Evaluation of the prognostic values of solute carrier (SLC) family 39 genes for patients with lung adenocarcinoma

The expression profiles of SLC39A genes in NSCLC

The mRNA expression profiles of total 14 SLC39A genes in patients with NSCLC from TCGA database was analyzed using UCSC Xena. As shown in Figure 1A, expression of SLC39A2, 4, 6, 7, 10, 11 and 14 was significantly higher in LUSC tissues than which in normal lung tissues, but SLC39A8, 12, and 13 were expressed higher in normal tissues. For patients with LUAD, mRNA levels of SLC39A1, 3, 4, 5, 6, 7, 9, 10, 11 and 14 were significant higher in cancer tissues than in relative normal samples, but SLC39A8 and SLC39A12 were expressed lower in cancer samples. (Figure 1B)

Figure 1. The box plots showed mRNA expression of total 14 SLC39A families in NSCLC tissues and in normal lung tissues were determined using UCSC database. Expression of SLC39A1-14 is shown in LUSC (A) and LUAD (B). * P<0.05; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; NSCLC, non-small cell lung cancer; SLC39A, solute carrier family 39.

As shown in Supplementary Figure 1, the mRNA expression profiles of SLC39A genes (GSE31210) in patients with LUAD were similar with the results of TCGA-LUAD dataset.

The prognostic evaluation of SLC39A genes in patients with LUSC

The prognostic values of SLC39A genes and LUSC were determined by using the Kaplan-Meier plotter with TCGA-LUSC data. As shown in Figure 2, high level of SLC39A3 and SLC39A4 were significantly associated with inferior OS in patients with LUSC. High expressed SLC39A7 was related to better OS in patients with LUSC. Expression of other SLC39A genes, including SLC39A1, 2, 5, 6, 8, 9, 10, 11, 12, 13 and 14 were not associated prognostic values of LUSC.

Figure 2. The prognostic values (OS) of SLC39A members in patients with lung squamous cell carcinoma using Kaplan-Meier plotter. SLC39A1 – SLC39A14 (A–N). OS, overall survival; SLC39A, solute carrier family 39.

The prognostic evaluation of SLC39A genes in patients with LUAD

The same prognostic analysis of SLC39A genes and LUAD were performed. As shown in Figure 3, high expression of SLC39A1, 2, 4, 5, 7, 8, 11 and 13 was significantly associated with inferior prognostic values in patients with LUAD. High expressed SLC39A6, 10 and 12 were significantly linked with preferable OS. Other SLC39A genes including SLC39A3, 9 and 14 showed no significant influence on prognosis of patients with LUAD. By analyzing the dataset of GSE31210, twelve out of fourteen survival results were consistent with TCGA-LUAD except for SLC39A5 and SLC39A10 (P>0.05, Supplementary Figure 2).

Figure 3. The prognostic values (OS) of SLC39A genes in patients with lung adenocarcinoma by using Kaplan-Meier plotter. SLC39A1 – SLC39A14 (A–N). OS, overall survival; SLC39A, solute carrier family 39.

Subgroup analysis of the prognostic evaluation of SLC39A genes in patients with LUAD

For more significant associations were found between prognostic values with expression levels of SLC39A genes in LUAD than LUSC, further explorations of OS with various clinicopathological features in LUAD based on clinical stages, genders and smoking statues were performed. As shown in Table 1, overexpressed SLC39A1, 4, 5, 7, and 11 was significantly associated with more undesirable prognostic value in patients with Stage I LUAD. In contrast, high expression of SLC39A2, 6, 8, 9, 10 and 12 indicated better prognosis. For patients with Stage II LUAD, high expression of SLC39A1, 2, 4 and 5 suggested inferior prognosis, but high expression of SLC39A6, 8, 9, 10, 12 and 14 were associated with better prognosis. In Stage III LUAD patients, only high expressed SLC39A9 suggested poorer prognosis, but high expressed SLC39A2, 4, 12 and 14 were associated with preferable prognosis.

Regarding the genders, SLC39A1, 2, 4, and 11 high expression suggested worse prognostic values, but SLC39A6, 8, 9, 10, 12 and 13 high expression indicated better prognostic information in female group (Table 2). For male patients with LUAD, SLC39A1, 2, 4, and 11 high expression was significantly linked with worse prognosis, but SLC39A6, 7, 8, 9, 10 and 12 high expression indicated more favorable prognosis.

