miR-106a overexpression and pRB downregulation in sporadic colorectal cancer

https://doi.org/10.1016/j.yexmp.2012.11.002Get rights and content

Abstract

Rb1 plays an important role in cell cycle progression and therefore may be involved in malignant transformation of colonic cells. The aim of our research was to define the potential role of Rb1 as a prognostic biomarker in tumorigenesis of sporadic colorectal cancer, and to examine the role of miR-106a in Rb1 regulation as it functionally binds to 3′UTR of transcribed mRNA. We examined LOH and promoter methylation status. Real-time PCR was used for Rb1 mRNA and miR-106a, and immunohistochemistry for protein expression analysis. All the results obtained from patients' samples were correlated with the clinicopathological parameters in order to determine its influence on the sporadic colorectal carcinogenesis. LOH showed no correlation with mRNA and pRb expression. 51.5% of tumor samples were scored negative for pRb staining. Despite this finding, we detected overexpression of Rb1 mRNA in tumor samples in comparison to the adjacent normal tissue (p = 0.023). mRNA overexpression was consistent with Rb1 promoter methylation analysis results, which showed no methylation in the investigated samples. Expression analysis of miR-106a in the patients samples showed its overexpression in colorectal cancer (p < 10 4). Negative pRb score was expected according to the definition of tumor suppressor genes and their proposed role in the malignant transformation of the cells. The observed discrepancy between mRNA and protein expression can be explained by a regulatory mechanism that inhibits translation, such as microRNA silencing. Our results suggest that miR-106a might have a regulatory role for Rb1 in sporadic colorectal cancer.

Highlights

► Rb1 LOH showed no correlation with mRNA and protein expression. ► No Rb1 promoter methylation was detected in colorectal tumor and normal tissue. ► Rb1 mRNA was higher in tumors in comparison to normal colonic tissue. ► miR-106a was significantly higher in tumor in comparison to normal colonic tissue. ► Loss of pRb was found in majority of tumors.

Introduction

Colorectal cancer is one of the most frequent malignant diseases in the world. The incidence is expected to become even higher due to increasing porportion of elderly people in the world population and to increasing exposure to cancerous substances. (Jemal et al., 2011). Colorectal cancer arises from the accumulation of mutations during progression from normal colon epithelium to adenoma and carcinoma (Fearon and Vogelstein, 1990). This process implies changes in different types of genes and their expression pattern. Hanahan and Weinberg (2000) postulated that the aquired biological capabilities are common to all cancer types, but the timeline of mutations that occur in cancer related genes varies. One of the gatekeeper genes that codes for a transcription factor involved in the regulation of the cell cycle is Rb1 (retinoblastoma gene) (DiCiommo et al., 2000). It is a gene located at chromosome 13q14.2 that spreads over 180 kb of genomic DNA and contains 27 exons (Lee et al., 1987). It encodes a 110 kDa protein product, pRb (Lee et al., 1987), which is involved in maintaining the chromatine structure, and acts as a negative regulator of the cell cycle progression (Indovina et al., 2013). It is a member of the Rb protein family, together with p107 and p130. All three proteins are involved in the same cellular pathway but have distinct functions (Dannenberg and te Riele, 2006, Indovina et al., 2013). It is considered that the high majority, if not all human tumors, have defective Rb pathway (DiCiommo et al., 2000, Manning and Dyson, 2012). Rb1 was first found to be altered in retinoblastoma and it was a prototype model for studying a potential tumor supressor gene (Friend et al., 1986). Later extensive research revealed its association with other malignancies like small cell lung cancer, melanoma, prostate, breast, bladder and other types of cancer (Burkhart and Sage, 2008). Changes in Rb1 were also implicated in all stages of tumor development (Burkhart and Sage, 2008). In the gastrointestinal tract, loss of Rb1 function was associated with the progression of tumorigenesis (Kucherlapati et al., 2008). Vast amount of data are available from the published literature but its precise role in this type of cancer has been inconclusive due to newly appreciated complexity of its function and interactions (Viatour and Sage, 2011).

The aim of this study was to define the potential role of Rb1 as a prognostic biomarker in tumorigenesis of sporadic colorectal cancer. Loss of heterozygosity, methylation status and expression of Rb1 mRNA and protein were examined. The expression of miR-106a was also interesting for our research because it was reported to be specific for Rb1 mRNA, and that it functionally binds to its 3′UTR (Volinia et al., 2006). Therefore, we analyzed miR-106a expression level, and evaluated its role by comparring it to pRb expression. The results obtained from all patients' samples were correlated with the clinicopathological parameters in order to evaluate its role in the progression of sporadic colorectal cancer.

Section snippets

Patients and tissue specimens

Our research was carried out on tumor samples and samples of corresponding normal tissue obtained from Croatian Human Tumor Bank (Spaventi et al., 1994). The tumor specimens were collected through a routine surgery. Fresh samples of resected colon carcinoma and normal colon mucosa adjacent to resected carcinoma (more than 15 cm from the tumor) were snap-frozen in liquid nitrogen and stored in Human Tumor Bank at − 80 °C until further use. Tissue samples, adjacent to the segments of tissue that

LOH at the Rb1 gene locus

To analyze allelic imbalance at the Rb1 gene, we investigated LOH at two polymophic loci, D13S153 and Rb1.20. Heterozygosity was detected in 261 of 300 patients (87.0%) and LOH was found in 56 informative tumors (21.5%).

We found no statistically significant correlation of LOH occurrence at investigated loci with clinicopathological parameters (Table 1).

