Thirty fresh tumor samples were collected after informed consent was obtained from patients who underwent brain operations for glioma at Xiangya Hospital (Hunan, People's Republic of China). The samples were snap-frozen immediately following resection and stored in liquid nitrogen until processing. The 17 male and 13 female patients were aged from 17 to 68 years (mean age at registration = 42 years). Tumors were graded and classified according to the World Health Organization (2007), including astrocytoma (grade I (1), grade II-III (17)), oligodendroglioma (grade II (5)), oligoastrocytoma (grade II (3)), and glioblastoma (grade IV (4)) 33. For comparison, normal human tissues from patients without cancer were obtained at the time of autopsy.

Human glioblastoma-derived cell lines SF126 and SF767 were obtained from the Cell Research Institute of Peking Union Medical College (Peking, China) and cultured in minimal essential medium (MEM). Cos7 and Hela were obtained from American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM). All cells were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 ug/mL streptomycin and cultured at 37C with 5% CO₂.

The LRRC4 promoter region in the 5' end of humans was predicted using the PromoterInspector and PromoterScan programs. The CpG island was found using CpGplot from the European Molecular Biology Open Software Suite. To obtain the 5' flanking region of the LRRC4 gene, PCR amplification was performed on human genomic DNA (forward primer 5'-ATTGGTACCGGCGAGCTCACAGGGCAGGG-3', reverse primer 5'-CTTAGATCTCTGGAGAAGGAGGTGGGGAG-3'). After an initial denaturation step (94°C for 5 min), the PCR reactions were carried out for 32 cycles at 94°C for 30 sec, 62°C for 30 sec, and 72°C for 2 min, with a final extension of 10 min at 72°C. The PCR product was purified using a gel Purification Kit and cloned into the T/A cloning vector pGEMT-Easy (Promega). Positive clones of pT/A-2475/-101 were isolated and sequenced.

The 5' upsteam regulatory region of the LRRC4 gene was subcloned into the KpnI and Bgl II restriction sites of the pGL3-enhancer vector (Promega). Construct naming is based on the position of the promoter fragments (pGL3-2475/-101). Four deletion constructs of the LRRC4 promoter region were created (pGL3-1483/-101, pGL3-835/-101, pGL3-293/-101 and pGL3-835/-293), originating from the construct pGL3 -2475/-101 by PCR amplification using the primers listed in Table 1. Sense primers for generating the reporter constructs described above contained an adaptor with a KpnI restriction site (GGTACC) at the 5' end. Anti-sense primers contained an adaptor with a Bgl II restriction site (AGATCT). The promoter fragments were then subcloned into the KpnI/Bgl II sites of the pGL3 enhancer vector and sequenced.

Transfection was performed with Lipofectamine™ Reagent (Invitrogen, Carlsbad, CA). 5×10⁵ cells were seeded in each well of 24-well tissue plates. When cells reached 50–80% confluence, they were cotransfected using 1 μg of each DNA construct in pGL3-enhancer and 0.25 μg SV40β-galactosidase vector for normalizing transfection efficiency per well according to manufacturer's instructions. Firefly luciferase activity was measured in cell lysates 48 h after transfection by using the Luciferase Assay System (Promega) and a luminometer. β-galactosidase activity was measured in cell lysates by the β-galactosidase Enzyme Assay System (Promega). Experiments were repeated at least three times with three replicates per sample for each experiment. Results are normalized against β-galactosidase activity.

pGL3-835/-293 report plasmid constructs were methylated in vitro by SssI methylase (New England Biolabs) treatment following the manufacturer's instructions. Cells were transfected by methylated or mock-methylated constructs as described above.

Genomic DNA from cells and tissues was prepared using a DNA Extraction Kit (TaKaRa) according to manufacturer's instructions. Five hundred nanograms of genomic DNA was modified and purified using an EZ DNA Methylation-Gold Kit (ZYMO RESEARCH), following the manufacturer's protocol. Modified DNA was used immediately or stored at -80°C for up to six months.

