Med Oncol (2014) 31:768 DOI 10.1007/s12032-013-0768-4
ORIGINAL PAPER
Reduced expression of Slit2 in renal cell carcinoma Wei-Jie Ma • Yu Zhou • Dan Lu • Dong Dong Xiao-Jun Tian • Jie-xi Wen • Jun Zhang
•
Received: 25 October 2013 / Accepted: 5 November 2013 / Published online: 15 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is downregulated in RCC partially due to promoter methylation. Keywords Slit2 Renal cell carcinoma Methylation Tumor suppressor
W.-J. Ma Y. Zhou D. Lu D. Dong J. Wen J. Zhang (&) Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology (Ministry of Health), Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191, China e-mail: [email protected] X.-J. Tian Department of Urology, Peking University Third Hospital, Beijing, China
Introduction The development of renal cell carcinoma (RCC) was suggested to be tightly associated with the accumulation of multiple genetic and epigenetic inactivation of tumor suppressor genes (TSGs) [1, 2]. The representative and most common genetic alteration in sporadic clear cell RCCs is inactivation of the Von Hippel-Lindau (VHL) by chromosome 3p deletion [3]. Promoter methylation is an important epigenetic mechanism for inactivation of TSGs in many cancers [4]. Recently, many TSGs have been identified as potential TSGs in RCC and inactivated by promoter methylation. The identified aberrantly methylated genes include VHL, RASSF1A, p16, Timp-3, E-cadherin, b-catenin, SFRP1, SFRP2, SFRP5, DAL-1, COLIA1, KRT19, UCHL1, UNC5C and UNC5D [5–12]. Slit2 is the human orthologue of the Drosophila Slit2 [13]. It is located at chromosome 4p15.2. Slit2 was initially identified as a critical guidance cue for axons in the nervous systems [14]. In recent years, Slit2 is found to be widely expressed in mammalian tissues and functions out of the nervous system [15]. Intriguingly, Slit2 has been reported to be a potential TSGs in diverse tumors [16]. Promoter methylation is an important mechanism for its down-regulation in these cancers [17]. Slit2 promoter region hypermethylation has been detected in various cancers such as breast cancer, lung cancer, colorectal cancer, glioma, cervical cancer, colorectal cancer and leukemia [17–22]. However, the expression and regulatory mechanism for Slit2 in renal cell carcinoma were not systematically studied. Here, we analyzed the mRNA and protein expression level of Slit2 in renal cell carcinoma. We also provide the evidence that may explain the downregulation in RCC. These data suggested that Slit2 may also be a potential tumor suppressor gene in RCC.
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Materials and methods Cell lines, tumor specimens and 5-aza-dC treatment A series of cancer cell lines were used for this study, including 5 RCCs (786-O, A498, ACHN, Caki-1 and OS-RC-2), 3 lung cancer (NCI-H460, NCI-H1299 and A549), 2 colon cancer (SW480 and SW620), 1 cervical carcinoma (Hela), 1 leukemia (Jurkat). Human immortalized embryonic kidney cell line HEK293T was also used. Renal carcinoma and corresponding non-cancerous tissues (n = 38) were obtained from the Peking University Third Hospital (Beijing, China) with patients’ consents and institutional ethical approval. All of the specimens were pathologically confirmed. For demethylation, cell lines were treated with 10 mmol/L of 5-aza-20 -deoxycytidine (5-Aza-dC, Sigma-Aldrich) for 3 days with exchange of reagents and medium every 24 h.
Med Oncol (2014) 31:768
polymerase (Takara) for 38 cycles. The MSP primer sequences are Slit2-MF: 50 -aggacgaattattagtgggagatc-30 and Slit2-MR: 50 -actactccaaacaaaaccaaacg-30 ; Slit2-UF:50 -aaggatgaattattagtgggagatt-30 and Slit2-UR:50 -actactccaaa0 caaaaccaaacaac-3 . The BGS primer sequences are Slit2-L: 50 -aggggttagaaggagaaggattatt-30 and Slit2-R: 50 -aacaaaaataaaaaaaacaaaaacc-30 . Statistical analysis Statistical analyses were performed with SPSS, version 13.0 (SPSS, Inc.). Differences between two independent groups were analyzed by the Student’s t test. v2 test was used to calculate differences in the patient’s age, gender, tumor stage, histological grade and clinical classification. Methylation and immunohistochemistry status was evaluated between paired tumor and adjacent non-malignant tissue samples. P \ 0.05 was considered significant.
