Cloning and characterization of the 5 ' flanking region of microRNA let-7 a-1 / let-7 f-1 gene cluster in human lung cancer cell

In order to elucidate the molecular basis of microRNA let-7a-1/let-7f-1 gene cluster, the transcription initiation site which was determined by 5’ rapid amplification of cDNA ends (5’RACE) and 2.1 kb of the 5' flanking region proximal to the pre-let-7a-1 was isolated and characterized. The promoter activity of the 2.1 kb fragment was analyzed by a firefly luciferase-encoding gene expression vector (pGL3) transiently transfected into lung cancer cell line A549. The 2.1 kb promoter of let-7a-1/let-7f-1 displayed a lower activity and was significantly enhanced by ectopic expression of c/EBPα or p53 and treatment with dexamethasone. Despite the induction of other let-7 family members such as let-7a-3, let-7c and let-7d, all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA) display little enhancement effect on 2.1 kb promoter of let-7a-1/let-7f-1, as well as 1,25-(OH)2D3.


INTRODUCTION
MicroRNAs (miRNAs) are non-coding small RNAs found in diverse organisms (Chen, 2005;Hammond, 2006).They are encoded in long primary forms in the nucleus (pri-miR).The pri-miRs will be transported into the cytoplasm after being processed to 70 to 80 nucleotide pre-miR with a characteristic hairpin structure.The pre-miR are then processed by Dicer into the 22 to 25 nt mature forms (Lee et al., 2002).miRNAs mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation and thus, play key roles in regulating a wide array of cell functions * Corresponding author.E-mail: chenweiwen@sdu.edu.cn.
#These authors contributed equally to this work.(Ambros, 2003).Concordant with this, aberrant expression of miRNA genes could lead to human disease.In recent years, many reports have shown that miRNAs regulate cell proliferation and apoptosis and play a role in cancer: microRNAs can act as oncogenes or tumor suppressors (Chen, 2005;Hammond, 2006).
There are accumulating evidences that let-7, the first discovered miRNA, is tumor suppressor.A number of studies have demonstrated that let-7 was implicated in various cancers, particularly in lung cancer (Eder and Scherr, 2005;Lu et al., 2007;Sampson et al., 2007;Motoyama et al., 2008).Takamizawa et al. (2004) first reported that the expression of the let-7 microRNA was reduced in human lung cancers and overexpression of let-7 in A549 lung adenocarcinoma cell line inhibited lung cancer cell growth in vitro.Next, Johnson et al. (2005 identified that ras is one of the target genes of let-7 family.Lee and Dutta (2007) also found that let-7 repressed the HMGA2 oncogene in human lung cancer cells.Therefore, let-7 is tumor suppressor microRNA and maybe a potential target for lung cancer therapy.Despite this interest, the underlying molecular mechanisms of let-7 reduction in lung cancer are not well understood.The qRT-PCR results from different investigators have shown the significant down-regulation of pri-let-7 in lung cancer tissues and cells (Takamizawa et al., 2004;Yanaihara et al., 2006), suggesting the inhibition on transcription level maybe one of the mechanisms for let-7 expression reduction in lung cancer.But our understanding of let-7 transcription pattern in lung cancer cells is limited.Thus, cloning and characterization of the 5' flanking region of let-7a-1 and let-7f-1, which are the most abundant species of let-7 family and clustered within a few hundred bases in the human genome (Lagos-Quintana et al., 2001), may well be a start point for elucidating the transcription regulation mechanism of let-7.

Cell culture
The human lung cancer cell line A549 (ATCC-American Type Culture Collection) were cultured with F-12K (Gibco, BRL Gaithersburg, MD, USA) medium containing 10% fetal bovine serum, plus 100 u/ml ampicillin and 100 u/ml streptomycin at 37°C with 5% CO2.Within 24 h of passage, cells with more than 70 to 80% confluence were used for transfection.

