| BackgroundThe CXCL12/CXCR4 signaling axis is a coupled molecular pair. Binding of the chemokine CXCL12 to its receptor CXCR4 activates a variety of intracellular signal transduction pathways and cell migration. CXCR4 exhibites high affinity and absolute specificity for its ligand CXCL12. So CXCL12 appears to be the only ligand for CXCR4. The CXCL12/CXCR4 signaling axis regulates a variety of immune responses, cell migration and embryogenesis in many normal and pathophysiological processes. Recently, it has been established that it can serve as tissue-specific attractant molecules for tumor cells, promoting tumor cell invasion to adjacent sites, including breast, hepatocellular, and basal cell carcinomas.It has emerged that epigenetic events can lead to gene inactivation that contributes to neoplasia. The most important mechanism in mammalian cells is DNA methylation. A typical CpG island on the start site of the transcripton is revealed in the 5' region of the CXCL12 gene. Moreover, this region of CXCL12 lacks a true TATA-box. It indicated that hypermethylation of the promoter region of CXCL12 likely plays an important role in the regulation of their mRNA levels in malignant tumors. In mammals, DNA methylation patterns are established and maintained by mainly three known functional DNMT genes, namely DNMT1, 3A, and 3B. There is a considerable level of cooperation and functional overlap among them. ObjectiveTo study the expressions of the CXCL12 and CXCR4 genes in primary astrocytoma and breast carcinoma, as well as their correlation with various clinical characteristics. To identify the methylation status of CXCL12, as well as their relationship with their mRNA levels. To study the relationship of DNA methyltransferases with DNA hypermethylation. To analyze the mechanism of DNA hypermethylation involved in tumor progression in primary astrocytortia and breast carcinoma.Methodsi DNA methylation-mediated regulation of CXCL12/CXCR4 axis in primary astrocytoma1. Seventy-six of primary astrocytoma samples (20 of WHO grade II, 26 of WHO grade III, and 30 of WHO grade IV) and 10 normal brain tissues were collected during surgery. All the patients were followed up after surgery.2. The mRNA expression levels of CXCL12 and CXCR4 genes were detected by conventional RT-PCR and SYBGreen Real-time PCR assays in these primary astrocytomas and normal brain tissues. The CXCL12 and CXCR4 mRNA expression levels among various histologic grades and age subgroups were evaluated by using the Kruskal-Wallis test. The comparisons of CXCL12 and CXCR4 mRNA levels between different sex teams were calculated using the Mann-Whitney test.3. After sodium bisulfite modification of genomic DNA, methylation specific PCR (MSP) was carried out to analyze the methylation status of promoter regions of the CXCL12 gene in these primary astrocytomas and normal brain tissues. Correlations of methylation alterations of the CXCL12 gene with clinicopathologic parameters, including histologic grade, sex and age were analyzed by x2 test. Comparisons of CXCL12 mRNA levels between different methylation status teams were also calculated.4. The mRNA expression levels of DNMT1, DNMT3A and DNMT3B genes were detected by conventional RT-PCR and SYBGreen Real-time PCR assays in these primary astrocytomas and normal brain tissues. The Spearman rank correlation test was used to determine the links between continuous mRNA values of each DNMT. Relationships of the mRNA expression levels of DNMT1, DNMT3A and DNMT3B genes with the CXCL12 methylation status were also analyzed.ii DNA methylation-mediated regulation of CXCL12/CXCR4 axis in primary breast cancer1. Sixty-three of primary breast carcinoma samples and 20 normal breast tissues were obtained from patients (9 of stage I, 25 of stage IIA, 13 of stage IIB, and 16 of stage IIIA) , when treated with curative resectional surgery. All the patients were followed up after surgery.2. The protein expression levels of ER, PR, Her-2, p53, and Ki-67 in primary breast carcinomas and normal breast tissues were determined by immunohistochemical detection.3. The mRNA expression levels of CXCL12 and CXCR4 genes were detected by conventional RT-PCR and SYBGreen Real-time PCR assays in these primary breast carcinomas and normal breast