MicroRNA-101 downregulation increases C-Fos expression and contributes to the pathogenesis of non-small cell lung cancer

Tissue samples and cell lines

Eleven pairs of primary NSCLC tissues and matched adjacent tissues were collected from Department of Thoracic Surgery, the Third People’s Hospital of Shenzhen, China from 2009 to 2010 with informed consent and approval. Our studies have been reviewed and approved by the Ethics Committee of the Third People’s Hospital of Shenzhen and all the experiments were carried out in accordance with relevant guidelines and regulations.

NSCLC cell line A549 was obtained from the Institute of Biochemistry and Cell Biology of Chinese Academy of Science (China) and originated from the ATCC (Manassas, VA, USA). All cells were cultured in DMEM with 10% fetal bovine serum, 2 μM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin sulfate.

RNA extraction and quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNAs were then stored at −80°C before analysis.

For miRNA expression, around 10 ng of RNA were converted to cDNA using the ABI miRNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA). U6 gene was used as a normalization control for analysis.

For C-Fos expression, synthesis of cDNA was performed with 1 μg of total RNA per sample with the ABI reverse transcription kit (Applied Biosystems, USA) according to the manufacturer’s manual. QRT-PCR was performed in triplicate for each sample using ABI SYBR Green Master kit (Applied Biosystems, USA) according to the manufacturer’s instructions. GAPDH was used as a housekeeping gene for normalization. The primer sequences used are: (1) C-Fos: 5′-CGTGCCAGACATGGACCTAT-3′ (forward) and 5′-CGGGGTAGGTGAAGACGAAG-3′ (reverse); (2) GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GCATGGACTGTGGTCATGAG-3′ (reverse).

The expression ratio calculation is based on the following calculation strategy.

• MiR-101 expression=2- CT value(miR-101)- CT value(U6).

• C-Fos expression=2- CT value(C-Fos)- CT value(GAPDH).

MiRNA mimics, C-Fos siRNA and transfection

The human miR-101 duplex mimics (miR-101) and negative control oligonucleotide duplex mimics (miR-NC) were designed and obtained from Ribobio (Guangzhou, China). C-Fos siRNA and control siRNA oligonucleotides were purchased from Santa Cruz (Santa Cruz, USA). MiRNA or siRNA transfection was performed using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s protocol.

MiRNA target prediction

Putative miRNA target genes were searched by the website program, www.microRNA.org.

Luciferase assay

A549 cells were transfected with a C-Fos 3′UTR plasmid, Renilla luciferase pRL-TK vector (Promega, Madison, WI, USA) and miR-101 mimics/miR-NC mimics. The transfection is by Lipofectamine 2000 reagent (Invitrogen, USA) following the instruction. Luminescence was then examined 48 h later on the Dual-Luciferase Reporter Assay System (Promega, USA).

Cell viability analysis

Cell viability was determined by the Cell Counting Kit-8 assay (Dojindo, Japan) following the manufacturer’s protocol.

Caspase 3/7 activity

Caspase 3/7 activity was analyzed following the manufacturer’s instructions (Pierce, USA). Briefly, A549 cells were cultured in a white-walled 96-well plate. Before testing, cells were washed with PBS and culture with Caspase-Glo^® 3/7 Reagent at room temperature for 1–2 h. The luminescence of each sample was measured in a plate-reading luminometer (Tecan Infinite M200, Switzerland).

Western blot analysis

For total protein isolation, cells were washed with 1× PBS, lysed with RIPA buffer for 15 min on ice. After centrifugation at 12,000 rpm for 15 min, the supernatants were collected. Proteins extracted from cells were immunoblotted with different antibodies following the protocol. The primary antibodies used were C-Fos (dilution 1:2000) and GAPDH (1:10,000; Cell Signaling Technology, USA).

Statistical analysis

Data were expressed as mean±SEM of three independent experiments. For all statistical tests, PRISM 5.0 (San Diego, CA, USA) was used. Analysis of intergroup differences between two groups was carried out using t-test, while analysis of more than two groups was by ANOVA. p-Values<0.05 were considered statistically significant.

Expression of miR-101 is downregulated in human NSCLC tissues

To investigate the miR-101 expression in NSCLC, 11 cases of human NSCLC tissues along with matched adjacent tissues were recruited and examined. As shown in Figure 1, miR-101 expression was significantly decreased in NSCLC tissues compared to their matched adjacent tissues (p=0.036).

Figure 1:

Expressions of miR-101 in human NSCLC tissues.

Mature miR-101 expression in primary NSCLC tissues and matched adjacent lung tissues were analyzed by real-time PCR and standardized to the endogenous control U6. Y axis represents the expression ratio of miR-101. It is calculated by 2^{- CT value(miR-101)- CT value(U6)} .

MiR-101 suppresses cell proliferation of A549 cells and induces apoptosis

The biological function of miR-101 in NSCLC A549 cells was next under investigation. Mature miR-101 mimics or negative control mimics were transiently transfected into A549 cells by Lipofectamine 2000. The expression of miR-101 exhibited around 18-times of increase of transfection of miR-101 at 24 h (p<0.01) (Supplemental Figure 1). Cell viability was analyzed by the CCK-8 proliferation assay after 48 h. As a result, A549 cells showed decreased cell proliferation after transfection with miR-101 compared with negative control (Figure 2A). These results suggest that miR-101 has an inhibitory effect on the proliferation of A549 cells.

