Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer

Paired specimens of non-small cell lung cancer (NSCLC) tissues including 41 squamous cell carcinomas (SCC), 18 adenocarcinomas AC) and adjacent morphologically normal tissues (conventional "normal" matched control samples) were obtained after surgical resection of primary lung cancer prior radiation or chemotherapy and stored in liquid nitrogen. "Normal" matched controls were obtained minimum at 2 cm distance from the tumor and confirmed histologically as normal lung epithelial cells. The diagnosis was verified by histopathology and only samples containing 70% or more tumor cells were used in the study. The samples were collected in accordance to the guidelines issued by the Ethics Committee of Blokhin Cancer Research Center, Russian Academy of Medical Sciences (Moscow). All patients gave written informed consent that is available upon request. The study was done in accordance with the principles outlined in the Declaration of Helsinki. All tumor specimens were characterized according to the International System of Clinico-Morphological Classification of Tumors (TNM), based on the tumor-node-metastasis and staging classification of 1989 [35] and WHO criteria classification of 1999 [36]. Relevant clinical and pathological characteristics of the patients with NSCLC included in this study are summarized in Table 1. Normal lung tissues (autopsy material) were obtained post mortally from ten healthy individuals (age 23-49 lacking cancer history with absence of chronic diseases).

DNA was extracted using the Dneasy Tissue kit (Qiagen, USA) and total RNA was isolated with Rneasy mini kit (Qiagen, USA) according to the manufacturer's recommendation. RNA quality was assessed with spectrophotometer NanoDrop ND-1000 (NanoDrop Technologies Inc. USA) and by gel electrophoresis. All RNA samples were treated with RNAse free DNase I (Fermentas, Lithuania) and cDNA was synthesized using MMLV reverse transcriptase and random hexamers according to standard manufacturer's protocol (Fermentas, Lithuania).

The sequences of primers and probes are shown in Table 2. All reactions were performed using ABI 7000 PRISM™ SDS (Applied Biosystems) with RQ software (PCR program: 10 min at 95°C, then 40 two-step cycles 15 s at 95°C and 60 s at 60°C) in total volume 25 μl in triplicate. All probes contained the dye FAM at 5'-end and RTQ1 at 3'-end. Final concentrations of primers and probes for target and reference genes were: RBSP3 cDNA primers- 350 nM, probe - 150 nM; RBSP3 DNA primers - 150 nM, probe - 100 nM; ACTB DNA primers- 200 nM, probe - 100 nM, NPRL2 cDNA primers - 500 nM, probe 300 nM; RASSF1A cDNA primers - 300 nM, probe 300 nM;GAPDH cDNA primers - 300 nM, probe - 150 nM; RPN1 cDNA primers - 350 nM, probe - 200 nM;RPN1 DNA primers - 200 nM, probe - 100 nM; GUSB cDNA primers - 350 nM, probe - 250 nM; GUSB DNA primers - 200 nM, probe - 200 nM. PCR products were analyzed in 1.8% agarose gels and nucleotide sequences of the amplicons were verified by sequencing with 3730 DNA Analyzer automated sequencer (Applied Biosystems).

qPCR data were analyzed using the relative quantification or ΔΔC_t-method [37, 38] based on mRNA (or DNA) copy number ratio (R) of a target gene versus reference gene in a given tumor sample relative to matched normal control sample (see above Tissue specimens section) according to the formula:

where E - efficiency of reaction, C _T - threshold cycle, ref - reference gene, tar - target gene.

All preliminary validation steps have been done: standardization of all assays, reproducibility of the qPCRs in parallel and in independent runs, selection of reference samples and testing of reference genes http://​www.​gene-quantification.​info/​.

Microarrays were constructed essentially as previously described [39, 40]. In brief, two oligonucleotides:

NotX: 5'-AAAAGAATGTCAGTGTGTCACGTATGGACGAATTCGC-3'

and NotY: 5'-GGCCGCGAATTCGTCGGTATGCACTGTGTGTGACATTCAAA-3"

were used to create the NotI linker. Annealing was carried out in a final volume of 100 μl containing 20 μl of 100 μM NotX, 20 μl of 100 μM NotY, 10 μl of 10×M buffer (Roche Molecular Biochemicals) and 50 μl of H₂O. Two micrograms of tumor and normal control DNA (50 μg/ml) were digested with 20 U of Sau3A (Roche Molecular Biochemicals) at 37°C for 5 h and then 0.4 μg of the digested DNAs were circularized overnight with the T4 DNA ligase (Roche Molecular Biochemicals) in the appropriate buffer in 1 ml reaction mixture. Then DNA was concentrated with ethanol, partially filled in and digested with 10 U of NotI at 37°C for 3 h. Following digestion, NotI was heat inactivated and DNAs were ligated overnight in the presence of a 50 M excess of NotI linker at room temperature. NotI- representation (NR) probes were labeled in a PCR reaction with NotX primer. The majority of products of the DNA amplification step were in the 0.2-1.0 kb range. Repeated PCR was conducted for labeling NR with fluorophores.