Reverse transcriptase PCR A human normal tissue cDNA panel was purchased from Clontech. Total RNA was isolated from cell lines and frozen stored tissue specimens by TRIZOL reagent (Invitrogen, Carlsbad, CA). RNA was reverse transcribed using the Reverse Transcription System (Promega, Madison, WI) according to the manufacture’s instruction. PCR amplifications of Slit2 were performed using the following primers: Slit2-F: 50 -tcagctgtttcctgagttgc-30 and Slit2-R: 50 tggttgaaacttgccacaga-30 . PCR products were resolved on 1 % agarose gels. Immunohistochemistry and scoring The tissue microarray (Shanghai Outdo Biotech Co. Ltd.) of 75 paired tumor and non-cancerous tissue was incubated with anti-Slit2 antibody (Santa Cruz) at 1:80 dilution in PBS overnight at 4 °C. Staining was evaluated by percent of tumor cell positivity and staining intensity. Slides were cored by 3 reviewers, and discrepancies were resolved by a urological pathologist. Methylation-specific PCR and bisulfate genomic sequencing Genomic DNA was extracted from cell lines and tissue samples by a commercial DNA extraction kit (Promega). Methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS) analysis were conducted as described. Amplified BGS PCR products were cloned into the pGEM-T Easy vector (Promega), and 8 random clones from each sample were sequenced. The bisulfite-treated DNA was amplified with the methylation-specific primer set and sequencing primer set by PCR using hot-start Taq
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Results Expression patterns of Slit2 in normal tissue and RCC cell lines In order to assess whether Slit2 is involved in RCC development, we first examined the expression profile of Slit2 in normal adult tissues and found that Slit2 was broadly expressed. It is expressed in brain, lung, kidney, placenta, spleen, thymus, prostate, testis, heart, ovary, etc. (Fig. 1a). Then, we also detected the expression level of Slit2 mRNA in multiple cancer cell lines. In renal cell carcinoma cell lines, the expression of Slit2 mRNA was weakly expressed in 4/5 RCC cell lines (786-O, A498, ACHN and Os-RC-2) (Fig. 1b). Notably, Slit2 expression was maintained in immortalized epithelial cell lines such as HEK293. Taken together, these data suggest that Slit2 was broadly expressed in normal tissues and down-regulated in RCC cell lines and it may be involved in RCC development. Down-regulation of Slit2 in primary renal cell carcinomas We further investigated whether Slit2 was down-regulated in renal carcinomas by RT-PCR. As shown in Fig. 2a, compared to adjacent non-malignant tissues, Slit2 mRNA was dramatically down-regulated in inactivation in 20 of 38 (52.6 %) RCC cases. We further examined Slit2 protein expression in a total of 75 paired RCC tissue sections by immunohistochemistry. Strong Slit2 staining was shown in non-cancerous renal tissue, mainly in the renal epithelium. On the contrary, Slit2 protein was weakly detected in RCC tissues (Fig. 2b). Histochemical studies showed that Slit2
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Fig. 1 Slit2 expression in normal tissues and cell lines. a RT-PCR analysis of Slit2 mRNA expression in 14 normal tissues. b RT-PCR analysis of Slit2 mRNA expression in multiple cell lines
A Slit2 GAPDH PBMC
Ovary
Colon
Testis
Prostate
Thymus
Lung
Spleen
Pancreas
Kidney
Kidney
Muscle
Liver
Lung
Placenta
Brain
Heart
B
Others
Slit2 GAPDH
A
Ca.
Adj.
Ca.
Adj.
Ca.
Adj.
Ca.
Adj.
Slit2 GAPDH
B
expression of Slit2 in these cell lines (Fig. 3a). To confirm this, we found that the methylation status of Slit2 promoter was changed after Aza treatment in A498 and 786-O cell lines (Fig. 3b). Further BGS also supported this. Therefore, Slit2 expression was significantly increased in renal cancer cell lines after demethylation agent treatment along with an increase in unmethylated alleles and a decrease in methylated alleles (Fig. 3c). Frequent Slit2 promoter methylation in primary RCC
Ca.
×200
Adj.