According to the smoking status, two groups were set as never smoking and smoking (included smoking history). As shown in Table 3, for never smoking patients, high mRNA expression of SLC39A1, 2, 4, 7, 11 and 13 indicated as inferior OS. While SLC39A8, 9 and 10 mRNA high expression was related with better prognostic value. Regarding to smoking patients, high level of SLC39A1, 2, 4, 5, 7, 11 and 13 suggested more undesirable prognosis, but increased expression of SLC39A6, 8, 9, 10 and 12 indicated better prognosis.

SLC39A7 plays an essential role for LUAD cell survival

The survival data of LUAD cell lines was downloaded from Project Achilles. As shown in Figure 4A, the CERES scores suggested total 27 out of 31 LUAD cell lines were less than -1 by silencing SLC39A7 gene expression with the CRISPR-Cas9 system (NCIH838, NCIH1944, NCIH2023, A549, HCC515, EKVX, NCIH1650, HCC827, NCIH1648, HCC827GR5, NCIH2122, LXF289, NCIH1666, HCC2935, SW1573, NCIH1437, NCIH2291, A427, PC14, NCIH1975, ABC1, NCIH1792, NCIH3122, HOP62, HCC44, HCC461, NCIH2030), which demonstrated that SLC39A7 gene is essential for cell survival in LUAD. Conversely, as shown in Supplementary Table 1, other SLC39A genes were not associated with the cell survival of LUAD (CERES scores > -1). To further investigate the relationship between SLC39A7 and cell growth of LUAD, a special knockdown assay targeting SLC39A7 was performed. Western bolt showed SLC39A7 was downregulated with siR-SLC39A7 transfected in A549 cells (Figure 4B). As shown in Figure 4C, the cell viability of A549 cell was significantly inhibited with siR-SLC39A7 treatment over the time (P<0.001). Furthermore, we investigate the role of siR-SLC39A7 in cell colony formation. When knocking down SLC39A7 expression, the size and number of cell colony was significantly inhibited (Figure 4D). Above all, these results indicated an important role played by SLC39A7 for LUAD cell survival.

Figure 4. SLC39A7 played a vital role in cell survival of lung adenocarcinoma. (A) CERES values of SLC39A7 in various lung adenocarcinoma cell lines, the score less than -1 means the gene is important for cell survival. (B) After 48 h transfected with control siRNA or siRNA-SLC39A7, A549 cells were harvested. The lysates were undergone for detecting SLC39A7 by Western blotting. (C) After seeding, A549 cells were transfected with control siRNA or siRNA-SLC39A7. The CCK-8 assay was performed at 0.5, 1.5, 2.5 and 3.5 days after transfection. The cell viability of siRNA-SLC39A7 treatment was normalized to control group. (D) A549 cells were transfected with control siRNA or siRNA-SLC39A7. Then the quantitative cells were placed and cultured for 14 day, and stained with crystal violet-glutaraldehyde solution. All experiments performed in triplicate, and the results are calculated using mean ± standard deviation. One-way ANOVA or Student t test was carried out for evaluating the statistical significance between groups. *** P<0.001. SLC39A, solute carrier family 39.

High expression of SLC39A7 is associated with smoking

Smoking is a critical inducing factor for lung cancer. To determine SLC39A7 expression was whether associated with smoking, the TCGA-LUAD data was analyzed. As shown in Figure 5A, 5B, SLC39A7 was found highly expressed in current smoker patients than those never smoking or only having smoking history. However, no significant association was found between lifelong non-smoker, history smoker (>15 y) and history smoker (≤15 y). (Figure 5C) In addition, SLC39A7 level was not associated with the current smoking years. (Figure 5D) These results suggest that expression of SLC39A7 could be provisionally increased by smoking.

Figure 5. SLC39A7 expression in lung adenocarcinoma was associated with smoking statues. TCGA-LUAD dataset was downloaded and analyzed. (A) Expression of SLC39A7 in current smokers or lifelong non-smokers. (B) Expression of SLC39A7 in current smokers or history smokers. (C) Expression of SLC39A7 between lifelong non-smokers, history smokers (>15 y) and history smokers (≤15 y). (D) SLC39A7 expression based on smoking duration time (y) analyzed by using UCSC databases. SLC39A, solute carrier family 39.

