TRIM31 serves as a potential tumor suppressor gene in Colorectal Cancer
The present study aims to investigate the biological roles of TRIM31 in human colorectal cancer. TRIM31 is a new member of RBCC proteinscomposed of RING finger, B-box and coiled-coil domains, involved in various cellular processes including tumour development and antiviral response.As overexpression of TRIM31 in gastricadenocarcinoma has been proved, we supposed TRIM31 possess similar properties in colorectal cancer. In our study,TRIM31 overexpression suppresses colony formation of SW480 cells while knockdown of its expression with short interfering RNAs (siRNAs). In addition, expression of cell cycle regulator cyclinD1 and cyclin E were decreased after TRIM31 transfection. Inconclusion, TRIM31 is a characteristic RBCC protein with the ability to regulate cellproliferation negatively and may be a potential oncogene in colorectal cancer.
Keywords colorectal cancer, RBCC, TRIM31, oncogene,
Colorectal cancer (CRC) has been taken as a global health challenge with high incidence rate and mortality. Characterized by discrete mutations and chromosomal alterations, patients with similar pathological features often showed different outcomes. More deep understanding of the molecular mechanisms could help providing new prognostic indicators of CRC.
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TRIM31 is a member of a subfamily of RING finger domain-containing proteins, which called the tripartite motif family (TRIM). TRIM proteins take part in many biological processes, as well as several pathological conditions such as tumorigenesis, disorders of immunological and developmental. As originally identified as a gene induced by growth-suppressive retinoid, TRIM31 has been reported in some diseaseså‚è€ƒæ-‡çŒ®.
However, The biological roles of TRIM31 in colorectal cancer have not been explored. In order to address these questions, we analyzed the potential molecular mechanism for and biological significance of TRIM31 were investigated in vitro. Materials and methods
Cell culture and transfection
Sw480 and 293T cell lines were obtained from shanghai cell center. The cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA,USA) supplemented with 10% fetal bovine serum and seeded in 6-well plate 24h before the transfection with pCMV6-TRIM31plasmid. The plasmid was was purchased from Origene, and the transfection were carried out using the attractene reagent (qiagen, germany) according to the manufacturer’s protocol.
Cell proliferation test
Cell proliferation test was performed by using Cell CountingKit-8 (Dojindo, Gaithersburg, MD) according to themanufacturer’s protocol. Briefly, 24 hours after transient transfectionwith TRIM31 plasmid, cells were seeded in 96-well plates at a concentrationof 5×103cells each well. Then, 10μl CCK-8 solution were added to each well and the cell density was measured at 450 nm using a microplate reader.
Colony formation assay
Cells were seeded in 6-well plates at the concentration of 1 × 103per well. After the transfection with TRIM31 plasmid, cells were cultured for one week to form the colony. After that, the plate were washed with PBS and stained with violet. Colonies contained with more than 50 cells were counted.
Western blot analysis
48h after transfection, cells were lysed and proteins were extracted and quantified. 60mg protein were separated by SDS-PAGE. After being transferred to the polyvinylidene fluoride (PVDF) membranes, proteins on the membrane combined with different antibodies. After incubation withperoxidase-coupled anti-mouse/rabbit IgG (Santa CruzBiotechnology) at 37 °C for 2 h, bound proteins werevisualized using ECL and detected using BioImagingSystems (UVP Inc., Upland, CA, USA). The relativeprotein levels were calculated based on β-actin as theloading control.
SPSS version 16.0 for Windows was used for all analyses. Student’s test was used to compare other data. A P value was based on the two-sided statistical analysis. P < 0.05 was considered to indicate statistical significance.
The upregulated level of TRIM31 in colorectal cancer cells
48h after transfection with TRIM31 plasmid,
TRIM31inhibits proliferation of colorectal cancer cells
By transfecting TRIM31 plasmid, we upregulated the protein level of TRIM31 in HT29 cells, which was shown in fig1. CCK-8 assay showed that the overexpression of TRIM31 led to a significant reduction of the proliferation rate.