×200
Fig. 2 Down-regulation of Slit2 in primary renal cell carcinoma. a RT-PCR analysis of Slit2 mRNA in renal cell carcinoma and paired adjacent non-cancerous tissues. b Immunohistochemistry analysis of Slit2 protein expression in renal cell carcinoma (left) and paired adjacent non-cancerous tissues (right)
protein was down-regulated in 56 (74.67 %) RCC tissues compared to paired adjacent non-cancer tissues (Table 1). Statistical analysis showed that Slit2 down-regulation did not correlate with age, gender and histological grading. Interestingly, Slit2 down-regulation correlated with tumor stage (P B 0.001) and clinical classification (P \ 0.05). It indicated that down-regulation of Slit2 mainly occurred in the early stages of RCC. Promoter methylation of Slit2 in renal cancer cell lines As we have mentioned above, the expression of Slit2 was down-regulated in several cancer cell lines. The region spanning the putative promoter of Slit2 and exon 1 and the beginning of intron 1 is a typical CpG island. We next analyzed whether demethylation agents can upregulate the expression of Slit2 in cancer cell lines. We treated several cancer cell lines which had lower Slit 2 expression including A498, 786-O, Os-RC-2, A549 and Hela with demethylation agent Aza and found that Aza treatment upregulated the
We also analyzed the Slit2 promoter methylation status in 38 primary RCC samples and paired adjacent non-malignant renal tissues. Of 38 tumor samples, 8 (21.1 %) showed methylation in the Slit2 promoter (Fig. 4a), as compared with no methylation in the corresponding normal tissues. Notably, eight tumor tissues with Slit2 promoter methylation were among the 20 tumor cases with down-regulated Slit2 expression. This demonstrated that methylation of Slit2 promoter can lead to the down-regulation of Slit2 in RCC. The methylation-specific PCR results were confirmed by direct sequencing of CpG islands in the Slit2 promoter region (Fig. 4b). We also evaluated the relationship of Slit2 methylation with clinicopathological features in patients with RCC. No correlation with age, gender, tumor stage and clinicopathological parameters was observed by statistical analysis. Although not statistically significant, it exhibited the trend that more methylation of Slit2 occurred at lower tumor stages (T1/T2) of RCC (Table 2).
Discussion Besides the representative tumor suppressor gene VHL, in recent years, many emerging genes have been demonstrated to be critical for RCC development [2, 8]. Although slits, netrins, semaphorins and the ephrins are conserved
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Table 1 Relationship between immunohistochemical and clinicopathologic characteristics in patients with RCC
Total N
ca [ adj N (%)
\ 50
14
0(0 %)
3(21.4 %)
11(78.6 %)
C 50
61
8(13.1 %)
8(13.1 %)
45(73.8 %)
Male
50
6(12 %)
8(14 %)
36(72 %)
Female
25
2(8 %)
3(12 %)
20(80 %)
Left
43
3(7 %)
5(11.6 %)
35(81.4 %)
Right
32
5(15.6 %)
6(18.8)
21(65.6 %)
I II
4 35
0(0 %) 2(5.7 %)
1(25 %) 3(8.6 %)
3(75 %) 30(85.7 %)
III
28
6(21.4 %)
5(17.9 %)
17(60.7 %)
IV
8
0(0 %)
2(25 %)
6(75 %)
11(17.7 %)
48(77.4 %)
Variables
v2
ca \ adj N (%)
ca = adj N (%)
P value
Age(y) 2.408
0.3
1.114
0.573
2.532
0.282
8.382
0.211
14.059
0.001
12.523
0.014
Gender
Site
Histological grade
Tumor stage T1/T2
62
3(4.8 %)
T3/T4
13
5(38.5 %)
I
33
0(0 %)
6(18.2 %)
27(81.8 %)
II
24
3(12.5 %)
5(20.8 %)
16(66.7 %)
III
18
5(27.8 %)
0(0 %)
13(72.2 %)
0(0 %)
8(61.5 %)
Clinical classification
A
Slit2
5-aza-2
−
+
GAPDH
B
−
5-aza-2
+
M
U
M
U
−
−
+
+
A498
A498
786-0
786-O A498(79.5%) OS-RC-2 A549
5-aza-2 deoxycytidine treatment A498(21.6%) Hela
Fig. 3 Pharmacological demethylation with Aza treatment upregulated Slit2 expression in cancer cell lines. a RT-PCR analysis of Slit2 expression in cancer cell lines with demethylation with Aza.
b Methylation-specific PCR analysis of Slit2 promoter methylation with or without Aza treatment
families of axonal guidance cues in the nervous system [23], the signaling mediated by these molecules have been involved in functions out of the nervous system including cancer development [24, 25]. Previously, our data show that one of the netrin-1 receptor UNC5C acts as a tumor suppressor in RCC and is down-regulated in RCC. We also demonstrated the expression of UNC5D; the latest identified member of netrin-1 receptors was attenuated in RCC cell lines and primary RCC. UNC5D expression in RCC
cell lines exhibited tumor-suppressive functions. DNA methylation and loss of heterozygosity contributes to the inactivation of UNC5C or UNC5D in renal cell carcinoma [11, 12]. Here, we systematically analyzed the expression of Slit2, the representative molecule of slits, in renal cell carcinoma and undermined the possible mechanism for its down-regulation in RCC. We first examined the expression pattern of Slit2 in normal tissues. Consistent with recent findings, Slit2
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A
Page 5 of 7 768
Ca. M
Adj.