GO enrichment analysis for SLC39A7

To explore cell functional change by SLC39A7 expression in LUAD cells, a GO Enrichment Analysis was performed. As shown in Figure 6A, total 301 DEGs were identified according to the expression level of SLC39A7 and the heat-map was given. As shown in Figure 6B, GO enrichment analysis based on cellular component (CC) was given. The most valuable top 10 were endoplasmic reticulum lumen, integral component of lumenal side of endoplasmic reticulum membrane, MHC class II protein complex, MHC protein complex, clathrin-coated endocytic vesicle membrane, clathrin-coated endocytic vesicle, lumenal side of endoplasmic reticulum membrane, clathrin-coated vesicle, endocytic vesicle, and endocytic vesicle membrane.(Table 4) For biological process (BP) analysis, high expression of SLC39A7 mainly enhanced multicellular organismal homeostasis, positive regulation of cell-cell adhesion, and maintenance of gastrointestinal epithelium. (Table 4)

Figure 6. GO enriched analysis, GSEA analysis and co-expression analysis of SLC39A7 in lung adenocarcinoma. (A) Heat-map showed the DEGs of SLC39A7. (B) GO enriched analysis (CC) for SLC39A7. (C) GSEA of Wnt/β-catenin signaling pathway. (D) Co-expression of SLC39A7 and RRBP1 by using cBioportal database. (E) RRBP1 expression of mRNA in LUAD tissues and relative normal tissues in Ualcan database. (F) Kaplan-Meier plotter was used to evaluate prognostic value (OS) of RRBP1 in lung adenocarcinoma. A549 cells were transfected with a control siRNA or siRNA-SLC39A7 (G)/ RRBP1 (H). Western blotting was performed to detect the expression of SLC39A7 and RRBP1. (I) Immunohistochemistry assays were performed to detect expression and distribution of SLC39A7 and RRBP1 proteins in lung adenocarcinoma tissues. DEG, differentially expressed genes; GSEA, Gene Set Enrichment Analysis; RRBP1, ribosome binding protein 1; SLC39A, solute carrier family 39.

The GSEA analysis of SLC39A7 in LUAD

The Hallmark effect gene sets was evaluated by GSEA analysis. As shown in Figure 6C, the Wnt beta-catenin was significantly associated with SLC39A7 high expression (NES=1.93, NOM p=0.023, FDR q=0.23). Other affected signal pathways included fatty acid metabolism (NES=6.37, NOM p<0.001, FDR q=0.01), estrogen response late (NES=6.38, NOM p=0.0014, FDR q=0.05) and coagulation (NES=4.73, NOM p=0.0014, FDR q=0.027).

SLC39A7 and RRBP1 were co-expressed in LUAD patients

To further study the potential mechanism of SLC39A7 in LUAD, a co-expressed data was analyzed by using cBioportal database. Totally, 9623 genes were identified with the co-expression profile of SLC39A7 in patients with LUAD, and RRBP1 was a correlated gene. (Figure 6D, p=2.565E-33) Further analysis using UALCAN database indicated that RRBP1 was also high expressed in LUAD tissues than in corresponding normal lung tissues. (P<1E-12, Figure 6E) In addition, over-expressed RRBP1 was associated with poorer prognosis in patients with LUAD. (P=2.2E-16, Figure 6F) To determine the expression relationship between SLC39A7 and RRBP1, siR-SLC39A7 or siR-RRBP1 were transfected into A549 cells for 48 h. As shown in Figure 6G, 6H, knocking down of SLC39A7 cannot inhibit the expression of RRBP1. On the contrary, knocking down of RRBP1 significantly reduce the expression of SLC39A7. To further certificate the above results, we investigate the expression and distribution of SLC39A7 and RRBP1 in LUAD patients. Totally 58 LUAD cases were included and investigated from Renmin Hospital of Hubei Province. As shown in Figure 6I, both SLC39A7 and RRBP1 were mainly expressed in cytoplasm. According to the expression strength of SLC39A7, all patients (58 cases) with LUAD were divided into SLC39A7-High (32 cases) and SLC39A7-Low groups (26 cases). In SLC39A7-High group, 25 samples showed RRBP1-positive. In SLC39A7-Low group, 4 samples presented RRBP1-positive. Protein expression of SLC39A7 was positively correlated with RRBP1 protein (P<0.001). These results suggested that SLC39A7 could be associated with the RRBP1 signaling pathways in LUAD.