Figure 2:

Effect of overexpression of miR-101 on cell proliferation and apoptosis of A549 cells.

(A) A549 cells were transfected with 50 nM of miR-101 mimics (miR-101) or negative control mimics (miR-NC). After 48 h, cell proliferation was measured by CCK-8 assay. (B) Caspase-3/7 activities were examined by caspase 3/7 assay (**p<0.01).

To further investigate the role of apoptosis in miR-101 function, caspase 3/7 activity in A549 cells was analyzed. Caspase 3/7 activity significantly increased in miR-101-transfected cells (Figure 2B). It indicated the increase of cell apoptosis. Together, these results indicated that the inhibition of cell growth by miR-101 was associated with increased cell apoptosis.

MiR-101 targets C-Fos

To further explore the mechanisms through which miR-101 suppresses A549 cell growth, we analyzed probable downstream genes related to cell proliferation or apoptosis. Computational prediction was used to search for miR-101 likely target genes. Among them, C-Fos was found to have a putative miR-101 binding site in its 3′UTR (Figure 3A). The 3′-UTRs of C-Fos bear binding sites of miR-101 with highly conservative sequences in mammals.

Figure 3:

miR-101 regulated the expression of C-Fos.

(A) Bioinformatic analyses (www.microrna.org) were performed to search for miR-101 target genes. Among them, C-Fos was found to have putative miR-101 binding sites within its 3′UTR. The sequences of predicted miR-101 binding sites are shown. (B) Relative luciferase activity was analyzed after co-transfection of miR-101 or miR-NC with the reporter plasmids along with an endogenous control Renilla luciferase pRL-TK vector in A549 cells. (C) A549 cells were transfected either with miR-101 or miR-NC for 48 h. C-Fos protein levels were examined by Western blots using GAPDH as a loading control (one of two similar blots is shown). Results are shown as mean±SEM (*p<0.05).

To determine whether C-Fos is directly regulated by miR-101 through 3′UTR binding, we performed a dual-luciferase assay to verify the relationship. The 3′UTR fragment with the predicted binding site, was cloned and inserted into the downstream region of a luciferase reporter gene in a pGL3-con vector. A549 cells were co-transfected with C-Fos-3′UTR-reporter plasmid and miR-101 or miR-NC mimic or miR-NC for luciferase activity analysis. As shown in Figure 3B, miR-101 reduced the activity of luciferase containing C-Fos 3′UTR compared to control ones. These results indicate that miR-101 may suppress C-Fos expression by targeting its 3′UTR.

To further investigate the effects of miR-101 on C-Fos gene expression, protein level of C-Fos was analyzed by Western blotting. A549 cells were transfected with miR-101 mimics for 48 h. In A549 cells transfected with miR-101 mimics, the protein expressions of C-Fos were significantly less than that of the negative control of cells (Figure 3C).

Taken together, these results suggest that miR-101 decreased the expression of C-Fos through direct 3′UTR interactions.

Inhibition of C-Fos reduces cell proliferation of A549 cells through apoptosis

Since miR-101 decreased C-Fos expression, we further investigated the function of such alteration. A549 cells were transfected with C-Fos siRNA for 72 h, and then cell viability was examined. As shown in Figure 4A, cell proliferation was inhibited after transfection with C-Fos siRNA. In addition, cell apoptosis was analyzed through caspase 3/7 activity. After knockdown of C-Fos by siRNA, caspase 3/7 activity significantly increased compared with the control cells. It suggests the increase of cell apoptosis.

Figure 4:

Effect of C-Fos on cell proliferation and apoptosis of A549 cells.

A549 cells were treated with C-Fos siRNA for 72 h. (A) Cell viability was detected by CCK-8 assay. (B) Caspase-3/7 activities were examined by caspase 3/7 assay (**p<0.01).

These results are similar to the effect of miR-101 overexpression (Figure 2A and B). It supports the involvement of C-Fos in miR-101-mediated cell growth inhibition and cell apoptosis induction of A549 cells.

C-Fos is significantly upregulated in NSCLC

To further explore the functional role of C-Fos in NSCLC, we examined its expression in clinical samples. A total of 11 cases of human NSCLC tissues along with matched adjacent tissues were examined. As shown in Figure 5, C-Fos expression was significantly (p=0.0086) increased in NSCLC tissues compared to the matched adjacent tissues. These results indicate the opposite expression pattern of C-Fos to that seen for miR-101.

Figure 5:

Expressions of C-Fos in human NSCLC tissues.

C-Fos expression in primary NSCLC tissues and matched adjacent lung tissues were analyzed by real-time PCR and standardized to the endogenous control GAPDH. Y-axis represents the expression ratio of C-Fos. It is calculated by 2^{- CT value(C-Fos)- CT value(GAPDH)}.