The methylation-specific PCR primers were designed according to the promoter-active DNA sequence using Methyl Primer Express v1.0. Modified DNA was amplified by two different primer pairs specific to the unmethylated (U) and methylated (M) LRRC4 promoter sequences, respectively. For the methylated (M) sequence, the forward and backward primers were 5'-AGCGTAGTATTTAGCGAGTGC-3' and 5'-TAAACCCTAACACCGACTCG-3'.

For the unmethylated (U) sequence, the primers were forward 5'-GGGAGTGTAGTATTTAGTGAGTGT-3' and backward 5'-TAAACCCTAACACCAACTCACTC-3'. PCR amplification was performed for a total of 35 cycles with an annealing temperature of 58°C. Methylation specific PCR products were analyzed by a 2% agarose gel and stained with ethidium bromide.

In order to cover the whole promoter region of LRRC4, two PCR regions were amplified using primers that avoided CpG sites. For Region 1, primers were 5'-GYGGATTGGAGAATTGATTT TT-3' and 5'-AACTATACAAATACATACCCCCCCC-3'. For Region 2, primers were 5'-GAGGGGGGGGTATGTATTTGTATAGT-3' and 5'-CCCACCCTCAAAACAAACCC TC-3'. The PCR amplification was performed for a total of 38 cycles with annealing temperature of 56°C. PCR products were gel-purified and cloned into the T/A cloning vector pGEMT-Easy (Promega). Ten subclones were isolated and identified by double digestion and sequencing.

The human glioblastoma-derived cell lines SF126 and SF767 were grown for 4 days in the presence of various concentrations of 5-Aza-dC (2.5, 5, 10 and 15 μM). Fresh drug was added every 24 h. RNA and DNA were separately isolated.

RNA was isolated from harvested cells with Trizol (Invitrogen) reagent and then treated with DNase (Roche) to eliminate contaminated DNA. Reverse transcription of the RNA was performed according to the instructions of Promega. To amplify LRRC4, 2 μl of cDNA was used for each PCR using the primers, 5'-CAACTTGGCCCACAAT AACC-3' (forward) and 5'-CATCCGACCCTCAGAAATGT-3' (reverse). The primers for GAPDH were 5'-GTCAGTGGTGGACCTGACC T-3' (forward) and 5'-AGGGGAGATTCAGTGTGGTG-3'(reverse). The GAPDH primers were added to the PCR at the end of the tenth cycle as control experiments. Ten microliters of each reaction was then run on 2% agarose gel and stained with ethidium bromide.

To clone the LRRC4 promoter region, a database was searched against the human genomic DNA using LRRC4 (Genbank accession No. AF196976) as the query http://www.ncbi.nlm.nih.gov/BLAST to reveal the 5'upstream sequence of the LRRC4 gene. Several bioinformatics tools were used to identify the potential promoter region of the LRRC4 gene. A 605-bp region spanning positions -814 to -210 was identified as the potential promoter region of the LRRC4 gene by using a PromoterInspector 20, whereas a 251-bp region located from -788 to -538 was identified as the LRRC4 promoter by using a PromoterScan program 21. We detected two CpG islands that spanned positions -2151 to -433 and -366 to -101 using the EMBOSS CpGplot program 22 (Figure 1). Finally, a genomic DNA fragment that spanned positions -2475 to -101 relative to the initiation codon ATG of the LRRC4 gene was amplified by PCR. The PCR product was cloned into the T/A cloning vector pGEMT-Easy (Promega) (Data not shown).

To identify the promoter region of the LRRC4 gene, five constructs of progressive deletion spanning positions -2475 to -101 were generated (pGL3-2475/-101, pGL3 -1483/-101, pGL3-835/-101 and pGL3-293/-101) and cloned to upstream of the luciferase reporter gene in the pGL3-enhancer vector (Promega). Transient transfection experiments were carried out using Cos7 and Hela cells, respectively. The luciferase activity driven by LRRC4 promoter constructs was measured 48 h after transfection. Expression levels were corrected for variable transfection efficiencies by cotransfection with a plasmid directing the β-galactosidase expression.