In order to test the effect of TRIM31 on tumorgenesis, we carried out colony formation assay. After transfection of a plasmid, cells were cultured in the medium, allowing for the colony formation 1 week later. Colony formation of HT29 was alleviated upon transfection with the wild-type TRIM31 gene (figure ).
TRIM31 inhibits expression of cyclin D1 and cyclin E
We searched for human colorectal cancer cell lines with a high TRIM31 expression.
We employed pancreatic adenocarcinoma cells for molecular characterization of endogenous TRIM31.
TRIM genes are frequently expressed as multiple alternative splicing forms.
by RT-PCR analysis but no cell line showed a high expression level.
TRIM31 is characterized by the presence of the RING finger, B box and coiled-coil domains, which belongs to the RBCC family. RBCC proteins are either positively or negatively linked to cell growth. TRIM19 repress cell growth while TRIM32 promote cancer cell proliferationæ’å…¥æ-‡çŒ®. The overexpression of TRIM 31 was detected in chronic gastritis and the cell apoptosis and proliferation was affected by TRIM31. and connected to various pathological conditions.
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RBCC proteins have been reported to be related with cell growth. For example, it is reported that TRIM25/Efp and TRIM32 promote tumorigenesis [Urano et al., 2002; Horn et al., 2004]. On the other hand, TRIM19/PML and TRIM35/MAIR repress tumor cell proliferation [Mu et al., 1994; Kimura et al., 2003]. In the present research TRIM31 expression was increased in colorectal cancer cells (Fig. 1A) where both apoptosis and proliferation are dysregulated [Jones et al., 1997]. These facts raise a question whether TRIM31 would stimulate or arrest cell growth. Overexpression of TRIM31 inhibited colony formation of 293T cells substantially (Fig. 7A), in accordance with induction of TRIM31 by growth-suppressive retinoids [Dokmanovic et al., 2002]. Knockdown of TRIM31 in AsPC-1 cells by specific siRNAs failed to regress cell growth but exhibited a consistent tendency to cause a slight increase in the cell number (Fig. 7B). Considering the moderate inhibition of colony formation by overexpression of TRIM31, the weak stimulating effect of its knockdown on cell growth was not surprising because, in general, the degree of cell growth enhancement by the knockdown of growth suppressors is not so remarkable even though their overexpression exerts a drastic anti-proliferative effect [Yang et al., 2004; Xiao et al., 2005]. Presumably multiple inhibitory proteins form the balance between cell growth arrest and proliferation. In any case, TRIM31 can be recognized as a growth suppressor. As expression of TRIM31 had no influence on colony formation of HeLa cells (data not shown), it is possible that the effect of TRIM31 is cell context-dependent and variable for distinct precursor cells during tumor development. In fact, the effects of oncogenes and tumor suppressors depend on the types of cell [Weinstein, 2000]. Alternatively, the restriction of TRIM31 expression in digestive tissues (Fig. 1B) where rapid turnover of epithelial cells occurs [Hall et al., 1994] may implicate the maintenance of the balance between proliferation and apoptosis of cells as its biological function. Naturally, the normal function of TRIM31 in these digestive tissues would also be the formation of the homeostatic feedback loop regulating cell growth. It is also known that p16/INK4, a tumor or growth suppressor, is overexpressed in cervical and prostate cancers. These cancers may have the distinct mechanism inactivating the senescence program elicited by the tumor suppressor [Bulten et al., 2006; Feng et al., 2007]. Likewise, TRIM31 could be involved in gastric carcinogenesis in a similar manner.
In summary, we demonstrated that TRIM31 is upregulated in gastric adenocarcinoma. TRIM31 has distinguishing properties of the RBCC protein family, including ubiquitin ligase activity. TRIM31 is capable of suppressing cell growth although its precise role remains to be known. TRIM31 may be a potential biomarker of stomach cancer since it appears to be overexpressed from the pre-cancerous stage.
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