to be statistically significant.

 Results



miR-17-92 host gene expression in cancerous and noncancerous gastric samples

The expression of MIR17HG and TBP genes was determined using qRT-PCR in 30 tumor and 30 non-tumor gastric tissues. The CT values obtained from RT-PCR reactions on MIR17HG gene were normalized to that of the TBP gene and relative changes in expression of MIR17HG were monitored. Statistical analysis of the real-time qRT-PCR experiment results demonstrated that the relative expression of the gene in pooled tumoral tissues is significantly lower than those in nontumoral ones (P < 0.005) [Table 1].{Table 1}

Correlation of miR-17-92 host gene expression with clinicopathological features in gastric carcinoma

In order to examine the clinical importance of the MIR17HG underexpression, the correlation between clinicopathological status of gastric tumor specimens and level of MIR17HG expression was examined. Clinicopathological variables of patients are listed in [Table 1]. Although analysis demonstrated that the relative expression of the gene did not significantly show any differences in different sizes of the tumors, that is, larger or smaller than 5 cm (P: 0.307), the size of the tumors in early stages of cancer (stage I and II) was inversely correlated with the expression of MIR17HG (P: 0.007). Different grades of gastric cancer showed a significant correlation with the gene expression (P < 0.005). Our results didn't show any significant association of MIR17HG expression with TNM staging of gastric cancer (P: 0.197). However, there was a significant inverse correlation between the gene expression and early-late stages of the cancer (P: 0.042). Although M classification did not reach statistical significance (P: 0.252), the presence or absence of metastasis was correlated with the expression of MIR17HG (P: 0.006). No significant correlation was found between the expression of this host gene and invasion and metastasis to lymph nodes [Table 1]. Furthermore, there was no statistical significant correlation between the expression of the gene and other clinicopathological features of gastric tumors [Table 1].

 Discussion



miRNAs of miR-17-92 cluster can both target tumor suppressor genes and oncogenes acting as cell cycle promoters and/or inhibitors.[8]

Our pilot study demonstrated that the MIR17HG expression decreased significantly in gastric cancer. Furthermore, there was an association between the expression levels of MIR17HG and gastric cancer grades and early-late stages. Moreover, the expression of MIR17HG was conversely associated with the size of tumoral specimens in the early stages (stages I and II). Finally, MIR17HG expression was negatively correlated with the metastasis.

pri-miR-17-92 is overexpressed in lymphoma. Of note, the amplification of chromosomal band 13q31-q32 is often observed in hematopoietic malignancies.[4] However, we observed underexpression of the pri-miR-17-92 in gastric adenocarinoma. Remarkably, loss and no gain of the chromosomal band 13q31-q32 have been observed in gastric adenocarcinoma.[9] Therefore, it is plausible that the underexpresion in the gastric tumors may be due to the loss of the corresponding chromosomal band.

Retrieving the corresponding data from ONCOMINE (http://www.oncomine.org) shows the underexpression of MIR17HG in gastric cancer tissues and cell lines. Furthermore, applying drug sensitivity sample filter on the corresponding data from ONCOMINE shows that there is a positive correlation between the underexpression of MIR17HG and drug sensitivity. Finally, MIR17HG is among the top 5% underexpressed transcripts in diffuse gastric adenocarcinoma. Taken together, MIR17HG underexpressed in gastric cancer tissues and may be used as a biomarker in personalized medicine.

Intronic miRNAs are usually expressed in coordination with the host gene mRNA.[10] Regarding Wei et al. study in pediatric malignancies [11] and Kim et al. in gastric cancer,[5] it was expected that MIR17HG showed overexpression in gastric cancer. However, the miRNAs may show host gene-independent gene expression.[12] In contrast to Kim study, Sikand et al. demonstrated that the expression of MIR17HG and miRNAs of miR-17-92 cluster don't reveal any statistically significant correlation; thus the host gene and resident miRNAs may be expressed independently.[13] Considering the putative promoter of MIR17HG located upstream of the OncomiR-1 locus,[4] it is plausible that MIR17HG and OncomiR-1 are transcribed from different promoters.

Owing to the functional relationships with their host genes, intragenic miRNAs may have unique negative regulatory feedbacks. To do this, intronic miRNAs may directly target their host transcripts or indirectly they regulate transcription factors, which regulate the transcription of their host genes.[14] Dysregulation of negative feedback control systems may be a reason for cancer development. Data released recently by ENCODE's project from the study of 118 transcription factors showed that 34 of the 118 studied transcription factors related to the miR-17-92 cluster;[4] Therefore, it is possible that dysregulation of the miR-17-92 control feedback systems has a role in gastric cancer development.

In gastric carcinoma, patients who have larger tumors had more invasion into the gastric wall and more frequent lymph node metastasis.[15] In the same vein, we did not observe a significant association between the expression of this host gene and tumor size and between the expression of the gene and invasion and metastasis.

 Conclusion



In summary, our study demonstrated that the MIR17HG is differentially expressed in tumoral and non-tumoral tissue specimens of gastric cancer. Furthermore, there was an association between the expression levels of MIR17HG and gastric cancer grades and stages. Moreover, the expression level of MIR17HG was conversely associated with the size of tumoral specimens in early stages (stages I and II). We also observed an association between the presence of metastasis and lower expression of MIR17HG. Finally, our results suggest that MIR17HG gene expression is dysregulated in the tumors in which it may indicate a tumor suppressive function of this miRNA cluster host gene in gastric cancer.

 Acknowledgments



This study was a part of FB' MSc thesis in Shahrekord University, Shahrekord, Iran.

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