Background
The
Polycomb group (
PcG) genes were first identified in Drosophila as a class of regulators responsible for maintaining homeotic gene expression throughout cell division [
1],
PcG genes are conserved from Drosophila to mammals, and the expression levels of mammalian
PcG genes differ between different tissues and cell types [
2],
PcG genes act as epigenetic silencers during embryo morphogenesis with a central role in the nervous system, heart, and skeleton development [
3‐
7].In addition,
PcG members have been involved in the regulation of such adult processes as the cell cycle, X-inactivation, and hematopoiesis [
8‐
14].
PcG expression is deregulated in some types of human cancer [
15].Moreover, several PcG genes may regulate the self-renewal of specific stem cell types, suggesting a link between the maintenance of cell homeostasis and carcinogenesis [
16,
17].
Bmi-1 is one of the key PcG proteins. It was initially identified as an oncogene that cooperated with c-Myc in the generation of mouse pre-B-cell lymphomas. It is also considered the first functional mammalian
PcG protooncogene to be recognized, and it has been implicated in axial patterning, hematopoiesis, cell cycle regulation, and senescence [
18‐
21]. Human
Bmi-1 gene is located at the short arm of chromosome 10p13 [
22], The region is involved in chromosomal translocations in leukemia and is amplified in non-Hodgkin's lymphoma as well as in solid tumors [
23]. Bmi-1 induces S-phase entry by inhibiting Rb function via repression of the INK4a/ARF locus [
24‐
26]. Moreover, overexpression of
Bmi-1 in mammary epithelial cells may activate telomerase and lead to immortalization [
27]. Overexpression of
Bmi-1 has been found in several human malignancies including breast cancer, colorectal cancer, nasopharyngeal carcinoma, melanoma, gastric cancer, and bladder cancer [
28‐
33]. Overexpression of
Bmi-1 often correlates with poorer prognosis and treatment failure [
30,
32‐
34].
Bmi-1 also plays an important role in self-renewal of hematopoietic stem cells, neural stem cells and mammary stem cells [
35‐
37].
In addition to
Bmi-1, mammalian cells also express a Bmi-1-related PcG protein Mel-18. The
Mel-18 gene product is structurally highly similar to Bmi-1 protein. Interestingly, we have found that
Bmi-1 is negatively regulated by
Mel-18 and expression of
Mel-18 negatively correlates with
Bmi-1 in breast tumors, and
Mel-18 overexpression in breast cancer cell line MCF7 results in downregulation of
Bmi-1 and reduction of transformed phenotype [
38]. Negative correlation between
Bmi-1 and
Mel-18 expression was also recently reported in hematopoietic stem cells [
39]. Lee et al. also recently reported that overexpression of
Mel-18 inhibits growth of breast cancer cells [
40]. These data suggested that
Mel-18 acts as a potential tumor suppressor. However, the function of
Mel-18 is still debatable. In few other studies, it was found that similar to
Bmi-1, Mel-18 can act as an oncogene [
41,
42]. So, the role of
Mel-18 in cancers other than breast cancers and different pathological conditions is still not clear and need to be clarified.
Gastric cancer is one of the most common malignancies throughout the world. It has been reported that
Bmi-1 is overexpressed in gastric cancer and is an independent prognosis factor [
32]. We have also studied the expression of Mel-18 and Bmi-1 in gastric tumors by immunohistochemistry (IHC). We found that gastric tumor tissues expressed significantly higher Bmi-1 and lower Mel-18, and the expression of Mel-18 negatively correlated with Bmi-1; there was a significant positive correlation between Bmi-1 expression with lymph node metastasis, or clinical stage, but there was no obvious correlation between Mel-18 expression and clinicopathological factors; downregulation of
Bmi-1 by
Mel-18 overexpression or knockdown of Bmi-1 expression was accompanied by decreased transformed phenotype and migration ability in gastric cancer cell lines in
in vitro study[
33]. So, the results of Bmi-1 expression correlated with lymph node metastasis or clinical stage in
in vivo study was accordance with the results in
in vitro study, while the results of no correlation was found between Mel-18 expression and clinicopathological factors in
in vivo study was not accordance with the results in
in vitro study, we suspected that one of the reason may due to the reliability of IHC method which was used to detect the expression of Bmi-1 and Mel-18 in tumor tissues in most paper of literature including our previous study. This method lacks standard procedure and evaluation criterion and its' reliability depends on the specific of antibody. The results of quantitative Real time RT-PCR (QRT-PCR) with specific primer is more reliable than that of IHC to measure the gene expression level especially for Mel-18, which lacks specific mouse monoclonal antibody till now. Here, we examine the expression of
Mel-18 and
Bmi-1 at mRNA level by using QRT-PCR method in a series of gastric cancer, and evaluate the correlation between
Mel-18 and
Bmi-1 expression levels. Furthermore, a correlation study between expression levels of both the analyzed genes and several clinical pathologic variables of the tumors was designed. In this study, we characterized the expression profile of
Mel-18 and
Bmi-1, and their clinical significance in gastric cancer.
Discussion
Mammalian PcG protein complexes are generally classified into two distinct types: Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Mel-18 protein product is a constituent of mammalian PRC1 together with M33, Bmi-1 or rae28/Mph-1, and Scmh1 [
1,
44‐
47]. In human tumors, some reports have showed alterations in PcG expression, in such human hematologic malignancies as nodal B-cell lymphomas [
48,
49], mantle cell lymphomas [
23,
50], and Hodgkin's lymphomas [
13,
51,
52].It has been reported that solid tumors, such as lung cancers [
53],medulloblastomas [
3], liver [
54], penis [
55], breast [
28,
56],colon [
57], and prostate carcinomas [
58], also display disturbed
PcG gene expression.