Hybridization of coupled normal/tumor NotI samples was carried out at 42°C for 15 h in a Lucidea Base device (Amersham Pharmacia Biotech) according to manufacturer's recommendations. Automatic washing of the microarrays was performed in the same device using manufacturer's protocol. The following solutions were sequentially used for the washing: 1) 0.2% SDS+1 SSC; 2) 0.2% SDS+0.1 SSC; 3) 0.1 SSC; 4) de-ionized water; 5) isopropyl alcohol. Then microarrays were scanned in the GenePix 4000 A and results were processed with GenePix Pro 6.0 software (Amersham Pharmacia Biotech).

Nonparametric Wilcoxon test was used to compare mRNA expression differences of target and reference genes for the same NSCLC sample. Then groups of samples were compared in respect to average level of mRNA decrease (LD_av) and the frequency of decrease (FD). The LD was calculated as 1/R and reflects the n-fold factor by which the mRNA content decreased in the tumor compared to normal tissue. Nonparametric Kruskal-Wallis and Mann-Whitney rank-sum tests were used to test mRNA differences (both LD_av and FD) for each target gene in NSCLC (AC, SCC) and with and without metastases. Nonparametric Spearmen's criterion was used to calculate the coefficient of correlation between the levels of mRNA decrease (LD_av) for each set of pairs of target genes. P-values < 0.05 were considered statistically significant. All statistical procedures were performed using the BioStat software [41].

Genomic structure and location of primers and probes is shown in Figure 1.

The results of mRNA level quantification for three genes and statistical analysis are shown in Figures 2 and 3 and Table 3. The variability of GAPDH and RPN1 mRNA was less than 2-fold in studied samples, therefore in this study we considered significant greater than or equal to 2-fold decrease of target genes expression.

As expression of all three target genes was frequently decreased in the same NSCLC samples we tested the probability that this decrease was not random. To do this we used 36 NSCLC samples and two control reference genes GAPDH and RPN1 (Figure 3, Table 4). Results demonstrated that this simultaneous down-regulation was very frequent and statistically valid: all three genes had reduced expression (≥2) in 39% (P < 0.001) of NSCLC specimens (36% in AC and 41% in SCC). Simultaneous decreased expression of RBSP3 and NPRL2 was observed in 61% of cases and for RBSP3 and RASSF1A this occurred in 50%. In the case of NPRL2 and RASSF1A simultaneous down-regulation was detected in 44% of tumors (Table 4). Spearmen's coefficient values r_s for RBSP3, NPRL2/G21, RASSF1A gene pairs were high (0.63 - 1.0, P < 0.001) in different groups especially in AC and SCC with metastases.

To understand the mechanism underlying the observed down-regulation of RBSP3 in NSCLC samples we used qPCR to test DNA copy number changes, i.e. genetic factors [9, 10] and NotI microarrays to examine methylation i.e. epigenetic factors [39]. Methylation was detected in 38% of AC (3 of 8) and in 80% of SCC (8 of 10) cases with decreased expression of RBSP3. Deletions were detected in 25% of AC samples and in 30% of SCC tumors (Table 5). Both methylation and hemizygous deletions were observed in two SCC specimens. Methylation or/and deletions of RBSP3 were detected in 63% of AC and in 90% of SCC cases. The data suggested that both genetic and epigenetic mechanisms are important for transcriptional inactivation of RBSP3 in NSCLC.

Using NotI microarrays we cannot test methylation of NPRL2 and RASSF1A promoters because they don't contain NotI sites. However we can check genes SEMA3F and GNAI2 that are located in the same LUCA sub-region as NPRL2 and RASSF1A. We tested how often deletions and methylations occurred in the same NSCLC sample at both LUCA and AP20 sub-regions and found that this occurred in 58% of all studied cases (15 of 26). Thus, most likely both genetic and epigenetic mechanisms are responsible for simultaneous down-regulation of expression of these three genes.