Hypomethylation-activated cancer-testis gene SPANXC promotes cell metastasis in lung adenocarcinoma

2.1 The expression of SPANXC in normal tissues and LUAD patients

GTEx databases were used to assess testis-specific expression of SPANXC at transcription level. The expression abundance (FPKM) of genes and transcripts was downloaded from the GTEx project (http://www.gtexportal.org/home/, dbGaP Accession phs000424.v6.p1). We obtained the expression of SPANXC in LUAD tissues by analysing RNA sequencing data of LUAD patients from TCGA projects (https://confluence.broadinstitute.org/display/GDAC/Home, 2014-07-13 release). The detailed characteristics of samples from GTEx and TCGA are shown in Table S1. Gene expression of SPANXC is dichotomized into above median and below median expression groups based on median cut-off. Overall survival analysis curves were generated using Kaplan-Meier method with Survival package in R. The survival difference between the dichotomized groups was evaluated using log-rank test. DNA demethylation data of LUAD from TCGA were analysed with the R-package.

2.2 RNA extraction, reverse transcription and the quantitative real-time PCR

The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen), according to the manufacturer's protocol. One microgram of total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCRs (SYBR, TaKaRa) according to the manufacturer's instructions. Results were normalized to the expression of GAPDH. The rest of primers are listed in Table S2.

2.3 Cell culture

Lung adenocarcinoma cell lines A549 and NCI-H1299 were cultured in 1640 medium and DMEM (GIBCO-BRL) supplemented with 10% foetal bovine serum (10% FBS), 100 U/mL penicillin and 100 mg/mL streptomycin in humidified air at 37°C with 5% CO₂.

2.4 Transfection of cell lines

The siRNAs and plasmid were transfected into LUAD cells using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. The sequences for siRNAs are listed in Table S1.

2.5 Cell proliferation assay

A count of 2000 cells per well was cultured in 96-well plates (Corning Inc), and proliferation was evaluated by CCK8 for every 24 hours. For colony formation assay, a certain number of transfected cells were placed in each well of 6-well plates and maintained in proper media containing 10% FBS for 2 weeks, replacing the medium every 4 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich) in PBS for 10 minutes. The colony formation was determined by counting the number of stained colonies. Triplicate wells were measured in each treatment group.

2.6 Cell migration and invasion assays

For cell migration and invasion assays, 24-well transwell chambers with 8-μm pore size polycarbonate membrane were used (Corning). Cells were seeded on the top side of the membrane pre-coated with Matrigel (BD) (without matrigel for cell migration assay). Incubation for 24 hours, cells inside the upper chamber were removed with cotton swabs, while cells on the lower membrane surface were fixed and then stained with 0.5% crystal violet solution. Five fields were counted randomly in each well. All assays were performed in triplicate, and the experiment was repeated three times.

2.7 Co-immunoprecipitation and mass spectrometry (LC-MS/MS) assays

Co-IP experiments were performed using a Pierce™ Classic Magnetic IP/Co-IP Kit (Thermo) according to the manufacturer's instructions. Briefly, cell pellet was resuspended in Buffer A (10 mmol/L Hepes pH7.5, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT and 1 mmol/L PMSF/Cocktail) for 10 minutes on ice, 0.25% NP-40 was added for 5 minutes, and cytosol fraction and nuclear pellets were obtained by centrifugation at 13 000 RPM for 10 minutes. Nuclear pellet was then resuspended in Buffer C (20 mmol/L Hepes pH 7.5, 10% glycerol, 0.42 mol/L KCl, 4 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT and 1 mmol/L PMSF/cocktail) 20 minutes on ice, and nuclear fraction was obtained after 13 000 RPM 10 minutes centrifugation. Cell fraction was mixed together, and 500 µg of lysate was used for one IP reaction. Antibodies was added, IP was performed on the rotating plate in 4°C for 3 hours, and 20 µL washed Magnetic beads was added and incubated for one hour. Quickly wash four times with Wash buffer (50 mmol/L TrisCl 7.9, 10% glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 5 mmol/L MgCl2, 10 mmol/L β-ME 0.1% NP-40). The expression plasmid of SPANXC was marked by flag. Antibody for Flag was from Sigma. The elutions of proteins were subjected to mass spectrometric analysis. LC-MS/MS experiments were performed with a LTQ linear ion trap mass spectrometer (Thermo Finnigan) equipped with a microspray source.16

2.8 In vivo assay

NCI-H1299 cells were stably transfected with overexpression of SPANXC and empty vector and harvested cells and washed with PBS, and resuspended at 2 × 10⁷ cells/mL. Then, the suspended cells were injected into the tail veins of nude mice, which were treated 60 days after injection. The presence of tumour nodules was determined and was counted. Our study was carried out in strict accordance with Guide for Care and Use of Laboratory Animals of the National Institutes of Health. Our project was approved by committee on the ethics of animal experiments of Nanjing Medical University.