As shown in Figure 2, in Cos7 and Hela cells, luciferase expression driven by the constructs pGL3-2475/-101, pGL3-1483/-101, pGL3-835/-101 and pGL3-835/-293 exhibited similarly high levels, but the reporter driven by construct pGL3-293/-101 showed little luciferase activity. Thus, the sequence spanning position -835 to -293 relative to the translation start site of LRRC4 functions as a promoter. MethPrimer program analysis shows that the LRRC4 promoter region has high G/C content (approximately 70%) and characteristics of a CpG island 23. In addition, this promoter region has no TATA box or CAAT box.

The presence of a CpG island suggested the possibility that the gene might be regulated through changes in the methylation status, which have been shown to cause gene silencing 2425. To analyze the effects of DNA methylation on promoter activity, LRRC4 promoter reporter construct pGL3-835/-293 was treated with or without SssI methylase to methylate the promoter in vitro. In vitro-methylated or mock-methylated LRRC4 promoter constructs were transfected into Cos7 and Hela cells along with SV40β-galactosidase vector. Firefly luciferase activity was measured in cell lysates as shown in Figure 3A. It can be seen that LRRC4 promoter activity was strongly suppressed by methylation in both cell lines. The same result was verified in SF126 and SF767 cell lines (Figure 3B).

Our previous study showed that the expression of LRRC4 was absent in glioma cell lines SF126 and SF767 18. Moreover, DNA sequence analysis failed to identify any mutation in the LRRC4 coding region, except for one Single Nucleotide Polymorphisms site hat does not affect amino acid sequences 18. To clarify the mechanism of LRRC4 inactivation in these two cell lines, methylation-specific PCR was used to examine the methylation status of the LRRC4 promoter. Both SF767 and SF126 cell lines showed methylation of the LRRC4 promoter. The methylated sequence was obtained and is shown in Figure 4A.

To investigate whether aberrant methylation of the LRRC4 in glioma cell lines reflects an epigenetic event occurring in primary glioma, we next examined 30 primary glioma biopsies and three tissue specimens of normal brain for LRRC4 methylation. Some methylation analyses are shown in Figure 4B. It was demonstrated that the LRRC4 promoter was free from methylation in the three normal brain tissue samples, but was methylated to different extents in the 30 primary gliomas.

To determine a more detailed map of the methylation in the LRRC4 promoter, we performed bisulfite sequencing around the promoter region of the LRRC4 gene in some of the glioma biopsies and cell lines studied above. In order to cover the whole promoter region of LRRC4, two PCR regions were amplified using primers that avoid CpG sites. Region 1 spans -934 to -529 relative to the LRRC4 translation start site, including 48 CpG sites. Region 2 covers -550 to -197 relative to the LRRC4 translation start site, including 21 CpG sites. Bisulfite sequencing of 10 individual clones of PCR products of both Region 1 and Region 2 from primary glioma biopsies (T5 and T10) and cell lines (SF126 and SF767) revealed densely methylated CpGs within the promoter regions compared to normal brain tissue (Figure 5). Whereas not all CpGs around the LRRC4 promoter are methylated, most of them are in SF126 and SF767 glioma cell lines. However, in the primary glioma tissue samples (T5 and T10), the methylation pattern appeared much more heterogeneous and varied in density in different clones of the same sample. This may be due to mixed cellularity in tissue samples.

In order to demonstrate a functional association between LRRC4 promoter methylation and its gene inactivation, a DNA demethylating agent, 5-Aza-2'-deoxycytidine (5-Aza-dC), was used to treat SF126 and SF767 cell lines. By RT-PCR analysis, LRRC4 expression was detected after treatment with 5-Aza-dC in both cell lines (Figure 6A). Moreover, LRRC4 expression increased with increasing dosage of 5-Aza-dC. LRRC4 expression was highest when induced by 15 μM 5-Aza-dC in SF767 cells, but in SF126 cells LRRC4 expression was highest when induced by 10 μM 5-Aza-dC.

To confirm that reactivation of LRRC4 mRNA expression in glioma cell lines was caused by demethylation of the LRRC4 promoter, methylation-specific PCR was used to detect methylation status changes in the LRRC4 promoter in the SF767 and SF126 cell lines after 5-Aza-dC treatment. Figure 6B shows that 5-Aza-dC results in the partial demethylation of LRRC4.