The protein expression of SLC39A genes in LUAD

The mRNA level could not be completed reflect associated protein expression. Therefore, we investigated the protein profiles in LUAD patients from CPTAC by using UALCAN database. SLC39A4, 7, 10, 11 and 14 in cancer tissues were significantly highly expressed compared with in normal tissues.(P<0.05, Figure 7A, 7C, 7E–7G) But, expression of SLC39A6 and SLC39A8 were higher in normal samples than in cancer samples. (P<0.05, Figure 7B, 7D) Other SLC39A genes were not investigated in this database. Except for SLC39A6, other results were consistent with which in mRNA levels. Representative immunohistochemical staining specimen of SLC39A7 in LUAD and normal lung tissue was presented in Figure 7H. The expression of SLC39A7 was significantly higher in tumor tissues than which in normal lung tissue.

Figure 7. Protein expression of SLC39A genes in patients with lung adenocarcinoma. (A) SLC39A4. (B) SLC39A6. (C) SLC39A7. (D) SLC39A8. (E) SLC39A10. (F) SLC39A11. (G) SLC39A14. (H) Immunohistochemical staining of SLC39A7 proteins in lung adenocarcinoma tissue specimens. * P<0.05; SLC39A, solute carrier family 39.

Kaplan-Meier plotter

Kaplan-Meier plotter (http://kmplot.com/analysis/index.php?p=background) provides prognostic evaluation of more than 54000 genes in total twenty-one cancer types from TCGA databases. [37] The association between overall survival (OS) of patients with NSCLC and mRNA expression profiles of SLC39A genes was analyzed using the Kaplan-Meier plotter. Then, a stratified analysis was performed based on relatively clinicopathological features to determine the association between the OS of patients with LUAD and SLC39A genes with this tool. A logrank P value and hazard ratio (HR) with 95% confidence intervals were calculated in this analysis. To perform the prognostic evaluation, all cases were divided into two groups automatically with selecting best cutoff.

Cancer virtual cohort discovery analysis platform (CVCDAP) analyzed tool

Key features of CVCDAP (https://omics.bjcancer.org/cvcdap/home.do) is a comprehensive online analyzed tool which contains twenty-nine inbuilt tools which provided analysis of mRNA and protein levels and clinicopathological data in TCGA datasets. The differentially expressed genes (DEGs) and visualized heatmap based on expression levels of SLC39A7 were performed in this web (Log2 fold change=1, FDR=0.05).

cBioportal for cancer genomics

The relative clinical and mRNA expression data of TCGA-LUAD were downloaded for cBioportal for Cancer Genomics (http://www.cbioportal.org/). Additionally, the co-expression data were also analyzed by using cBioportal for Cancer Genomics.

GEO datasets analysis.

The expression data as well as clinical information of GSE31210 (contain 226 primary LUADs and 20 normal lung tissues) were obtained from Gene Expression Omnibus (GEO) datasets (http://www.ncbi.nlm.nih.gov/gds/) and used to evaluate the relative mRNA expression profiles of SLC39A genes in LUAD patients and normal lung tissues. Sangerbox (http://sangerbox.com/AllTools?tool_id=9698927), a comprehensive tool, was utilized to perform single factor survival analysis (Cox regression).

Gene set enrichment analysis (GSEA) of SLC39A7

To determine the molecular function of SLC39A7 in LUAD, a GSEA analysis (http://www.broad.mit.edu/gsea) with the Hallmark effector gene sets was performed based on the mRNA level of SLC39A7 in the TCGA-LUAD dataset. [38]

Gene ontology (GO) enrichment analysis. GO

Enrichment Analysis were performed by using R for Windows version 3.5.2. The library packages included “DOSE”, “clusterProfiler”, “stringr”, and “GOplot”.

Scores of project Achilles

By using CRISPR-Cas9 technology, Project Achilles systematically explores and identifies numbers of genes which are essential for survival of cancer cell lines, and provides CERES dependence scores. [39] To explore the importance of SLC39A genes for LUAD cell survival, we obtained CERES dependence scores from Depmap portal (https://depmap.org/portal).

Cell lines and reagents

A549 cell line was kindly provided by Prof. Xiaoping Sun of Wuhan University. The A549 cell line was cultured at humidified atmosphere of 5% CO₂ and 37° C and grown in DMEM media (HyClone; USA) with 10% FBS (Gibco; USA) and 1% antibiotic (penicillin/ streptomycin, Sigma; USA).