U
M
Ca. M
B
U
Ca. M
Adj. U
M
U
M
U
M
Adj.
U
Ca.(86.9%)
M
U
Adj. U
M
Ca. M
Adj. U
M
Categorization
Age
\50
9
2(22.2 %)
7(77.8 %)
C50
29
6(20.7 %)
23(79.3 %)
Male
17
5(29.4 %)
12(70.6 %)
Female
21
3(14.3 %)
18(85.7 %)
T1/T2
32
8(25 %)
24(75 %)
T3/T4
6
0(0 %)
Tumor stage Histological grade
U
tissues. Results from 6 representative pairs are shown. b Detailed BGS analysis of the Slit2 promoter in a representative paired tissue
Clinicopathological characteristic
Gender
U
Adj.(11.9%)
Fig. 4 Promoter hypermethylation contributes to the down-regulated expression of Slit2 in primary RCCs. a Slit2 promoter methylation was assesses by MSP in primary RCCs and adjacent non-cancerous
Table 2 Relationship between methylation and clinicopathologic characteristics in patients with RCC
M
Ca. U
Ca.
Adj.
n
Gene inactivation Methylation (%)
Demethylation (%)
11
2(18.2 %)
9(81.8 %)
II
20
5(25.0 %)
15(75.0 %)
7
1(14.3 %)
6(85.7 %)
38
8(21.1 %)
30(78.9 %)
III
expression was not restricted to the nervous system [26]. It is widely expressed in various tissues [27]. Then, we detected the expression of Slit2 in diverse tumor cell lines and found that Slit2 expression was very weak in many cell lines including renal cell carcinoma. Although the promoter methylation of Slit2 was reported, the expression of Slit2 in RCC was not systematically analyzed [28]. In 38 cases of paired RCC and non-malignant tissues, Slit2 mRNA was down-regulated in 20 cases of RCC. Furthermore, immunohistochemical studies in 75 cases also showed that the protein of Slit2 was down-regulated in RCC. These data together suggest that similar to that in other cancers, the expression of Slit2 was also down-regulated in RCC. Then, we treated the cancer cell lines with weak expression of Slit2 by demethylation drug, then the expression of Slit2 was up-regulated. The up-expression of Slit2 in renal cancer cell lines after treatment with the demethylating agent exhibited good concordance with the methylation status, suggesting the expression of Slit2 was
P value
0.01
0.922
1.293
0.255
1.900
0.168
0.435
0.805
0(100 %)
I
Total
v2
down-regulated mainly by hypermethylation in these cell lines. Then, we went further and analyzed the methylation status of Slit2 in primary RCC and found that the methylation frequency of Slit2 promoter in primary RCC was 21.1 % (8/38). The eight cases of RCC which has the Slit2 promoter methylation were among the 20 cases of RCC, which had down-regulated expression of Slit2 mRNA. Thus, promoter methylation may be responsible for its down-regulation in RCC. However, we cannot exclude other mechanisms such as loss of heterozygosity which may also be involved in the down-regulation in RCC. For example, *63 % of breast tumors also show loss of heterozygosity at the 4p 15.1–15.3 region where Slit2 was located [29]. It is worth mentioning that the down-regulation of Slit2 occured at the early stages of tumor development (T1/T2). It may be involved in the initiation of RCC development. Besides, it is possible that Slit2 serve as an early diagnostic marker for RCC.
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In summary, we reported the expression pattern of Slit2 on paired RCC and demonstrated the down-regulation of Slit2 on RCC tissues. We also explored the mechanism for its down-regulation in RCC. Further studies will focus on the tumor-suppressive function of Slit2 in RCC. Studying the expression and regulatory mechanism of Slit2 in RCC will facilitate the deep understanding the development of RCC and help find novel therapeutic strategies for RCC. Acknowledgments This work received supports from Beijing Municipal Natural Science Foundation (7122104) and the National Natural Science Foundation of China (81072395). Conflict of interest interest.
13. 14.