tissues. Correlations of the CXCL12 and CXCR4 mRNA expression levels with clinicopathologic parameters were analyzed using the Kruskal-Wallis test or the Mann-Whitney test, including age, menopausal status, lymph node status, histologic grade, tumor type, tumor diameter and immunohistochemical staining status (ER, PR, Her-2, p53, and Ki-67).4. After sodium bisulfite modification of genomic DNA, methylation specific PCR (MSP) was carried out to analyze the methylation status of promoter regions of the CXCL12 gene in these primary breast carcinomas and normal breast tissues. Correlations of methylation alterations of the CXCL12 gene with clinicopathologic parameters were analyzed by x2 test, including age, menopausal status, lymph node status, histologic grade, tumor type, tumor diameter and immunohistochemical staining status (ER, PR, Her-2, p53, and Ki-67). Relationships of the CXCL12 mRNA expression levels with CXCL12 methylation status were also analyzed.5. The mRNA expression levels of DNMT1, DNMT3A and DNMT3B genes were detected by conventional RT-PCR and SYBGreen Real-time PCR assays in these primary breast carcinomas and normal breast tissues. The Spearman rank correlation test was used to determine the links between continuous mRNA values of each DNMT. Relationships of the mRNA expression levels of DNMT1, DNMT3A and DNMT3B genes with the CXCL12 methylation status were also analyzed.6. The protein expression levels of CXCL12 and CXCR4 in primary breast carcinoma and normal breast tissues were determined by immunohistochemical detection. Correlations of the CXCL12 and CXCR4 protein expression levels with clinicopathologic parameters were analyzed by x2 test, including age, menopausal status, lymph node status, histologic grade, tumor type, tumor diameter and immunohistochemical staining status (ER, PR, Her-2, p53, and Ki-67). Relationships of the CXCL12 protein expression levels with CXCL12 methylation status were also analyzed.Resultsi DNA methylation-mediated regulation of CXCL12/CXCR4 axis in primary astrocytoma1. Quantitative analysis of the CXCR4 transcript revealed significantly (P < 10-7) higher levels in astrocytomas (6.00±4.77, Mean±SD) than in normal brain tissues (0.53±0.24). The expression of CXCR4 increased with increasing tumor grades (P = 1.2×10-5). Other parameters such as sex and age of the patients did not show significant correlations (P > 0.05) with CXCR4 mRNA status.2. Quantitative real-time PCR showed that 16 of 76 (21.1%) astrocytoma samples exhibited no or low expression of CXCL12 mRNA. These 16 samples were all in WHO grade II. Meanwhile, 47 of 76 (61.8%) astrocytomas displayed elevated transcription of CXCL12, with 20 of WHO grade III, and 27 of WHO grade IV. The expression levels of CXCL12 mRNA in WHO grade IV astrocytomas (7.88±6.12) were significantly (P = 4.74×10-5) higher than in normal brain tissues (1.86±0.62). There were no significantly difference between WHO grade II / III and normal brain tissues (P =0.534, 0.113, respectively). Other factors such as sex and age showed no links (P > 0.05) with the CXCL12 mRNA levels.3. Hypermethylation of the CXCL12 gene was detected in 34.2% of astrocytomas. Both methylated and unmethylated products were present in 21 of CXCL12-methylated tumors. The methylation frequency of CXCL12 in astrocytomas of WHO grade II was 55.0% (11/20), in WHO grade III was 34.6% (9/26) and in WHO grade IV was 20.0% (6/30). No methylation of CXCL12 was observed in the ten normal brain tissues. A significant inverse relationship (r = - 1, P = 10-6) between CXCL12 methylation and WHO grades were found, but no statistical (P > 0.05) differences were found among different sexes and ages.In WHO grades II - III astrocytomas, the Mann-Whitney test showed a significant difference (P = 0.001) of CXCL12 mRNA levels between CXCL12 methylated and unmethylated groups. However, such statistical difference was not observed in WHO grades IV astrocytomas samples (P = 0.550).4. Quantitative real-time PCR showed that the mRNA levels of DNMT1, DNMT3A and DNMT3B correlated strongly with each other, as follows: DNMT1 with DNMT3A (r = +0.344, P = 0.002), DNMT1 with DNMT3B (+r = 0.389, P = 0.001), and DNMT3A with DNMT3B (r = +0.305, P = 0.007). Furthermore, the expression levels of DNMT1, DNMT3A