Abstract
Purpose
Down-regulation of let-7 microRNA (miRNA) plays an important role in the pathogenesis of lung cancer. k-Ras and c-Myc, two key oncogenes in lung cancer, have been found to be targeted by let-7 in vitro. However, the in vivo relevance of these findings is unknown. The aim of the present study is to determine the effect of let-7a, a member of let-7 family, on the growth of lung cancer in vivo and to investigate whether let-7-induced suppression of k-Ras and c-Myc is involved in lung cancer.
Methods
A549-let-7a cell line and A549-control cell line, two stable transfected cell lines over-expressing let-7a and the control miRNA, were established and preserved in our lab. A549, A549-control, and A549-let-7a cells were injected subcutaneously into nude mice, respectively. After 30 days, the mice were killed; the xenografts were excised and weighed. The expression of let-7a in tumor xenografts was assessed by real-time reverse transcription-PCR (RT-PCR). The expression of k-Ras and c-Myc in xenografts were determined by western blot and immunohistochemistry detection.
Results
Real-time RT-PCR showed the expression of let-7a was increased significantly in A549-let-7a cells-injected group, compared with A549-control cells-injected group and A549 cells-injected group (P < 0.01). In the xenografts of A549-let-7a cells-injected group, a significant depression in tumor weight (P < 0.05) and significant decrease of k-Ras and c-Myc protein were observed (P < 0.01), compared to A549 cells-injected group and A549-control cells-injected group.
Conclusion
Overexpression of let-7a can inhibit the growth of lung cancer transplanted subcutaneously in nude mice by suppression of k-Ras and c-Myc.
Keywords: Let-7a, MicroRNA, Lung cancer, Nude mice, Tumor treatment
Introduction
Lung cancer is the most common cause of cancer deaths around the world. Its incidence continues to increase at a rate of approximately 0.5% per year, and the number of cancer deaths caused by this disease expected to rise to 50% by 2020 (Jemal et al. 2008). Despite recent progress in diagnosis and multimodality therapies for lung cancer, the prognosis still remain unsatisfactory with 5-year survival rates of less than 15%. They highlight the fact that new strategies based on molecular mechanisms are required for lung cancer treatment. Although several genetic or epigenetic changes in critical genes have been implicated to lung cancer, the mechanism relevant to lung carcinogenesis is not fully understood.
MicroRNAs (miRNAs) comprise a class of endogenous, noncoding RNA of 19–24 nt that are found in diverse organisms from plants to animals. Most animal miRNAs are thought to regulate gene expression by imprecisely binding to complementary sequences in the 3′-untranslated regions (UTRs) of their target mRNAs through a currently unknown mechanism (Bartel 2004; Croce and Calin 2005). Recently, it has been reported that miRNAs play a critical role in human cancers including lung cancer (Lu et al. 2005).
The let-7 miRNA, the second member of the miRNA family found in 2000, was initially identified to control developmental timing in C. elegans (Pasquinelli et al. 2000; Reinhart et al. 2000). In humans, the let-7 family, consists of 11 highly conserved miRNAs including let-7a, has been reported to locate in the genomic regions deleted in human cancers (Calin et al. 2004). Microarray analysis of miRNAs demonstrated that down-regulation of let-7 expression occurred in lung cancer cell lines and lung cancer tissues (Takamizawa et al. 2004). Moreover, reduced let-7 expression was significantly associated with shortened postoperative survival and overexpression of let-7 inhibited the growth of a lung cancer cell line in vitro (Takamizawa et al. 2004). Subsequently, Ras oncogene was identified to be targeted by let-7 using reporter gene assay in two cell lines (Johnson et al. 2005). In the following years, c-Myc, HMGA2, and a large number of cell cycle oncogenes were found to be modulated directly by the member of let-7 family in vitro (Koscianska et al. 2007; Lee and Dutta 2007; Mayr et al. 2007; Johnson et al. 2007). Additionally, our previous work showed that NIRF is also a target for let-7 in lung cancer (He et al. 2009). Taken together, previous studies suggested that down-regulation of let-7 is a key event in lung cancer, and the reduction of let-7 caused derepression of some oncogenes, such as Ras, c-Myc, HMGA2, and so on, may contribute to the pathogenesis of lung cancer. However, the in vivo relevance of these findings is not yet clear.
Here, we focused our study on let-7a, a member of the let-7 family. Our data showed that overexpression of let-7a inhibited the tumor growth of human lung cancer transplanted subcutaneously in nude mice. We also showed that reduction of k-Ras and c-Myc protein may be involved in the let-7a mediated growth-inhibitory effect in vivo. The present study may provide the basis information of lung cancer for future therapies.