15.
The authors disclose no potential conflicts of
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ORIGINAL PAPER
Reduced expression of Slit2 in renal cell carcinoma Wei-Jie Ma • Yu Zhou • Dan Lu • Dong Dong Xiao-Jun Tian • Jie-xi Wen • Jun Zhang
•
Received: 25 October 2013 / Accepted: 5 November 2013 / Published online: 15 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is downregulated in RCC partially due to promoter methylation. Keywords Slit2 Renal cell carcinoma Methylation Tumor suppressor
W.-J. Ma Y. Zhou D. Lu D. Dong J. Wen J. Zhang (&) Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology (Ministry of Health), Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191, China e-mail: [email protected] X.-J. Tian Department of Urology, Peking University Third Hospital, Beijing, China
Introduction The development of renal cell carcinoma (RCC) was suggested to be tightly associated with the accumulation of multiple genetic and epigenetic inactivation of tumor suppressor genes (TSGs) [1, 2]. The representative and most common genetic alteration in sporadic clear cell RCCs is inactivation of the Von Hippel-Lindau (VHL) by chromosome 3p deletion [3]. Promoter methylation is an important epigenetic mechanism for inactivation of TSGs in many cancers [4]. Recently, many TSGs have been identified as potential TSGs in RCC and inactivated by promoter methylation. The identified aberrantly methylated genes include VHL, RASSF1A, p16, Timp-3, E-cadherin, b-catenin, SFRP1, SFRP2, SFRP5, DAL-1, COLIA1, KRT19, UCHL1, UNC5C and UNC5D [5–12]. Slit2 is the human orthologue of the Drosophila Slit2 [13]. It is located at chromosome 4p15.2. Slit2 was initially identified as a critical guidance cue for axons in the nervous systems [14]. In recent years, Slit2 is found to be widely expressed in mammalian tissues and functions out of the nervous system [15]. Intriguingly, Slit2 has been reported to be a potential TSGs in diverse tumors [16]. Promoter methylation is an important mechanism for its down-regulation in these cancers [17]. Slit2 promoter region hypermethylation has been detected in various cancers such as breast cancer, lung cancer, colorectal cancer, glioma, cervical cancer, colorectal cancer and leukemia [17–22]. However, the expression and regulatory mechanism for Slit2 in renal cell carcinoma were not systematically studied. Here, we analyzed the mRNA and protein expression level of Slit2 in renal cell carcinoma. We also provide the evidence that may explain the downregulation in RCC. These data suggested that Slit2 may also be a potential tumor suppressor gene in RCC.
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Materials and methods Cell lines, tumor specimens and 5-aza-dC treatment A series of cancer cell lines were used for this study, including 5 RCCs (786-O, A498, ACHN, Caki-1 and OS-RC-2), 3 lung cancer (NCI-H460, NCI-H1299 and A549), 2 colon cancer (SW480 and SW620), 1 cervical carcinoma (Hela), 1 leukemia (Jurkat). Human immortalized embryonic kidney cell line HEK293T was also used. Renal carcinoma and corresponding non-cancerous tissues (n = 38) were obtained from the Peking University Third Hospital (Beijing, China) with patients’ consents and institutional ethical approval. All of the specimens were pathologically confirmed. For demethylation, cell lines were treated with 10 mmol/L of 5-aza-20 -deoxycytidine (5-Aza-dC, Sigma-Aldrich) for 3 days with exchange of reagents and medium every 24 h.
Med Oncol (2014) 31:768
polymerase (Takara) for 38 cycles. The MSP primer sequences are Slit2-MF: 50 -aggacgaattattagtgggagatc-30 and Slit2-MR: 50 -actactccaaacaaaaccaaacg-30 ; Slit2-UF:50 -aaggatgaattattagtgggagatt-30 and Slit2-UR:50 -actactccaaa0 caaaaccaaacaac-3 . The BGS primer sequences are Slit2-L: 50 -aggggttagaaggagaaggattatt-30 and Slit2-R: 50 -aacaaaaataaaaaaaacaaaaacc-30 . Statistical analysis Statistical analyses were performed with SPSS, version 13.0 (SPSS, Inc.). Differences between two independent groups were analyzed by the Student’s t test. v2 test was used to calculate differences in the patient’s age, gender, tumor stage, histological grade and clinical classification. Methylation and immunohistochemistry status was evaluated between paired tumor and adjacent non-malignant tissue samples. P \ 0.05 was considered significant.