Amplifying and cloning a 2.1 kb fragment of the 5' flanking region of the let-7a-1/let-7f-1 gene cluster
Human genomic DNA was extracted from white blood cells using the method of rapid isolation of mammalian DNA.The primer pair PF (5'-CCGCTCGAGACCCAGCCATGTTCAGTTCT -3'; with an Xho I site at its 5' end) and PR (5'-CCCAAGCTTCAGTGAA GAGAACATCCAGG-3'; with a Hind III site at its 5' end) were used to amplify the 5' flanking region of the let-7a-1/let-7f-1 gene cluster from the extracted human genomic DNA by PCR.The PCRamplified fragment was 2123 bp (-1999 to +124 bp) and it was excised with Xho I and Hind III (TaKaRa) and ligated into the equivalent site of the pGL3-Basic vector (Promega, Madison WI, USA) to form the let-7a-1/let-7f-1 cluster promoter-luciferase reporter construct, designated pGL3-2123.The resulting construct was confirmed by restriction enzyme digestion and sequence analysis using the general primers Rvprimer3 and GLprimer2.

Treatment of the transfected cells with hormones
The stocks of all-trans retinoic acid (ATRA), 9-cis-retinoic acid (9cRA) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were prepared in dimethylsulfoxide (DMSO); the stock of dexamethasone (DEX) were prepared in ethanol.All of the hormones were purchased from Sigma (St. Louis, MO, USA).After the transfection of pGL3-2123 in 24-well plates, the cells were treated for 48 h with 10 -6 ~10 -8 M of ATRA, 9cRA, 1,25(OH)2VitD3 or DEX in 10% FBS-F-12K medium, respectively.The controls received the DMSO or ethanol vehicle at a concentration equal to that of the treated cells.All the cells underwent the dual-luciferase reporter assay 48 h after the completion of the treatment procedure.

Dual-luciferase reporter assay
The activities of firefly luciferase in pGL3 and Renilla luciferase in pRL-TK (Promega) were determined following the dual-luciferase reporter assay protocol recommended by Promega.The cells were rinsed with PBS after harvest and cell lysates were prepared by manually scraping the cells from the culture plates in the presence of 1×PLB (passive lysis buffer).20 µl of cell lysate was transferred into luminometer tubes containing 100 µl LAR.Firefly luciferase activity (M1) was measured first and then Renilla luciferase activity (M2) was measured after the addition of 100 µl of Stop and Glo Reagent.

Statistical analysis
Data are expressed as mean ± SD of at least three independent experiments.Statistical significance of differences between groups  The results are expressed as the relative luciferase activities (M1/M2), that is, the ratio of firefly luciferase activity (M1) in the pGL3 plasmid and Renilla luciferase activity (M2) in the pRL-TK plasmid.
was analyzed by unpaired Student's t test and P < 0.05 was considered to be statistically significant.

