Restoration of mutant K-Ras repressed miR-199b inhibits K-Ras mutant non-small cell lung cancer progression

In Situ Cell Death Detection Kit, 5-azacytidinecytidine, anti-caspase 3 antibody, anti-actin antibody, anti-p70S6K (Thr389), fetal bovine serum (FBS) and cell culture medium were obtained from Sigma (St. Louis, MO). Dual-Luciferase Assay Kit and Invasion Assay Kit were purchased from Promega (Madison, WI) and BD Biosciences (San Jose, CA), respectively. TRIzol, cDNA reverse transcription kit, miRNA luciferase reporter vector, primer sets for miR-199b and U6, Lipofectamine 2000, SYBR Green PCR kit, miR-199b mimics, control oligonucleotides, and miRNA assay kit were purchased from Life Technologies (Carlsbad, CA). Human miRNA Array v2.0 and Human genome U133 Plus 2 array were obtained from Arraystar (Rockville, MD) and Affymetrix (Santa Clara, CA), respectively. shRNA of K-Ras, Renilla Luciferase, and K-Ras (G12D) expression constructs were kindly provided by Dr. Cheng (Moffitt Cancer Center). Antibodies against K-Ras, KSR2, PIK3R1, Rheb1, Akt1, phospho-Akt (Ser473), phospho-ERK (Thr202/Tyr204), phospho-mTOR (Ser2448), and Ki-67 were purchased from Abcam (Cambridge, MA). K-Ras (G12D) antibody was obtained from Cell Signaling Technology, Inc.

H157, H1975, H2172, HCC827, H2122, H441, A549 and H460 were purchased from American Type Culture Collection (Manassas, VA). PC-9 and H125 were kindly provided by Dr. Shen (Jilin University, China). All cell lines were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS. NSCLC specimens were collected before treatment from patients with newly diagnosed NSCLC at Daping Hospital, Third Military Medical University. This experiment was approved by the ethical review committees of Daping Hospital, Third Military Medical University.

Total RNA was isolated from tissues and cells using TRIzol according to the manufacturer’s instructions. miR-199b and U6 were analyzed using a TaqMan miRNA Assay Kit. The relative expression of miR-199b was normalized against U6 expression using the 2^-△Ct method, and the miR-199b expression fold-change in NSCLC tissue was matched to nontumor control samples for evaluation. For other genes, RT and PCR were performed with the cDNA RT Kit and SYBR Green PCR Kit, respectively. Primer sequences are listed in Additional file 1: Table S1. miRNAs affected by K-Ras were detected using Human miRNA Array v2.0 and genes affected by miR-199b were detected using Human genome U133 Plus 2 array.

The 3`-UTR segments of genes that were predicted to interact with miR-199b were amplified by PCR from human genomic DNA and inserted into MluIand HindIII sites of the miRNA Expression Reporter Vector. Luciferase assay was performed using HEK293 cells as previously described [11].

Western blotting and immunohistochemistry were performed as previously described [12]. Western blot band intensity was quantified using Image J software (National Institutes of Health, Bethesda, MD).

Cells were transfected with the indicated oligonucleotides for 24 h and then subjected to analysis. Invasion, cell viability and cell proliferation assays were performed as previously described [11].

After 24 h of oligonucleotide transfection, cells were trypsinized and resuspended in 0.5 ml 0.35% agar in growth medium at a density of 2500 cells/well (6-well plate). Then, the agar-cell mixture was plated on the top of a solid layer of 0.8% agar in growth medium. Colonies were counted 12 days later.

miR-199b effects on K-Ras mutation-driven lung tumorigenesis were examined using 6-week-old female K-Ras^LA1 transgenic mice at Seoul National University, Korea. miR-199b expression plasmids or empty vectors were mixed with the gene delivery nanoparticle, PCA_mH_n, as previously described [13], and this mixture was then delivered to mice using an aerosol-based, nose-only exposure chamber system as previously described [14]. Briefly, mice were randomly divided into three treatment groups (6 mice per group), and then mice were exposed to aerosol containing 10 mg of PCA_mH_n with or without 1 mg plasmid (miR-199b plasmid or empty vector). K-Ras^LA1 mice were exposed to aerosol twice a week for one month.

For the subcutaneous tumor growth assay, 2 × 10⁶ indicated cells in 0.1 ml of phosphate-buffered saline were subcutaneously injected into 6-week-old male nude mice. One month after cell injection, these mice were sacrificed.

For lung metastasis experiments, 5 × 10⁵ indicated cells were suspended in 0.1 ml of PBS and injected into the lateral tail vein of 6-week-old male nude mice. One month after injection, mice were sacrificed, and lung surface tumor foci were counted. This experiment was conducted at the Daping Hospital and Research Institute of Surgery, Third Military Medical University. All animal experiments were approved by the Animal Care and Use Committees of the appropriate institutions.