Reverse transcriptase PCR A human normal tissue cDNA panel was purchased from Clontech. Total RNA was isolated from cell lines and frozen stored tissue specimens by TRIZOL reagent (Invitrogen, Carlsbad, CA). RNA was reverse transcribed using the Reverse Transcription System (Promega, Madison, WI) according to the manufacture’s instruction. PCR amplifications of Slit2 were performed using the following primers: Slit2-F: 50 -tcagctgtttcctgagttgc-30 and Slit2-R: 50 tggttgaaacttgccacaga-30 . PCR products were resolved on 1 % agarose gels. Immunohistochemistry and scoring The tissue microarray (Shanghai Outdo Biotech Co. Ltd.) of 75 paired tumor and non-cancerous tissue was incubated with anti-Slit2 antibody (Santa Cruz) at 1:80 dilution in PBS overnight at 4 °C. Staining was evaluated by percent of tumor cell positivity and staining intensity. Slides were cored by 3 reviewers, and discrepancies were resolved by a urological pathologist. Methylation-specific PCR and bisulfate genomic sequencing Genomic DNA was extracted from cell lines and tissue samples by a commercial DNA extraction kit (Promega). Methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS) analysis were conducted as described. Amplified BGS PCR products were cloned into the pGEM-T Easy vector (Promega), and 8 random clones from each sample were sequenced. The bisulfite-treated DNA was amplified with the methylation-specific primer set and sequencing primer set by PCR using hot-start Taq
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Results Expression patterns of Slit2 in normal tissue and RCC cell lines In order to assess whether Slit2 is involved in RCC development, we first examined the expression profile of Slit2 in normal adult tissues and found that Slit2 was broadly expressed. It is expressed in brain, lung, kidney, placenta, spleen, thymus, prostate, testis, heart, ovary, etc. (Fig. 1a). Then, we also detected the expression level of Slit2 mRNA in multiple cancer cell lines. In renal cell carcinoma cell lines, the expression of Slit2 mRNA was weakly expressed in 4/5 RCC cell lines (786-O, A498, ACHN and Os-RC-2) (Fig. 1b). Notably, Slit2 expression was maintained in immortalized epithelial cell lines such as HEK293. Taken together, these data suggest that Slit2 was broadly expressed in normal tissues and down-regulated in RCC cell lines and it may be involved in RCC development. Down-regulation of Slit2 in primary renal cell carcinomas We further investigated whether Slit2 was down-regulated in renal carcinomas by RT-PCR. As shown in Fig. 2a, compared to adjacent non-malignant tissues, Slit2 mRNA was dramatically down-regulated in inactivation in 20 of 38 (52.6 %) RCC cases. We further examined Slit2 protein expression in a total of 75 paired RCC tissue sections by immunohistochemistry. Strong Slit2 staining was shown in non-cancerous renal tissue, mainly in the renal epithelium. On the contrary, Slit2 protein was weakly detected in RCC tissues (Fig. 2b). Histochemical studies showed that Slit2
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Fig. 1 Slit2 expression in normal tissues and cell lines. a RT-PCR analysis of Slit2 mRNA expression in 14 normal tissues. b RT-PCR analysis of Slit2 mRNA expression in multiple cell lines
A Slit2 GAPDH PBMC
Ovary
Colon
Testis
Prostate
Thymus
Lung
Spleen
Pancreas
Kidney
Kidney
Muscle
Liver
Lung
Placenta
Brain
Heart
B
Others
Slit2 GAPDH
A
Ca.
Adj.
Ca.
Adj.
Ca.
Adj.
Ca.
Adj.
Slit2 GAPDH
B
expression of Slit2 in these cell lines (Fig. 3a). To confirm this, we found that the methylation status of Slit2 promoter was changed after Aza treatment in A498 and 786-O cell lines (Fig. 3b). Further BGS also supported this. Therefore, Slit2 expression was significantly increased in renal cancer cell lines after demethylation agent treatment along with an increase in unmethylated alleles and a decrease in methylated alleles (Fig. 3c). Frequent Slit2 promoter methylation in primary RCC
Ca.
×200
Adj.