Materials and methods
Reagents
RPMI 1640 medium and fetal bovine serum were obtained from Gibco (Grand Island, NY, USA). RIPA lysis buffer and BCA protein assay kit were provided by the Beyotime Institute of Biotechnology (Jiangsu, China). PVDF membrane was obtained from Millipore (Billerica, MA, USA). The polyclonal rabbit anti-human antibody for k-Ras, c-Myc and β-actin, were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA). All the other reagents were of analytical grade and obtained from commercial sources.
Cell culture and mice
The human lung adenocarcinoma cell line A549 was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and incubated in 5% CO2 at 37°C. Stable transfected cell lines over-expressing let-7a and the control miRNA, respectively, A549-let-7a and A549-control cells, were derived from the A549 lung cancer cell line as previously described (He et al. 2009). BALB/C nude (nu/nu) mice (6–7 weeks old, SPF degree, 20 ± 3 g) were purchased from Peking University Laboratory Animal Center (Beijing, China). All procedures were conducted according to the internationally accepted ethical guidelines (NIH publication no. 85-23, revised 1985).
In vivo tumor xenograft model
Twenty-four nude mice were divided into three groups randomly. Each group was injected subcutaneously in the backs with the suspension of 200 μl containing 2 × 106 A549 cells, A549-control cells, and A549-let-7a cells, respectively. The mice were observed for the diameters and the growth of tumor xenografts. After 30 days, the mice were killed; the xenografts were excised and weighed. The inhibitory rates of xenografts were calculated according to the formula: inhibitory rate (%) = 1 − mean weight (A549-let-7a cells-injected group or A549-control cells-injected group)/mean weight (A549 cells-injected group) × 100%.
Real-time reverse transcription (RT)-PCR
RNA was extracted from tumor xenografts by Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. TaqMan miRNA assays (Applied Biosystems, CA, USA) were used to measure let-7a expression following manufacturer’s instructions. Briefly, 10 ng of total RNA was reverse transcribed using miRNA-specific looped RT primer, MultiScribe reverse transcriptase, RT buffer, dNTPs, and RNase inhibitor in the GeneAmp 9700 PCR system (Applied Biosystems) under the following conditions: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min. Real-time PCR was performed on the resulting complementary DNA using miRNA-specific forward PCR primers, specific reverse PCR primer, miRNA-specific TaqMan probe and TaqMan Universal PCR Master Mix in a 7500 real-time PCR system as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. RNU24 served as an internal control. Relative fold changes of let-7a expression were calculated by the ΔΔCT method (Livak and Schmittgen 2001) and the values were expressed as 2ΔΔCT.
Western blot analysis
Tumor tissues were washed thrice with ice-cold PBS (pH 7.4), and lysed with ice-cold RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF). The lysate was clarified by centrifugation at 15,000×g for 30 min at 4°C. The resulting supernatant was stored at −80°C until further analysis. And the protein content was determined using the BCA protein assay kit with BSA as the standard. Proteins (20 μg) were denatured at 100°C in loading buffer for 10 min and separated by SDS-PAGE. After electrotransfer, the membrane was probed with rabbit anti-human k-Ras, c-Myc (both 1:500 dilution), and β-actin (1:400 dilution), respectively. Then, blots were incubated with a goat anti-rabbit secondary antibody (1:1,000 dilution) (Zhongshan Goldenbridge Biotechnology, Corp. Beijing, China). After washing, the bounding antibody was visualized using the enhanced chemiluminescence assay kit (Tiangen Biotechnology, Corp. Beijing, China). The intensities of bands indicated by western blot were semi-quantified using Quantity-One 4.4.0 software (Bio-Rad, Hemel Hempstead, UK).
Immunohistochemistry assay
The tumor xenografts were embedded in paraffin and sectioned at 5 μm. After deparaffinization of the sections, antigen retrieval was done by boiling the samples in citrate buffer for 15 min at 92–98°C and trypsinizing with 0.125% trypsin for 15 min at 37°C. The sections were treated with 30 g/l BSA in PBS for 20 min at 37°C to block non-specific staining, and then incubated with rabbit anti-human k-Ras antibody or c-Myc (both 1:50 dilution) at 37°C for 2 h. After incubation with goat anti-rabbit secondary antibodies (IgG/HRP, Ready to use) for 30 min at 37°C, the sections were treated using DAB staining (Zhongshan Goldenbridge Biotechnology, Corp. Beijing, China). The nucleus was counterstained with hematoxylin. The rest of the procedures were performed in accordance with the manufacturer’s instructions. The position where brown color was expressed was positive. The positive signals were measured from ten randomly different fields under the microscope (400×) using the Image-Pro PLUS V6.0.0.260 (Media Cybernetics, Silver Spring, USA).