Genomic DNA was isolated using the QIAGEN DNA extraction kit, and 1 μg of genomic DNA was treated with sodium bisulfite. The bisulfite-treated DNA was desalted and eluted in 40 μl of elution buffer; then, 2 μl of DNA was amplified with the forward primer 5′-TTAAAGAGGTTGGGTATGAG-3′ and the reverse primer 5′-ATCCTCTAATCCATCCAAAC-3′. PCR products were ligated into the TA cloning vector, and the DNA sequences were determined using the following primers: forward primer: 5′-GGAGGAGAGGAGGAAGTT-3′; reverse primer: 5′-CCAACCTATATCCCCCTAAC-3′.

To identify miRNAs that are regulated by mutant K-Ras in NSCLC, we performed miRNA array assays using the K-Ras (G12D)-overexpressing NSCLC cell lines H1975 and H522, as well as their vector control cells. As shown in Fig. 1a, we detected a total of 46 miRNAs that were significantly decreased by K-Ras (G12D) overexpression in both NSCLC cell lines compared to the respective control cells. Among them, miR-199b was the most significantly inhibited. Consistent with miRNA array results, clinical samples and NSCLC cell line analysis results showed that miR-199b was dramatically decreased in K-Ras-mutated NSCLC specimens and cell lines compared to wild- type K-Ras NSCLC specimens and cell lines (Fig. 1b and c). Additionally, we demonstrated decreased expression of miR-199b in K-Ras-mutated NSCLC specimens compared to their adjacent tissues (Fig. 1d). However, we did not observe differences between adjacent tissues and tumors in K-Ras wild-type NSCLC patients (Fig. 1e). These data strongly suggested that mutant K-Ras may be involved in the negative regulation of miR-199b in NSCLC. To test this hypothesis, we measured the expression of miR-199b after knockdown or overexpression of mutant K-Ras (G12D) (Additional file 1: Figure S1) in NSCLC cells with mutant or wild-type K-Ras, respectively. Our data show that knockdown of K-Ras significantly increased miR-199b expression, while overexpression of mutant K-Ras suppressed miR-199b expression (Fig. 1f and g). Taken together, our results support the idea that mutant K-Ras suppresses the expression of miR-199b in NSCLC.

To further validate the effects of downregulated miR-199b expression on NSCLC progression, we performed cell-based experiments where we expressed antisense oligonucleotides in the high-expressing miR-199b NSCLC cell lines H522 and H1975. As expected, inhibition of miR-199b (Additional file 1: Figure S2a) strongly promoted cell proliferation (Fig. 2a), soft agar colony formation (Fig. 2b) and invasion (Fig. 2c) in both H522 and H1975 cells. To further confirm these in vitro experimental results in vivo, nude mice were subcutaneously inoculated with miR-199b-silenced H522 cells and the corresponding control cells. Consistent with the in vitro results, animal experiments showed that inhibition of miR-199b (Additional file 1: Figure S2b) significantly stimulated tumor growth (Fig. 2d) and cancer cell proliferation (Fig. 2e) compared to the vector control.

We also investigated the effects of downregulated miR-199b on lung cancer metastasis by tail vein injection of H522 cells that stably express miR-199b-antisense oligonucleotides into nude mice, and we assessed tumor nodule burden after 1 month. Our data show that inhibition of miR-199b dramatically increased NSCLC cell lung metastasis (Fig. 2f). These in vivo and in vitro data suggest that miR-199b may affects clinical outcome of NSCLC patients. In fact, TCGA dataset analysis showed that decreased miR-199b expression levels were significantly correlated with poor survival rates in patients with lung adenocarcinoma (Fig. 2g). Taken together, these findings support a model where inhibition of miR-199b significantly contributes to NSCLC progression.

Given that the K-Ras mutation inhibits the expression of miR-199b and that the inhibition of miR-199b promotes NSCLC progression, we hypothesized that restoring miR-199b could suppress K-Ras-mutated NSCLC. Cell-based assays on the K-Ras-mutated NSCLC cell lines A549 and H2122 with overexpressed miR-199b (Additional file 1: Figure S2c) showed significant suppression of cell proliferation (Fig. 3a), soft agar colony formation (Fig. 3b) and invasion (Fig. 3c) in both cell lines. Then, we tested the effects of miR-199b overexpression on K-Ras-mutated NSCLC growth in xenograft models that were generated by stably expressing miR-199b in A549 cells (Additional file 1: Figure S2d). We found that overexpression of miR-199b significantly inhibited tumor growth (Fig. 3d) and cancer cell proliferation (Fig. 3e) compared to the vector control. We also confirmed the in vivo anti-metastatic effects of miR-199b using a lung metastasis model that was generated by stably expressing miR-199b in A549 cells. As shown in (Fig. 3f), miR-199b-overexpressing A549 cells formed fewer tumor nodules in the lung than the vector control, demonstrating that miR-199b can inhibit the metastasis of K-Ras-mutated NSCLC cells.