Cloning and activity assay of the 2.1 kb promoter fragment of let-7a-1/let-7f-1 cluster
Based on the result of 5'RACE, starting 2.1 kb upstream of pre-let-7a-1, a genomic fragment including positions -1999 to +124 relative to the transcription start site was cloned into the pGL3-Basic to form pGL3-2123, which was confirmed by DNA sequencing.To evaluate the promoter activity, the pGL3-2123 was transfected into lung cancer cell line A549, pGL3-Control and pGL3-Basic were also introduced into parallel wells, respectively, as positive and negative controls.The luciferase activity was assayed and normalized to pRL-TK (Figure 2).After 48 h of transfection into A549 cells, pGL3-2123 yielded a result of 5.24 by dual-luciferase reporter assay (M1/M2).Comparing with respective 38.76 of pGL3-Control and0.26 of pGL3-Basic, the 2.1 kb promoter of let-7a-1/let-7f-1 cluster display a lower activity in A549 cells, which was consistent with the fact that A549 cell line has the reduced expression of endogenous let-7a-1/let-7f-1 (Takamizawa et al., 2004), suggesting the inhibition of promoter may be one of the reasons for reduced expression of let-7a-1/let-7f-1 in lung cancer cells.
c/EBPα, one member of the c/EBP family, is a gene thought to be a tumor suppressor (Timchenko et al., 1996;Lekstrom-Himes and Xanthopoulos, 1998;Pabst et al., 2001).As a transcription factor, c/EBPα is discovered controlling the transcription of some tumor suppressor microRNA.Recently, the c/EBPα is reported to directly interacted with the mir-122 (hepatocyte-specific microRNA) promoter and transactivate it (Zeng et al., 2010).Studies in acute myeloid leukemia (AML) have shown that mir-34a and mir-223 were transcriptional targets of c/EBPα (Pulikkan et al., 2010(Pulikkan et al., , 2010)).In A549 and H1299 lung cancer cells, the ectopic c/EBPα expression increases expression of mir-1 6.1-fold and 4.92-fold, respectively (Nasser et al., 2008).mir-661, which can inhibit metastatic tumor antigen 1, is another c/EBPα target (Reddy et al., 2009).In our experiment, the expression of ectopic c/EBPα significantly increase the promoter activity of let-7a-1/ let-7f-1 cluster (3.6-fold) in A549 cells with little endogenous c/EBPα expression (Li Figure 3.The effects of some transcription factors on promoter activities of let-7a-1/ let-7f-1 cluster.A549 cells were co-transfected with pGL3-2123 and eukaryotic expression plasmids of c/EBPα, PPARγ2, NF-κB p50, Sp1, p53, ras or myc.The control cells were co-transfected with pGL3-2123 and corresponding empty eukaryotic expression plasmids.All the cells were harvested for the dual-luciferase reporter assay after 48 h of transfection.The results were expressed as ratio of relative activity, that is, the M1/M2 value from pGL3-2123 co-transfected with eukaryotic expression plasmids was divided by the M1/M2 value from controls.**P < 0.01, compared with the control.et al., 1995), suggesting that the let-7a-1/ let-7f-1 cluster is a new transcriptional target of c/EBPα.p53 is a broad-spectrum tumor suppressor gene that plays very important roles in various cancers.The P53 protein functions as transcription factor to transactivate expression of a number of target genes to promote tumor suppression and genome integrity.Recently, a number of studies have shown that many miRNAs are also members of p53-network.For example,all of which function as tumor suppressors and play a key role in control of tumor progression, angiogenesis and metastasis, are shown to be direct transcriptional targets of p53 (Hermeking, 2007;Tarasov et al., 2007;Boominathan, 2010).Besides mir-34a, Tarasov et al. (2007) also observed that activation of p53 in lung cancer cells results in increased expression of mature-let-7c (2.7-fold), mature-let-7e (2.1-fold) and mature-let-7a (1.9-fold).Our results further suggest that p53 increase the expression of let-7a-1/let-7f-1 at transcriptional level, but maybe due to the endogenous expression of wt p53 by A549 cells (Jia et al., 1997), the ectopic expression of p53 had a weaker increase effect on let-7a-1/let-7f-1 promoter than that of c/EBPα.