×200
Fig. 2 Down-regulation of Slit2 in primary renal cell carcinoma. a RT-PCR analysis of Slit2 mRNA in renal cell carcinoma and paired adjacent non-cancerous tissues. b Immunohistochemistry analysis of Slit2 protein expression in renal cell carcinoma (left) and paired adjacent non-cancerous tissues (right)
protein was down-regulated in 56 (74.67 %) RCC tissues compared to paired adjacent non-cancer tissues (Table 1). Statistical analysis showed that Slit2 down-regulation did not correlate with age, gender and histological grading. Interestingly, Slit2 down-regulation correlated with tumor stage (P B 0.001) and clinical classification (P \ 0.05). It indicated that down-regulation of Slit2 mainly occurred in the early stages of RCC. Promoter methylation of Slit2 in renal cancer cell lines As we have mentioned above, the expression of Slit2 was down-regulated in several cancer cell lines. The region spanning the putative promoter of Slit2 and exon 1 and the beginning of intron 1 is a typical CpG island. We next analyzed whether demethylation agents can upregulate the expression of Slit2 in cancer cell lines. We treated several cancer cell lines which had lower Slit 2 expression including A498, 786-O, Os-RC-2, A549 and Hela with demethylation agent Aza and found that Aza treatment upregulated the
We also analyzed the Slit2 promoter methylation status in 38 primary RCC samples and paired adjacent non-malignant renal tissues. Of 38 tumor samples, 8 (21.1 %) showed methylation in the Slit2 promoter (Fig. 4a), as compared with no methylation in the corresponding normal tissues. Notably, eight tumor tissues with Slit2 promoter methylation were among the 20 tumor cases with down-regulated Slit2 expression. This demonstrated that methylation of Slit2 promoter can lead to the down-regulation of Slit2 in RCC. The methylation-specific PCR results were confirmed by direct sequencing of CpG islands in the Slit2 promoter region (Fig. 4b). We also evaluated the relationship of Slit2 methylation with clinicopathological features in patients with RCC. No correlation with age, gender, tumor stage and clinicopathological parameters was observed by statistical analysis. Although not statistically significant, it exhibited the trend that more methylation of Slit2 occurred at lower tumor stages (T1/T2) of RCC (Table 2).
Discussion Besides the representative tumor suppressor gene VHL, in recent years, many emerging genes have been demonstrated to be critical for RCC development [2, 8]. Although slits, netrins, semaphorins and the ephrins are conserved
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Table 1 Relationship between immunohistochemical and clinicopathologic characteristics in patients with RCC
Total N
ca [ adj N (%)
\ 50
14
0(0 %)
3(21.4 %)
11(78.6 %)
C 50
61
8(13.1 %)
8(13.1 %)
45(73.8 %)
Male
50
6(12 %)
8(14 %)
36(72 %)
Female
25
2(8 %)
3(12 %)
20(80 %)
Left
43
3(7 %)
5(11.6 %)
35(81.4 %)
Right
32
5(15.6 %)
6(18.8)
21(65.6 %)
I II
4 35
0(0 %) 2(5.7 %)
1(25 %) 3(8.6 %)
3(75 %) 30(85.7 %)
III
28
6(21.4 %)
5(17.9 %)
17(60.7 %)
IV
8
0(0 %)
2(25 %)
6(75 %)
11(17.7 %)
48(77.4 %)
Variables
v2
ca \ adj N (%)
ca = adj N (%)
P value
Age(y) 2.408
0.3
1.114
0.573
2.532
0.282
8.382
0.211
14.059
0.001
12.523
0.014
Gender
Site
Histological grade
Tumor stage T1/T2
62
3(4.8 %)
T3/T4
13
5(38.5 %)
I
33
0(0 %)
6(18.2 %)
27(81.8 %)
II
24
3(12.5 %)
5(20.8 %)
16(66.7 %)
III
18
5(27.8 %)
0(0 %)
13(72.2 %)
0(0 %)
8(61.5 %)
Clinical classification
A
Slit2
5-aza-2
−
+
GAPDH
B
−
5-aza-2
+
M
U
M
U
−
−
+
+
A498
A498
786-0
786-O A498(79.5%) OS-RC-2 A549
5-aza-2 deoxycytidine treatment A498(21.6%) Hela
Fig. 3 Pharmacological demethylation with Aza treatment upregulated Slit2 expression in cancer cell lines. a RT-PCR analysis of Slit2 expression in cancer cell lines with demethylation with Aza.
b Methylation-specific PCR analysis of Slit2 promoter methylation with or without Aza treatment
families of axonal guidance cues in the nervous system [23], the signaling mediated by these molecules have been involved in functions out of the nervous system including cancer development [24, 25]. Previously, our data show that one of the netrin-1 receptor UNC5C acts as a tumor suppressor in RCC and is down-regulated in RCC. We also demonstrated the expression of UNC5D; the latest identified member of netrin-1 receptors was attenuated in RCC cell lines and primary RCC. UNC5D expression in RCC
cell lines exhibited tumor-suppressive functions. DNA methylation and loss of heterozygosity contributes to the inactivation of UNC5C or UNC5D in renal cell carcinoma [11, 12]. Here, we systematically analyzed the expression of Slit2, the representative molecule of slits, in renal cell carcinoma and undermined the possible mechanism for its down-regulation in RCC. We first examined the expression pattern of Slit2 in normal tissues. Consistent with recent findings, Slit2
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A
Page 5 of 7 768
Ca. M
Adj.