Statistical analysis
The statistical analysis was performed with SPSS 11.5 software for Windows. Results were expressed as mean ± standard deviation (SD). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically significant.
Results
Overexpression of let-7a in tumor xenografts
To up-regulate the expression of let-7a in tumor xenografts, the mice were randomized into three groups with different treatments, including A549 cells-injected group, A549-control cells-injected group and A549-let-7a cells-injected group. After 30 days, the mice were killed, and their tumors were excised. Then, the expression of let-7a in tumor xenografts was determined by real-time RT-PCR. As shown in Fig. 1, let-7a was significantly increased in the tumor xenografts of A549-let-7a cells-injected group compared to A549 cells-injected group (P = 0.008). No significant differences in the levels of let-7a were observed between the tumor xenografts injected with A549 cells and A549-control cells (P = 0.34). The expression of let-7a was approximately increased by four times in tumor xenografts of A549-let-7a cells-injected group compared with A549 cells-injected group and A549-control cells-injected group. These data indicated that let-7a was over-expressed in the tumor xenografts of mice injected with A549-let-7a cells.
Fig. 1.
Real-time RT-PCR analysis of let-7a expression. Data were expressed as mean ± SD (n = 3). **P < 0.01 versus A549 cells-injected group
Growth-inhibitory effect of let-7a in tumor xenografts
We also investigated the effect of let-7a on the growth of tumor xenografts in nude mice. Mean tumor weight was (1.961 ± 0.124) g for A549-let-7a cells-injected group, (2.894 ± 0.112) g for A549-control cells-injected group and (2.758 ± 0.076) g for A549 cells-injected group (Table 1). A significant depression in tumor weight was observed in the xenografts of A549-let-7a cells-injected group, compared with A549 cells-injected group (P = 0.034). The inhibitory rate of A549-let-7a cells-injected group on tumor growth was 32.24%. No significant difference in tumor weight was found between the A549-control cells-injected group and A549 cells-injected group (P = 0.575). These findings displayed that overexpression of let-7a significantly suppressed tumor growth in vivo.
Table 1.
Mean tumor weight and inhibitory rate in tumor xenografts (mean ± SD, n = 8)
| Group | Mean tumor weight (g) | Inhibitory rate (%) |
|---|---|---|
| A549 cells-injected group | 2.894 ± 0.112 | 0 |
| A549-control cells-injected group | 2.758 ± 0.076 | 4.70 |
| A549-let-7a cells-injected group | 1.961 ± 0.124* | 32.24* |
*P < 0.05 versus A549 cells-injected group
Inhibitory effects of let-7a on k-Ras and c-Myc expression in vivo
We next evaluated the k-Ras and c-Myc protein levels in tumor xenografts. western blot data shown included at least three independent experiments. As shown in Fig. 2, a significant decrease of k-Ras and c-Myc protein were observed in xenografts of A549-let-7a cells-injected group, as compared with A549 cells-injected group (P = 0.001 for k-Ras, P = 0.003 for c-Myc). No significant differences in the levels of k-Ras and c-Myc protein were found between the A549-control cells-injected group and A549 cells-injected group (P = 0.468 for k-Ras, P = 0.397 for c-Myc).
Fig. 2.
Western blot analysis of k-Ras and c-Myc protein. a Representative western blot analysis of k-Ras and c-Myc protein. 1 A549 cells-injected group. 2 A549-control cells-injected group. 3 A549-let-7a cells-injected group. b Quantification of expression data shown in (a). Data were expressed as mean ± SD (n = 3). **P < 0.01 versus A549 cells-injected group
To further confirm the effect of let-7a on k-Ras and c-Myc expression in tumor xenografts, immunohistochemistry assay was performed. Higher gray scale and stronger positive signal by DAB visualization were found in tumor xenografts of A549 cells-injected group and in tumor xenografts of A549-control cells-injected group, on the contrary, weak brown staining were observed in tumor xenografts of A549-let-7a cells-injected group (Fig. 3a). k-Ras protein was localized in the cell membrane and cytoplasm, while c-Myc protein was stained in cell nucleus. Consistent with the results of western blot, 64.67% decrease in k-Ras protein (Fig. 3b) and 73.35% reduction (Fig. 3c) in c-Myc protein were observed in tumor xenografts of A549-let-7a cells-injected group compared to A549 cells-injected group (P = 0.006 for k-Ras, P = 0.003 for c-Myc). These data provided a strong indication that let-7a significantly inhibited the expression of k-Ras and c-Myc in vivo.
Fig. 3.