The effects of hormones on promoter activity of let-7a-1/ let-7f-1 cluster
Besides the transcription factors mentioned earlier, two RAR-RXR heterodimer binding sites (-46/-22 and +90/+114), four VDR-RXR heterodimer binding sites (-1955/-1931, -1691/-1667, -1600/-1576 and -1592/-1568), three glucocorticoid responsive and related elements (GRE) (-1877/-1859, -1588/-1561 and -1416/-1398) were detected by MatInspector 2.0 software.The RAR (retinoic acid receptor), RXR (retinoid X receptor) and VDR (Vitamin D3 receptor) belong to the nuclear receptor superfamily of ligand-inducible transcription (Stunnenberg, 1993).The RAR and RXR mediate the effect of retinoids signals such as all-trans-RA (ATRA) and 9-cis-RA (9cRA), which influence processes such as growth and differentiation by regulation of target gene expression at the cellular level (Gudas, 1994).Retinoids suppress carcinogenesis in diverse epithelial tissues including lung.Clinical trials have demonstrated the efficacy of retinoids in suppressing lung cancer (Aapro, 1995).Several lines of evidence have shown that treatment with retinoic acid not only down-regulate miRNAs that function as oncogenes, but also up-regulate tumor suppressor miRNAs including members of let-7 family (Garzon et al., 2007;Careccia et al., 2009;Weiss et al., 2009).For example, acute promyelocytic leukemia (APL) successfully treated with ATRA showed upregulation of let-7c (Careccia et al., 2009), as well as Garzon et al. (2007) found up-regulation of let-7a-3, let-7c and let-7d by miRNA microarrays and qRT-PCR in APL patients and cell lines during ATRA treatment.To explore whether the retinoic acids are implicated in Figure 4.The effects of 9cRA and ATRA on promoter activity of let-7a-1/ let-7f-1 cluster.A549 cells transfected with pGL3-2123 were treated with 10 -8 M~10 -6 M of 9cRA and ATRA for 48 h, as well as parallel wells transfected with pGL3-Basic as negative control.The cells were harvested for the dual-luciferase reporter assay to detect the effects of hormones on promoter activity of the let-7a-1/ let-7f-1 cluster.The results were expressed as ratio of relative luciferase activity.controlling expression of let-7a-1/ let-7f-1 cluster, the A549 cells transfected with pGL3-2123 were treated with 10 -8 ~ 10 -6 M of 9cRA or ATRA for 48 h.As shown in Figure 4, the 2.1 kb promoter activity of let-7a-1/ let-7f-1 cluster did not display significant increase after treatment with 9cRA as well as ATRA.
The VDR, upon activation by 1,25(OH) 2 D 3 , forms a heterodimer with the RXR and binds to corresponding hormone response elements on DNA resulting in expression of specific geneproducts (Stunnenberg, 1993).1,25-(OH) 2 D 3 exerts important effects on cellular proliferation and differentiation and has been shown to decrease the growth of many cancers (Walters, 1992;Campbell et al., 1997).To date, a few studies have revealed the interaction between 1,25-(OH) 2 D 3 signal pathway and miRNAs (Mohri et al., 2009;Essa et al., 2010;Xi et al., 2010).In our study, 1,25-(OH) 2 D 3 , like retinoic acids, have no significant effect on 2.1-kb promoter of let-7a-1/ let-7f-1 cluster (Figure 5), suggesting the little possibility that the expression of let-Figure 6.The effects of DEX on promoter activity of let-7a-1/ let-7f-1 cluster.A549 cells transfected with pGL3-2123 were treated with 10 -8 M~10 -6 M of DEX for 48 h, as well as parallel wells transfected with pGL3-Basic as negative control.The cells were harvested for the dualluciferase reporter assay to detect the effects of hormones on promoter activity of the let-7a-1/ let-7f-1 cluster.The results were expressed as ratio of relative luciferase activities (M1/M2).**P < 0.01, compared with the control.
Despite the 1,25-(OH)2D3 and retinoic acids, the glucocorticoid signal dexamethasone enhanced the promoter activity of let-7a-1/ let-7f-1 1.91-fold, 2.38-fold and 2.54-fold at concentration of 10 -8 M, 10 -7 M and 10 -6 M, respectively (Figure 6).Dexamethasone, as a confirmed induction agent for apoptosis, has broad application in treating cancers (De Bosscher et al., 2000;Riccardi et al., 2000;Almawi et al., 2002).Dexamethasone act transcriptionally by binding the glucocorticoid receptor (GR) and subsequent binding to the promoter region of target genes on sites compatible with GRE motifs, which in turn directly or indirectly regulated gene expression (Almawi et al., 2002).Now the relationship between the glucocorticoid signal pathway and miRNAs has aroused the interest of investigators.For example, Liao and Lonnerdal (2010) found increase of mir-30e in dexamethasone-induced IEC-6 cells.Smith et al. (2010) studied the microRNA expression and processing during lymphocyte apoptosis induced by dexamethasone and found that mir-17-92 was repressed significantly (Smith et al., 2010).But so far, there is no evidence to support that dexamethasone directly activates the transcription through GRE within the promoter of microRNA.Thus, in our experiment, whether the dexamethasone enhance the promoter activity of let-7a-1/let-7f-1 by activate the GRE within its promoter need further work to confirm.

~10
-6 M of dexamethasone significantly increase the promoter activity of let-7a-1/let-7f-1, further research should be done to identify the responsive functional cis-elements within the let-7a-1/let-7f-1 promoter and elucidate their regulatory mechanisms, thus, providing a foundation for understanding the transcription pattern of let-7a-1/let-7f-1 and their application in target therapy for lung cancer.

Figure 2 .
Figure 2. Activity assay of pGL3-2123 in A549 cells.pGL3-2123 was transfected into A549 cells along with a negative control pGL3-Basic and a positive control pGL3-Control.The promoter activities were determined via the dual-luciferase reporter assay.The results are expressed as the relative luciferase activities (M1/M2), that is, the ratio of firefly luciferase activity (M1) in the pGL3 plasmid and Renilla luciferase activity (M2) in the pRL-TK plasmid.

Figure 5 .
Figure5.The effects of 1,25(OH)2D3 on promoter activity of let-7a-1/ let-7f-1 cluster.A549 cells transfected with pGL3-2123 were treated with 10 -8 M~10 -6 M of 1,25(OH)2D3 for 48 h, as well as parallel wells transfected with pGL3-Basic as negative control.The cells were harvested for the dual-luciferase reporter assay to detect the effects of hormones on promoter activity of the let-7a-1/ let-7f-1 cluster.The results were expressed as ration of relative luciferase activities.