Influence of silencing the MC 4 R gene by lentivirus-mediated RNA interference in bovine fibroblast cells

Melanocortin receptor 4 (MC4R) is a key element in the mechanisms used to regulate both aspects of keeping the balance between energy uptake and energy expenditure. MC4R was knocked down by lentivirus-mediated shRNA expressing plasmids, which were controlled by the U6 promoter in bovine fibroblast cells, and the expression of MC4R was examined by the real time-PCR and Western blot analysis. Real time-PCR analysis was used to characterize the expression of Leptin, POMC, AGRP, MC3R and NPY gene. The relative genes [leptin, proopiomelanocortin (POMC), agouti-related peptide (AGRP), MC3R and neuropeptide Y (NPY)] expression level seemed to be closely associated with the MC4R gene in bovine fibroblast cell lines (BFCs). The levels of both MC4R mRNA and protein were significantly reduced by RNA interference (RNAi) mediated knockdown of MC4R in BFCs cells transfected with plasmid-based MC4R-specific shRNAs. The finding of this study demonstrated that vector based siRNA expression systems were an efficient approach to the knockdown of the MC4R gene expression in bovine fibroblast cells and they provided a new molecular basis for understanding the relationship of MC4R and other genes, which were responsible for the regulation of energy homeostasis by the melanocortin system.


INTRODUCTION
The melanocortin receptor 4 (MC4R), a G-protein coupled receptor, plays a pivotal role in controlling meal size and energy homeostasis in mammals (Govaerts et al., 2005;Adan et al., 2006).The accumulating evidence implicated the components of the CNS melanocortin system, which encompassed several receptors and their peptide ligands in the central regulation of feeding and energy balance (Lu et al., 1994;Fan et al., 1997).MC4R was one of the five melanocortin receptors (MCRs) and was shown to be widely distributed throughout numerous *Corresponding author.E-mail: simmenta@yahoo.com.cn.
#These authors contributed equally to this work.brain regions (Mountjoy and Wong, 1997).In spite of the evidence, presented about the role of several important factors in energy metabolism, the regulation and integration of the satiety pathway and the energy homeostasis have not been completely elucidated.These important factors are neuropeptide Y (NPY), leptin, proopiomelanocortin (POMC), Agouti-related peptide (AGRP) and their respective receptors (Pelleymounter et al., 1995;Stephens et al., 1995;Hu et al., 1996;Boston et al., 1997;Clement et al., 1998;Friedman and Halaas, 1998), whereas only few researches have been conducted on the relationship of MC4R with the expression of upstream regulatory genes in bovine.
RNA interference (RNAi) is a wide and powerful tool which will lead to sequence-specific gene silencing at the post-transcriptional level.In mammalian cells, synthetic siRNAs can be transferred into the cells to induce transient gene silencing, whereas short hairpin RNAs (shRNAs) could be driven by RNA polymerase III promoters and are subsequently cleaved by the Dicer to produce siRNAs in order to introduce mRNA degradation; but there are lots of problems related to the method of delivery, especially low efficiency of transfection in primary cells.However, lentivirus can stably transfect to many cells, such as primary cells, suspension cells and stem cells, at high efficiency, and also sustain long-term gene expression by integrating itself into the host genome.Therefore, lentiviral vector is considered as the most suitable delivery method for this study.
In the recent studies (Chen et al., 2000;Ste et al., 2000), knockout of the MC4R gene was usually carried out in vitro or in vivo of mice.However, differences may exist between the bovine MC4R mRNA and the other species mRNA in terms of the length and spatial structure, so as to affect the effectiveness of shRNAs screening and relative regulation genes.Therefore, it is necessary to perform shRNAs screen in primary bovine cells which naturally knockdown the MC4R gene expression.
In this study, a shRNA-expressing lentiviral vector was constructed to target the bovine MC4R gene, and the resulting interference effect was analyzed in bovine fibroblast cells (BFCs).Subsequently, the resulting induction along with the expression of leptin, POMC, AGRP and NPY genes were studied.