2.9 Statistical analysis

All statistical analyses were performed using SPSS 20.0 software (IBM, SPSS). The significance of differences between groups was estimated by Student's t test, chi-squared test or Wilcoxon test, as appropriate. Two-sided P-values were calculated, and a probability level of 0.05 was chosen for statistical significance.

3.1 CT gene SPANXC is activated by promoter hypomethylation and is associated with poor prognosis in LUAD

Firstly, by utilizing transcription data from the GTEx, we found that SPANXC was a typical restricted expression in testis tissues (Figure 1A). Through integrating expression data of LUAD from the TCGA data, we found that SPANXC is frequently activated in LUAD patients (Figure 1B). These results indicated that SPANXC was CT gene and it was activated in LUAD.

Next, we inquired into the association between SPANXC activation and tumorigenesis of LUAD. Further analysis found that patients with higher SPANXC expression had a significantly poorer prognosis than those with lower SPANXC expression (HR = 1.74, P = 0.014, Figure 1C). Based on the methylation data of LUAD from TCGA, intriguingly, we found that the activation of SPANXC was due to the promoter hypomethylation of SPANXC (Figure 1D).

3.2 CT gene SPANXC regulates LUAD cell metastasis in vitro

To determine the role of SPANXC in tumorigenesis of LUAD, firstly, we determined the SPANXC expression levels in LUAD cell lines (Figure 2A). Then, we performed knockdown of SPANXC via siRNA in A549 cell lines which originally expressed relatively high SPANXC. After transfection with the specific siRNA (Figure 2B), there was no significant difference between treatment group and control group for cell proliferation and colony formation assay in A549 cells (Figure 2C and 2D). However, transwell assays found that knockdown of SPANXC resulted in significantly decreases in migration and invasion in A549 cells (Figure 2E). Conversely, overexpression of SPANXC can significantly promote cell migration and invasion in NCI-H1299 cells (Figure 3A and 3B).

3.3 SPANXC promotes cell metastasis in vivo

To further determine whether SPANXC affects cell metastasis in vivo, firstly, NCI-H1299 cells stably transfected with SPANXC expression vector by lentivirus (Figure 3C). We found that lentivirus-mediated stably transfected overexpression of SPANXC could promote cell migration and invasion in NCI-H1299 (Figure 3D). Then, NCI-H1299 cells with overexpression of SPANXC or control vector were injected into the tail veins of nude mice. Metastatic nodules on the surface of the liver were counted after 60 days. The mice injected with overexpression of SPANXC cells succumbed to death more rapidly than the control cells (Figure 4A). And the number of metastatic nodules on the surface of the liver was significantly higher than the control cells (Figure 4B and 4C). Taken together, SPANXC exhibits a pivotal role in cell metastasis of LUAD.

3.4 CT gene SPANXC could promote cell metastasis by binding to ROCK1

Our data suggested that SPANXC may play an important role in cell metastasis of LUAD. To explore the mechanism for SPANXC in metastasis of LUAD, we performed co-immunoprecipitation (IP) assays after overexpression of SPANXC, followed by mass spectrometry (MS), to assess the proteins interacting with SPANXC. The results showed that ROCK1 gene was obviously enriched after overexpression of SPANXC than that in control cells (Figure 5A). Then, we further validate the physical interaction between SPANXC and ROCK1 by Co-IP and Western blotting assays (Figure 5B). Moreover, overexpression of SPANXC could indeed induce ROCK1 protein expression (Figure 5C). And we also found that ROCK1 has significant higher expression in LUAD samples after analysing from TCGA unpaired 488 LUAD tumour and 57 adjacent normal tissues (Figure 5D). ROCK1 has been participated in multiple cellular processes and pathologies, especially in metastasis process of tumorigenesis including LUAD.17-19 Our results indicated that SPANXC could promote cell metastasis by binding to ROCK1.

DATA AVAILABILITY

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.