Immunohistochemisty detection of k-Ras and c-Myc in tumor xenografts (400×). a Representative immunohistochemisty assay of k-Ras and c-Myc protein. b Quantification of expression data shown in (a). The average value was measured from ten randomly different fields under the microscope (400×) using the Image-Pro PLUS V6.0.0.260. Data were expressed as mean ± SD (n = 3). **P < 0.01 versus A549 cells-injected group
Discussion
To date, more than 700 human miRNAs have been identified. Accumulating data demonstrated that alterations in their expression participate in the pathogenesis of human cancer. However, for most miRNAs, the functional consequences of miRNA abnormalities are not clear. Despite the fact that a great deal of computational approaches are being developed to predict regulatory targets of the miRNAs, the results are not fully coincident. The reason for this is mainly due to the differences in certain important details among the computational approaches (Ambros 2004). It is impossible to know which method is more accurate at the present stage. Thus, to gain a better understanding of the role for the altered miRNAs in human tumorigenesis, experimental support is required for the validation of the predicted target genes of the miRNAs, in which let-7 is one of the well-studied miRNAs.
The let-7 miRNA family consists of 11 highly conserved miRNAs in human genome. Let-7 is poorly expressed in lung cancer cell lines and in lung cancer tissues, and overexpression of let-7 can inhibit the growth of lung cancer cells in vitro, indicating that down-regulation of let-7 is a key event in lung cancer (Takamizawa et al. 2004). Supporting the relevance of this is the fact that the 3′-UTR of Ras oncogene (n-Ras and k-Ras) contains multiple let-7 complementary sites, allowing let-7 to regulate the expression of Ras (Johnson et al. 2005). Ras protein levels were inversely correlated with let-7 levels in lung cancer (Johnson et al. 2005), suggesting reduced expression of let-7 and concomitant enhanced expression of Ras may contribute to lung carcinogenesis. Subsequently, c-Myc, another key oncogene of proliferation signal in lung cancer (Robinson et al. 1995) was found to be targeted by let-7 using reporter assay (Koscianska et al. 2007). Although the study showed that let-7c, a member of the let-7 family, directly regulated the expression of c-Myc by binding to the 3′-UTR of c-Myc, other members of the let-7 family may also have similar effect due to the highly conserved sequences (Koscianska et al. 2007). This functional redundancy indicates the existence of a very fine mechanism by which the let-7 miRNA exert gene silencing effects in proliferation pathway. These studies emphasized the potential of let-7 in the target therapy of lung cancer. However, little is known about the in vivo relevance of these findings.
In our previous work, we established A549-let-7a cell line and A549-control cell line, two stable transfected cell lines over-expressing let-7a and the control miRNA, respectively (He et al. 2009). Real-time RT-PCR displayed that the expression of let-7a in A549-let-7a cells was more than five times higher than that in A549 cells, and no significant difference of let-7a expression was found between the A549-control cells and A549 cells (He et al. 2009). In the present study, to investigate the anti-tumor effect of let-7a in vivo, lung tumor xenografts models were generated by subcutaneous injection with A549 cells, A549-control cells and A549-let-7a cells. Mice injected with A549-let-7a cells showed a significant inhibition of tumor xenografts, compared with those injected with A549 cells and A549-control cells (Table 1), implying that overexpression of let-7a can inhibit the tumor growth of human lung cancer transplanted subcutaneously in nude mice. More importantly, decreased k-Ras and c-Myc protein levels were detected in the xenografts of A549-let-7a cells-injected group, compared with those of other two groups (Figs. 2, 3). Our data as well as other previous studies (Johnson et al. 2005; Koscianska et al. 2007) indicated that overexpression of let-7a could suppress the expression of k-Ras and c-Myc, thus leading to the growth inhibition of lung cancer in vivo.
Although k-Ras and c-Myc, two important oncogenes with the ability to induce cell proliferation, have been shown to be critical to let-7-mediated growth-inhibitory effect in lung cancer, it is not eliminated that other genes may also be targeted by let-7 and contributes to survival signaling, considering multiple targets for a given miRNA (Lewis et al. 2005). Indeed, HMGA2, several cell cycle oncogenes and NIRF were also found to be modulated directly by the member of let-7 miRNA in lung cancer (Lee and Dutta 2007; Johnson et al. 2007; He et al. 2009). Therefore, the regulatory pathways controlled by let-7 in lung cancer could be involved in a very complex and intricate network. Future studies are needed to further analyze the crosstalk of the network over the targets for let-7 in the complicated contexts.
In summary, our study showed that let-7a prevented the tumor growth of human lung cancer transplanted subcutaneously in nude mice by suppression of k-Ras and c-Myc expression. It could become a perspective therapeutic target for human lung cancer treatment.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 30800534).
Conflict of interest statement
We declare that we have no conflict of interest.
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