Methylation analysis

We analyzed 39 pairs of tumor tissue and corresponding normal tissue obtained from patients and no methylation was detected in the Rb1 promoter

Discussion

In this study, we investigated the changes in the Rb1 gene and its mRNA and protein expression patterns in sporadic colorectal carcinogenesis. Rb1 is one of the key regulators of the cell cycle, it interacts with numerous binding partners in the cell, and influences the transcription of several downstream targets (DiCiommo et al., 2000, Viatour and Sage, 2011). On the basis of the Knudson two-hit theory, it is expected to be lost during the early stages of tumorigenesis (Friend et al., 1986).

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Role of the funding source

This research was funded by Croatian Ministry of Science, Education and Sports (098-0982464-2508).

The study sponsor had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Acknowledgments

We thank Gordana Jurinic for technical assistance.

References (57)

  • J. Zou et al.

    Protein kinase CK2α is overexpressed in colorectal cancer and modulates cell proliferation and invasion via regulating EMT-related genes

    Journal of Translational Medicine

    (2011)
  • A.A. Ali et al.

    RB1 protein in normal and malignant human colorectal tissue and colon cancer cell lines

    The FASEB Journal

    (1993)
  • N.J. Arhel et al.

    The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines

    International Journal of Cancer

    (2003)
  • D.L. Burkhart et al.

    Cellular mechanisms of tumor suppression by the retinoblastoma gene

    Nature Reviews. Cancer

    (2008)
  • W.G. Cance et al.

    Altered expression of the retinoblastoma gene product in human sarcomas

    The New England Journal of Medicine

    (1990)
  • E. Cassinotti et al.

    DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps

    International Journal of Cancer

    (2012)
  • F. Cetani et al.

    A reappraisal of the Rb1 gene abnormalities in the diagnosis of parathyroid cancer

    Clinical Endocrinology

    (2004)
  • N.K. Clemo et al.

    The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumor cell survival

    Biochemical Society Transactions

    (2005)
  • J.H. Dannenberg et al.

    The retinoblastoma gene family in cell cycle regulation and suppression of tumorigenesis

    Results and Problems in Cell Differentiation

    (2006)
  • G.T. Deans et al.

    Prognostic factors in colorectal cancer

    The British Journal of Surgery

    (1992)
  • C.A. Eads et al.

    Epigenetic patterns in the progression of esophageal adenocarcinoma

    Cancer Research

    (2001)
  • S.H. Friend et al.

    A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma

    Nature

    (1986)
  • S. Ghosh et al.

    LIMD1 is more frequently altered than RB1 in head and neck squamous cell carcinoma: clinical and prognostic implications

    Molecular Cancer

    (2010)
  • R. Gope et al.

    Abundance and state of phosphorylation of the retinoblastoma susceptibility gene product in human colon cancer

    Molecular and Cellular Biochemistry

    (1992)
  • R. Gope et al.

    Increased expression of the retinoblastoma gene in human colorectal carcinomas relative to normal colonic mucosa

    Journal of the National Cancer Institute

    (1990)
  • V. Greger et al.

    Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma

    Human Genetics

    (1994)
  • M. Guy et al.

    Transcriptional down-regulation of the retinoblastoma protein is associated with differentiation and apoptosis in human colorectal epithelial cells

    British Journal of Cancer

    (2001)
  • P. Indovina et al.

    Emerging roles of RB family: new defense mechanisms against tumor progression

    Journal of Cellular Physiology

    (2013)
  • Cited by (35)

    • Experimental study on the inhibition effect of miR-106a inhibitor on tumor growth of ovarian cancer xenografts mice

      2016, Asian Pacific Journal of Tropical Medicine
      Citation Excerpt :

      In the occurrence and development of malignant tumor, some fragile area in chromosomes can change, and more than 50% of the miRNA genes are in the fragile area of chromosomes and with abnormal expression, resulting in changes in the expression of a variety of downstream target genes and causing corresponding changes in cell biological behavior [12,13]. miR-106a is a class of miRNA with proto-oncogene properties found in recent years, and it shows the trend of high expression in lung cancer [7], gastric cancer [8], colon cancer [9] and many other malignant tumor tissues. Experimental cell research of domestic LI Min and others [14] confirms that miR-106 is highly expressed in metastatic ovarian cancer cells SKOV-3 and miR-106a mimic can promote ovarian cancer cell migration and invasion in vitro.

    • MicroRNA-induced drug resistance in gastric cancer

      2015, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Regarding the importance of miRNAs as a new type of the gene expression regulatory mechanism and the broad range of miRNAs’ targets, it is accepted that other miRNA molecules could also contribute to the regulation of MDR in gastric cancer. MiR-106a is one of the important pro-oncogenic members of the miR-17 family[26] and is overexpressed in many cancers [27,28], including gastric cancer [29]. A recent report investigated the function of the miR-106a in the establishment of MDR in gastric cancer [30].

    • Upregulated miR-106a plays an oncogenic role in pancreatic cancer

      2014, FEBS Letters
      Citation Excerpt :

      SW-1990, a highly invasive pancreatic cancer cell line, exhibited the highest level of miR-106a of the four cell lines, which could indicate that overexpression of miR-106a may be involved in pancreatic cancer invasion. In studies conducted by Wang and Ivkovic et al. [12,13], miR-106a was found to be an oncogene in gastric and colorectal cancers. MiR-106a plays two roles in the development of tumors; Wang et al. found that miR-106a could inhibit the extrinsic apoptotic pathway by targeting the FAS cell surface receptor to further promote cell proliferation and repress apoptosis.

    • Insight of microRNA role in Colorectal Cancer

      2020, Progress in Microbes and Molecular Biology
    View all citing articles on Scopus
    View full text