Isolation and culture of cell
Auris tissues, blocked from 20 month-old male local yellow bovine in the Animal Center of Institute of animal science -Chinese academy of agriculture science, were resected under aseptic conditions.Bovine fibroblast cells and human embryonic kidney 293 T cells (HEK-293T) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco BRL, USA) at 37°C under 5% CO2.Cell cultures, at 80% confluence, were detached with trypsin-EDTA and plated into 12-well plates or 6-well plates at a density of 5 x 10 4 cells/cm 2 for transfection only.

Real-time PCR analysis
Total RNA was extracted by Tiangen RNeasy mini kit (Tiangen, China) according to the manufacturer's instructions.About 2 to 5 µg of the total RNA were reversely transcribed by Quant one step RT-PCR kit (Tiangen, China) with oligo(dT)18 primers.Real-time RT-PCR was performed by an ABI Prism TM 7900HT Sequence Detection System (Applied Biosystems, USA).Then, each PCR reaction with a total volume of 25 µL contained 2 µL of cDNA, 0.5 µL of 10 µmol L -1 primers, 9.5 µl ddH2O and 12.5 µl SYBR Premix Ex Taq (2×) (TaKaRa, China).The relative expression levels of all genes were normalized to 18S rRNA expression levels, respectively, and all primers are shown in Table 1.

Western blot analysis
The entire cellular protein was obtained with BCA Protein Assay Kit based on the manufacturer's instructions (Bestbio, China).The aliquots were separated on SDS-PAGE (10%) and transferred to nitrocellulose membranes.The MC4R antibody (SC6879-C19) was purchased from Santa Biotechnology (Santa Cruz Biotechnology Inc., USA).
Then, the membranes were incubated with goat anti-MC4R protein antibody at room temperature for 2 h and stained by the secondary antibody mouse anti-goat (SC2354) immunoglobulin G, horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology Inc., USA) for 1 h.Anti-GAPDH antibody was obtained from Sigma Biotechnology as a positive control (Sigma, USA).

Statistical analysis
Statistical analyses were performed by student's t-test using SPSS statistical software (SPSS Inc., USA).P < 0.05 was considered as statistically significant.

Detection of MC4R silencing of Lvsh-MC4R in FBCs
The Auris tissue block was minced into pieces and digested in the presence of type II collagenase.Then, the resulting pellet was cultured for 96 h, in which most of the cells were slender with fibroblast shape under microscope.Generated lentivirus was further used to infect FBCs cells to obtain the most effective target virus (Figure 2a).The total RNA was extracted after 72 h later, and the Real-time PCR results that infection with Lvsh-MC4R-1,-2,-3 or -4 could inhibit MC4R mRNA expression by approximately 71, 8, 11 or 15%, respectively, in contrast to the infection with Lvsh-MC4R-NC (Figure 2b).As the transfection efficiency of the bovine fibroblast cells was around 80%, the result suggested that Lvsh-MC4R-1 markedly suppressed MC4R mRNA expression, while Lvsh-MC4R-2, -3 and -4 did not influence the levels of MC4R mRNA.Subsequently, the result from the western blot, when compared to the infection of LvshMC4R-NC (Figure 2c), further confirmed, that the Lvsh-MC4R-1 could significantly suppress the expression of MC4R at both levels of mRNA and protein, and maintain satisfactory silencing effect in BFCs.

Effect of Lvsh-MC4R on expression of regulatory genes
In order to observe the effect of MC4R silencing on the other genes of energy metabolism, Lvsh-MC4R1 was selected as the knockdown group (KD), which was compared with the negative control group (NC) to detect the expression level of the four upstream regulatory genes in all the samples.As shown in Figure 3, the Leptin, POMC and NPY genes expression in the KD group was detected to be appreciably lower than that of the NC group; but the expression of MC3R gene in NC group was found to be significantly lower than the KD group, especially, where it was found that the expression of AGRP gene in the KD group was much significantly higher than that of the NC group.