Cell transfection

The special small interfering RNA (siRNA, SLC39A7, 5’-ACAAGAAAGGCAACAAUUCCAGAAUUGUUGCCUUUCUUGUCG-3’; ribosome binding protein 1(RRBP1), 5’-UGUUUUCUCCUUCUUUUUCUUGAAAAAGAAGGAGAAAACAGU-3’ GenePharma, Co., Ltd.) or a non-specific control was transfected into A549 cells using X-treme GENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions.

Cell viability assay

A CCK-8 assay (Dojindo Molecular Technologies; Japan) was performed to determine the survival rate of A549 cells treated with or without siRNA-SLC39A7. The A549 cells were added to 96-well plates and cultured in 5% CO2 at 37° C. After seeding, the cells were transfected with siRNA. After 0.5, 1.5, 2.5, 3.5 days transfection, 10 μl CCK-8 solution was added and cultured with these cells at 37° C for 1 h. The OD value was measured at 450 nm by using an absorbance microplate reader (ELx800, BioTek Instruments; USA).

Colony-formation assay

A549 cells were transfected with control siRNA or siRNA-SLC39A7. Then cells were digested and seeded in 35 mm dishes. After culturing in 5% CO2 at 37° C for 14 days, cells were stained with 0.6% crystal violet-glutaraldehyde solution for 1 min. Lastly, the number of cell colony was counted and photographed.

Western blot analysis

After 48 h transfected with siRNA, A549 cells were undergone for a Western blot assay. [40, 41] Cells were collected and homogenized in modified RIPA lysis buffer (Beyotime, China) with 0.5% protease inhibitor cocktail (Roche Diagnostics) and stayed on ice for 10 min. After centrifuging at 12,000 rpm, the supernatants were obtained and sonicated for 15 sec. The supernatants were quantified by BCA assay (BioRad Laboratories, USA). Then, the extracts were boiled with 5X loading buffer [Chunfeng Lv, China]. Twenty 20 μg total protein were electrophoresed through 10% SDS-PAGE gels and, then transferred onto the PVDF membrane. After blocking nonspecific binding sites with 5% milk in PBS for 1 h, membranes were incubated with primary antibodies of GAPDH (cat no. 5174; 1:2,000; Cell Signaling Technology, Inc; USA); RRBP1 (cat no. ab224354, 1:1000, Abcam, UK) or SLC39A7 (cat no. ab254566; 1:1000; Abcam, UK) overnight at 4° C. Then, the membranes were cultured with secondary antibodies conjugated with horseradish peroxidase at room temperature for 1 h. Last, the membranes were treated with ECL reagent for developing films at darkness.

Tissue sample and immunohistochemistry

Totally, 58 cases of LUAD were included in this study. The involved tumor patients were come from the Renmin Hospital of Wuhan University and definitely diagnosed in department of pathology in 2017. Immunohistochemistry was performed to detect the expression and distribution of SLC39A7 or RRBP1 in LUAD. Briefly, the paraffin-embedded sections were deparaffinized, restored and quenched. After blocking with goat serum for 1 h, sections were incubated with primary antibodies against SLC39A7 (cat no. ab254566, 1:1000, Abcam, UK) and RRBP1 (cat no. ab224354, 1:1000, Abcam, UK) overnight at 4° C. Then, the sections were washed with PBS and incubated with secondary antibody (MaxVision™ Kits, MXB, China) with horseradish peroxidase-conjugated polymer for 15 min. Subsequently, tissue section was stained with a DAB for 1 min. Finally, sections were counterstained with Harris hematoxylin for 20 s.

The interpretation of immunohistochemical staining results for SLC39A7 and RRBP1 were performed by two independent and unwitting researchers (HZ and JY) in our group. According to the staining intensity of SLC39A7 in tumor cells, all cases were classified into 4 catalogues as following: scores 0 = lacking, 1 = mild, 2 = moderate and 3 = strong. For analyzing, all patients were divided into two groups according to the scores of SLC39A7: SLC39A7-L (score value = 0, 1 and 2) and SLC39A7-H (score value = 3). The expression levels of RRBP1 were directly divided into two groups: positive group (expressed) and negative group (absent).

Statistical analysis

All results were calculated with three repeated experiments and presented as mean ± standard deviation (SD). Student t test or one way ANOVA (Newman-Keuls post analysis) was selected to evaluate statistical significance in two groups or more than two groups, respectively. The statistical significance between SLC39A7 and RRBP1 expression profiles in LUAD samples was determined using Chi-square test. The threshold of statistical significance was limited as P-value <0.05. All statistical analysis was performed with SPSS software.