Next, we investigated the effects of miR-199b on K-Ras mutation-driven lung tumorigenesis. To verify the effects of miR-199b on K-Ras mutation-driven lung tumorigenesis, we used aerosol-based, nose-only exposure methods to deliver miR-199b to the lungs of K-Ras^LA1 transgenic mice. To enhance the aerosol gene delivery efficiency, we used the gene carrier particle PCA_mH_n. As shown in Additional file 1: Figure S3a, we delivered a green fluorescent protein (GFP) expression plasmid with the gene carrier, which significantly enhanced the GFP signal compared with plasmid-only controls, suggesting that PCA_mH_n is an efficient gene delivery carrier. In fact, miR-199b expression plasmid delivery with the gene carrier PCA_mH_n significantly increased miR-199b levels in the lung tissues of K-Ras^LA1 transgenic mice compared to the carrier and vector control groups (Additional file 1: Figure S3b). Notably, such delivery of miR-199b to the lungs of K-Ras^LA1 transgenic mice dramatically decreased the numbers of tumors in the lung (Fig. 3g). Histopathologic examination also indicated that pulmonary tumor formation was significantly suppressed by miR-199b delivery (Fig. 3g). In addition, miR-199b delivery significantly inhibited the expression of the cell proliferation marker protein Ki-67 compared to other groups (Fig. 3h). Taken together, restoration of miR-199b can suppress K-Ras mutation-driven lung tumorigenesis, and restoration of miR-199b may be a useful strategy for treating K-Ras-mutated NSCLC.

To investigate the anticancer mechanism of miR-199b, we performed gene array analysis using miR-199b-overexpressing H1299 cells and their control cells. As shown in Fig. 4a, we detected a total 72 genes that were downregulated more than 1.5-fold in miR-199b-overexpressing H1299 cells compared to control cells. Among them, we found that 11 genes contained putative miR-199b target sites in their 3`-UTRs (www.​targetscan.​org) (Fig. 4b). Interestingly, 5 of the genes are involved in the activation of the K-Ras downstream signaling pathway, including K-Ras, Akt1, Rheb1, PIK3R1 and KSR2 (Fig. 4c). To test whether miR-199b regulates the expression of candidate target genes, we examined the expression levels of these targets in stably expressing miR-199b or miR-199b-antisense NSCLC cells. Our results show that miR-199b negatively regulated the expression of all candidate genes, as indicated by both the mRNA (Fig. 4d) and protein levels (Fig. 4e and Additional file 1: Figure S4a). We also observed downregulation of miR-199b candidate target genes in the lung tissues of K-Ras^LA1 transgenic mice in which miR-199b was delivered compared to the lungs of control mice (Figs. 4f and Additional file 1: Figure S4b). Furthermore, we demonstrated a direct interaction between miR-199b and the 3′-UTR of target genes using a miRNA luciferase reporter assay. We expressed luciferase constructs containing the 3′-UTR of miR-199b target genes. In all cases, luciferase activity was reduced in the presence of miR-199b mimics (Fig. 4g). In contrast, luciferase activity was unchanged in negative controls in which putative seed sequences were mutated (Fig. 4g).

We then tested whether miR-199b negatively regulates Akt and ERK signaling in NSCLC. As shown in Fig. 5a and Additional file 1: Figure S5a, overexpression of miR-199b significantly inhibited the phosphorylation of Akt, mTOR, S6K and ERK in NSCLC cell lines, and cleaved caspase-3 levels increased. Consistent with the in vitro results, K-Ras^LA1 transgenic mice experiments also showed that delivery miR-199b can significantly inhibit ERK and Akt signaling activation and induce cancer cell apoptosis (Fig. 5b). In contrast, inhibition of miR-199b significantly stimulated Akt and ERK signaling in NSCLC cells (Figs. 5c and Additional file 1: Figure S5b). Taken together, our findings indicated that miR-199b inhibited Akt and ERK signaling by directly targeting K-Ras and multiple coactivators of Akt and ERK signaling in NSCLC.

Previous studies showed that K-Ras can regulate gene expression by altering DNA methylation [15] and that the expression level of miR-199b is determined by its promoter methylation status [16]. We therefore suspected that mutant K-Ras-dependent suppression of miR-199b expression in NSCLC cells might be due to increased methylation in the miR-199b promoter. As expected, miR-199b expression levels were restored after treatment with the DNA methylation inhibitor 5-azacytidinecytidine in NSCLC cells containing K-Ras mutations (Fig. 6a). Consistent with these results, mutant K-Ras-dependent downregulation of miR-199b was blocked by 5-azacytidinecytidine treatment in wild-type K-Ras NSCLC cells (Fig. 6b). Moreover, bisulfite sequencing and quantitative methylation-specific PCR of the miR-199b CpG island region confirmed that methylation levels of the miR-199b promoter are increased in NSCLC cells overexpressing mutant K-Ras (Fig. 6c). In contrast, the silencing of mutant K-Ras decreased methylation levels (Fig. 6d). These findings indicated that mutant K-Ras inhibits miR-199b expression by increasing the methylation of the miR-199b promoter.