U
M
Ca. M
B
U
Ca. M
Adj. U
M
U
M
U
M
Adj.
U
Ca.(86.9%)
M
U
Adj. U
M
Ca. M
Adj. U
M
Categorization
Age
\50
9
2(22.2 %)
7(77.8 %)
C50
29
6(20.7 %)
23(79.3 %)
Male
17
5(29.4 %)
12(70.6 %)
Female
21
3(14.3 %)
18(85.7 %)
T1/T2
32
8(25 %)
24(75 %)
T3/T4
6
0(0 %)
Tumor stage Histological grade
U
tissues. Results from 6 representative pairs are shown. b Detailed BGS analysis of the Slit2 promoter in a representative paired tissue
Clinicopathological characteristic
Gender
U
Adj.(11.9%)
Fig. 4 Promoter hypermethylation contributes to the down-regulated expression of Slit2 in primary RCCs. a Slit2 promoter methylation was assesses by MSP in primary RCCs and adjacent non-cancerous
Table 2 Relationship between methylation and clinicopathologic characteristics in patients with RCC
M
Ca. U
Ca.
Adj.
n
Gene inactivation Methylation (%)
Demethylation (%)
11
2(18.2 %)
9(81.8 %)
II
20
5(25.0 %)
15(75.0 %)
7
1(14.3 %)
6(85.7 %)
38
8(21.1 %)
30(78.9 %)
III
expression was not restricted to the nervous system [26]. It is widely expressed in various tissues [27]. Then, we detected the expression of Slit2 in diverse tumor cell lines and found that Slit2 expression was very weak in many cell lines including renal cell carcinoma. Although the promoter methylation of Slit2 was reported, the expression of Slit2 in RCC was not systematically analyzed [28]. In 38 cases of paired RCC and non-malignant tissues, Slit2 mRNA was down-regulated in 20 cases of RCC. Furthermore, immunohistochemical studies in 75 cases also showed that the protein of Slit2 was down-regulated in RCC. These data together suggest that similar to that in other cancers, the expression of Slit2 was also down-regulated in RCC. Then, we treated the cancer cell lines with weak expression of Slit2 by demethylation drug, then the expression of Slit2 was up-regulated. The up-expression of Slit2 in renal cancer cell lines after treatment with the demethylating agent exhibited good concordance with the methylation status, suggesting the expression of Slit2 was
P value
0.01
0.922
1.293
0.255
1.900
0.168
0.435
0.805
0(100 %)
I
Total
v2
down-regulated mainly by hypermethylation in these cell lines. Then, we went further and analyzed the methylation status of Slit2 in primary RCC and found that the methylation frequency of Slit2 promoter in primary RCC was 21.1 % (8/38). The eight cases of RCC which has the Slit2 promoter methylation were among the 20 cases of RCC, which had down-regulated expression of Slit2 mRNA. Thus, promoter methylation may be responsible for its down-regulation in RCC. However, we cannot exclude other mechanisms such as loss of heterozygosity which may also be involved in the down-regulation in RCC. For example, *63 % of breast tumors also show loss of heterozygosity at the 4p 15.1–15.3 region where Slit2 was located [29]. It is worth mentioning that the down-regulation of Slit2 occured at the early stages of tumor development (T1/T2). It may be involved in the initiation of RCC development. Besides, it is possible that Slit2 serve as an early diagnostic marker for RCC.
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In summary, we reported the expression pattern of Slit2 on paired RCC and demonstrated the down-regulation of Slit2 on RCC tissues. We also explored the mechanism for its down-regulation in RCC. Further studies will focus on the tumor-suppressive function of Slit2 in RCC. Studying the expression and regulatory mechanism of Slit2 in RCC will facilitate the deep understanding the development of RCC and help find novel therapeutic strategies for RCC. Acknowledgments This work received supports from Beijing Municipal Natural Science Foundation (7122104) and the National Natural Science Foundation of China (81072395). Conflict of interest interest.
13. 14.
15.
The authors disclose no potential conflicts of
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