DISCUSSION
In this study, lentiviral vectors are used to silence bovine MC4R gene expression not only in HEK-293T cells, butalso in bovine fibroblast cells.Human U6 promoter was used to drive siRNA expression from the DNA templates and four target sites were designed.Finally, it was only one effective target site (Lvsh-MC4R1) that could down-regulate the MC4R gene expression in bovine fibroblast cells.Lentiviral vectors were chosen as the shRNA delivery vehicle to suppress the MC4R gene in bovine fibroblast cells, for the reason that lentivirus can integrate itself into the genome of host cells and sustain long-term gene expressions (Gropp and Reubinoff, 2007).Generally, a lentiviral vector expression system consists of three plasmid vectors, which include lentiviral plasmid (transfer vector), packaging vector and VSVG Fig. 1 The identities of pGCSIL-GFP-shMC4R vectors and LvshMC4R.A. The recombinant pGCSIL-GFP-shMC4R-1,-2,-3,-4 plasmids were identified by PCR.Lane 1, 3, 4, PCR products of the recombinant pGCSIL-GFP-shMC4R-1,-2,-3,-4 plasmids, Lane 2, PCR products of the empty vector; Lane 5, DNA ladder (DL 2000), 500 bp (up) and 250 bp (down); the positive clones were 352bp and the negative clones were 318 bp.B. 293T cells were infected with Lv-shMC4R (KD) or -NC (NC), and phase contrast or GFP expression which was examined after 72 h.expression plasmid, and a packaging cell line (pseudovirion producing cell line).In this study, there are four lentiviral plasmids that are successfully constructed to target MC4R and a negative control using the three plasmid expression system.The result, shown In Figure 2, suggested that the lentiviral vector expression system could effectively drive siRNA and transcribe it from the DNA templates into the bovine fibroblast cells.
In contrast to all other hormone systems known so far, the melanocortin system was the only one that was not only regulated by agonistic peptides, but also by two endogenously occurring neuropeptides that were blocked from ligand-induced MCR signalling: agouti and "agoutirelated protein" (AGRP).Agouti binds to almost all MCR with high affinity, whereas AGRP exhibits rather selective binding towards the MC3R and MC4R (Ollmann et al., 1997).This study indicated that AGRP impaired the appetite controlled in a melanocortin-independent manner, and it was defined as a classic competitive antagonist, since binding of AGRP prevented or displaced the binding of melanocortins to the MC3R or MC4R (Tolle and Low, 2008;Wu et al., 2008).However, this study herein found out that the expression of AGRP gene, presented significantly, was high after MC4R gene was silenced.This result supports the suggestion that mechanisms, other than competitive antagonism of MC4R signaling, should also be considered when discussing the physiological effects of AGRP on the molecular level.As the receptors for NPY and MC3R are known, the members of the G-protein-coupled receptor family were down regulated in hypothalamic regions under conditions that were known by enhancing the local NPY release (Widdowson et al., 1997).In this study, mRNA level of NPY gene was down regulated, and oppositely, that of MC3R gene was up regulated, suggesting that MC3R and MC4R genes were competitively bound with NPY.Adipose tissue-derived hormones, such as leptin, increase POMC expression in α-MSH-releasing neurons located in the arcuate nucleus of the hypothalamus (Shimizu et al., 2007).Various peripheral and central factors could signal energy balance to the POMC neurons, as ARC POMC neurons express the functional OB-Rb leptin receptor, while leptin apparently stimulates these neurons (Cheung et al., 1997;Schwartz et al., 1997).However, the expression level of leptin and POMC went down as the MC4R was knocked down in this study, suggesting that they might be the medium in the signalling pathway of the melanocortin system.
In summary, one out of the four designed target sites reduced the MC4R gene expression by lentivirus mediated in bovine fibroblast cells, and the expressions of a series of genes of the CNS melanocortin system were detected.The results of this study provided a new molecular basis for understanding the mechanism of energy homeostasis, and also laid the foundation for further study of the biological functions of MC4R gene and other related genes.

Fig. 2
Fig.2The effect of infection with Lv-shMC4R and LvshMC4R-NC in BFCs. A. BFCs were infected with Lv-shMC4R (KD) and LvshMC4R-NC (NC), and representative phase contrast or GFP expression was examined after 72 h.B. BFCs were infected with Lv-shMC4R or Lvsh-NC, and the level of MC4R mRNA was detected by Real-time PCR 72h later.mRNA levels in NC were taken as 1.0.C. MC4R protein levels were detected by western blot 72h later.Each treatment was established in duplicate and each sample was examined in duplicate.All data were expressed as means ± SEs

Table 1 .
Parameters of oligo-nucleotide primer pairs for PCR primers.