Bmi-1 is one of the most important PcG proteins that is known to regulate proliferation and senescence in mammalian cells, and plays an important role in self-renewal of stem cells. It can not only immortalize human mammary epithelial cells (HMECs) [
27], but also can cooperate with H-Ras to transform HMECs and transform keratinocytes [
59,
60]. Abnormal expression of Bmi-1 has been found in several human cancers and its overexpression is often correlated with poor prognosis in many types of malignances [
28‐
34]. Overexpression of Bmi-1 in gastric cancer has been previously reported[
32,
61]. It was found that Bmi-1 overexpression was highly correlated with tumor size, clinical stage, lymph node metastasis and T classification [
32]. In another study, Bmi-1 expression was closely related with the Lauren's and Borrmann's classification and clinical stage in gastric cancer [
61]. We also found that gastric tumor tissues expressed significantly higher Bmi-1, and Bmi-1 overexpression correlated with lymph node metastasis, or clinical stage, which was accordance with the results in
in vitro study that knockdown of Bmi-1 expression was accompanied by decreased transformed phenotype and migration ability in gastric cancer cell lines [
33]. In these studies Bmi-1 was detected at protein level by IHC method. Here we detected
Bmi-1 at mRNA level by QRT-PCR method and found that
Bmi-1 is overexpressed in gastric tumors and
Bmi-1 overexpression correlates with tumor size, depth of invasion (T classification), or lymph node metastasis (N classification), which confirms previous observation of Bmi-1 at protein level. It suggests that
Bmi-1 may play a crucial role and act as an oncogene in gastric cancer, and associated with the carcinogenesis, progression, and metastasis of gastric cancer.
Mel-18 was originally cloned from B16 mouse melanoma cells [
62].
Mel-18 may bind to the nucleotide sequence 5'-GACTNGACT-3', which is present in the promoter region of certain genes. One of the unique target genes of
Mel-18 is c-Myc transcriptionally repressed by
Mel-18. In mature resting B cells,
Mel-18 negatively regulates B cell receptor-induced proliferation through the down-regulation of the c-Myc/cdc25 cascade [
63,
64]. Our previous studies suggest that
Mel-18 is a physiologic regulator of
Bmi-1 expression and transcriptionally down-regulates
Bmi-1 expression during senescence in human fibroblasts and acts as a tumor suppressor in breast cancer [
38,
43]. Our previous data also showed an inverse correlation between Bmi-1 and Mel-18 expression at protein level in breast cancer and gastric cancer [
33,
38]. However, there was no correlation between Mel-18 expression at protein level and clinicopathological factors in
in vivo study, which was not accordance with the results in
in vitro study that Mel-18 overexpression was accompanied by decreased transformed phenotype and migration ability in gastric cancer cell lines[
33]. One of the reasons may due to the reliability of IHC method depends on the specific of antibody. Mel-18 antibody is rabbit polyclonal and it's specific is not so good as Bmi-1 antibody which is mouse monoclonal. So we suspect the results of Mel-18 expression in tumor tissues at protein level detected by IHC may be not too reliable. To clarify this problem and further explore the role of Mel-18 in gastric cancer, we detected it's expression at mRNA level by QRT-PCR in the present study. We found that most gastric tumor tissues (64.79%) expressed decreased mRNA levels of
Mel-18, and there was a strong negative correlation between
Bmi-1 and
Mel-18 expression at mRNA level. The results confirm the expression of Mel-18 and its' relationship with Bmi-1 at protein level in our previous study. More important, we also found that decreased expression of
Mel-18 correlated with lymph node metastasis or the clinical stage, which was accordance with the results in
in vitro study that
Mel-18 overexpression was accompanied by decreased transformed phenotype and migration ability in gastric cancer cell lines in our previous study[
33]. It provides more convincing
in vivo data to suggest that
Mel-18 may play a crucial opposite role to
Bmi-1 and act as a tumor suppressor in gastric cancer, and associated with the carcinogenesis, progression, and metastasis of gastric cancer.
In the current study we demonstrated that neoplastic cells in gastric cancer can't normally express Bmi-1 and Mel-18. We propose that abnormal PcG expression results in an altered composition of the PRC1 in gastric cancer cells, which probably affects expression of target genes involved in regulation of senescence and/or the cell cycle. Our observations add to the increasing evidence that PcG genes are very important contributors to the carcinogenesis and progression of human tumors. We additonally found that both Mel-18 and Bmi-1 correlated with lymph node metastasis. The mechanisms that they regulate cancer cells metastasis need to be further studied.
This research is the first time to study the correlation between Mel-18 or Bmi-1 expression at mRNA level and clinicopathological characteristics of gastric cancer by quantitative method. The expression of Bmi-1 and Mel-18 was correlated with gastric cancer progress, advanced gastric cancer more likely expressed higher Bmi-1 and lower Mel-18. Its clinical value deserves further study in a larger patient population.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LYW performed the experiment and prepared the manuscript; LJ supervised the experiment; GWJ designed the experiment and supervised the project. All authors have read and approved the final manuscript.