and DNMT3B were significantly (P = 0.025, 0.003, 0.043, respectively) higher in the CXCL12-methylated astrocytomas than those in the CXCL12-unmethylated ones .ii DNA methylation-mediated regulation of CXCL12/CXCR4 axis in primary breast cancer1. Quantitative analysis of the CXCR4 transcript revealed significantly (P = 0.001) higher levels in breast carcinoma tissues (1.22±0.80) than in normal mammary tissues (0.65±0.59). CXCR4 overexpression in primary breast carcinomas was significantly related to lymph node metastasis (> 3 positive lymph nodes metastasis, P = 0.013), and strong Her-2 expression (P = 0.031).2. Quantitative analysis of the CXCL12 transcript revealed significantly (P = 0.031) lower levels in breast carcinoma tissues (1.41±1.18) than in normal mammary tissues (2.02±0.71). Furthermore, significantly (P = 0.017) lower expression levels of CXCL12 were found in tumors with > 3 positive lymph nodes metastasis. A significant difference (P = 0.047) in CXCL12 mRNA levels was also found between estrogen receptor negativity and positive group. However, no links (P > 0.05) were found between CXCL12 mRNA expression levels and age, menopausal status, histological grade, tumor type, macroscopic tumor size, or other standard immunohistochemical parameters (ER, PR, Her-2, p53, and Ki-67).3. The rate of breast carcinoma specimens stained positive for CXCR4 was 79.4%. All normal breast specimens resulted in negative staining for CXCR4. There was significant correlation (P = 0.035) between aberrant CXCR4 expression and lymph node metastasis (> 3 positive lymph nodes metastasis).Only 38.1% of breast cancers showed CXCL12 positive staining. All normal breast specimens exhibited positive staining for CXCL12. None of the clinicopathological factors revealed a correlation (P > 0.05) with CXCL12 protein, including age, menopausal status, lymph node metastasis status, histological grade, tumor type, macroscopic tumor size, and immunohistochemical parameters (ER, PR, Her-2, p53, and Ki-67).4. There were 52.4% of primary breast tumors hypermethylated in the CXCL12 promoter region, whereas hypermethylation of CXCL12 was not observed in any of the 20 normal breast tissues. Methylated and unmethylated products were present in 20 of 33 CXCL12-methylated tumors. CXCL12 methylation was significantly associated with > 3 positive lymph nodes metastasis (P = 0.032), and estrogen receptor negativity (P = 0.011). A significant inverse relationship (r = -0.568, P =1.2×10-6) between CXCL12 methylation status and their mRNA levels was observed. A strong inverse relationship (P = 0.001) between CXCL12 methylation and protein expression was also observed.5. Quantitative real-time PCR showed that the mRNA levels of DNMT1, DNMT3A and DNMT3B correlated strongly with each other, as follows: DNMT1 with DNMT3A (r = +0.272, P = 0.031), DNMT1 with DNMT3B (r = +0.303, P = 0.016), and DNMT3A with DNMT3B (r = +0.389, P = 0.002). The expression levels of DNMT1 (P = 0.012) and DNMT3B (P = 0.026) were significantly higher in the CXCL12-methylated primary breast carcinomas than in the CXCL12-unmethylated ones. But no significant difference (P = 0.185) of DNMT3A was observed between the CXCL12-methylated and CXCL12-unmethylated breast carcinomas. Conclusions1. DNA hypermethylation of CXCL12 is implicated in part of astrocytomas, mainly in low-grade ones, via DNA hypermethylation by DNMTs. The chemokine receptor CXCR4 is overexpressed in astrocytoma, increasing with increasing WHO grade.2. Both CXCL12 and its receptor CXCR4 are up-regulated in astrocytomas of WHO grade IV (glioblastoma). The presence of CXCL12 and CXCR4 in tumor cells, which constitutes autocrine paracrine CXCL12/CXCR4 signaling, promotes invasion ofglioblastomas.3. DNMTs-mediated DNA hypermethylation of CXCL12 plays an important role in the down-regulation of CXCL12 expression in breast carcinomas. Both up-regulation of CXCR4 and down-regulation of CXCL12 are observed in primary breast carcinomas, which are significantly related to lymph node metastasis status.4. DNA hypermethylation plays an important role in the down-regulation of the CXCL12 gene of astrocytoma and breast carcinoma. Other mechanisms might be involved in its regulation.
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