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RECOMBINANT LENTIVIRUS VECTOR TARGETING BOVINE LXR MRNA AND ITS SILENCING EFFECTS ON BOVINE MUSCLE SATELLITE CELLS.

Byline: Y. Liu, P. Lib, L. Zhao, Z. Zhao, Y. Li, M. Liu, X. Yang and L. Zan

Abstract

The liver X receptor [alpha] (LXR[alpha]) is a member of the nuclear hormone receptor superfamily which could regulate the transcription of the genes involved in cholesterol transportation. In this study, Lentiviral vector containing LXR[alpha] shRNAs were constructed and transferred in bovine muscle satellite cells. The results indicated that LXR[alpha] down regulated PPAR[alpha], PPARI3, ABCA1, LPL, ApoE genes after 48 h post-pLenti-03 viruses' infection. The silencing effects of bovine LXR[alpha] gene on related were significant. This work identifies bovine LXR[alpha] role in cholesterol metabolism.

Key word: bovine LXR[alpha] gene, lentivirus, vector construction, gene silencing.

INTRODUCTION

Obesity, hypertension, dysmetabolism are diseases caused by unhealthy diet custom which influenced human's living quality. Thus, a low-cholesterol lifestyle is necessary. Currently, people have paid much attention to balancing their diet by low-intake of cholesterol. Producing good quality meat with low-fat (beef, mutton, pork, etc.) is a vital way to provide people with healthy meals condition.

The liver X receptor [alpha] (LXR[alpha], NR1H3) and LXR[beta] (NR1H2) are members of the nuclear hormone receptor superfamily. LXR[alpha] is abundantly expressed in the liver, intestine, adipose tissue, kidney and immune macrophages, whereas LXR[beta] is ubiquitously expressed (Thomas et al., 2013; Savkur and TP Burris 2004). Liver X receptors (LXRs) are activated in response to intracellular lipid accumulation, which could regulate transcription of an array of genes involved in the regulation of cholesterol homeostasis and reverse cholesterol transport (Zelcer and Tontonoz 2006; Lund et al., 2003).

Cholesterol homeostasis is intricately regulated by a battery of transcription factors among which LXRs are nuclear receptors that play a crucial role in transcriptional regulation of lipid metabolism and inflammation (Ostlund, 1993) Activated LXRs form a heterodimer with the retinoid X receptor [alpha] (RXR[alpha]), which binds to LXR responsive elements (LXREs) and consequently promotes target gene expression (Zelcer and Tontonoz 2006). LXR/RXR heterodimers are characterized by the ability to be activated by ligand in an independent manner. Thus, LXR/RXR heterodimers are activated by the RXR ligand, e.g., 9-cisretinoic acid, and the LXR ligands, e.g. oxysterols, or are activated synergistically in the presence of ligands for both receptors (Willy et al., 1995).

Recent studies showed that LXR[alpha] is considered as a critical regulator in cholesterol homeostasis in macrophages, which could regulate several genes involved in cholesterol transport, such as the ATP-binding cassette trans-porters (ABCs), ABCA1, ABCG1, apolipoprotein E (ApoE) and lipoprotein lipase (LPL) (Vinoda et al., 2014).

ABCA1, ABCG1 and ApoE are all target genes regulated by LXR (Ishimoto et al., 2006). Besides, peroxisome proliferator-activatedreceptor-alpha (PPAR[alpha]) can activate the cytochrome p450 enzymes, resulting in some of the hydroxyl cholesterol as LXR[alpha] endogenous ligand further activate LXR[alpha], which further activating ABCA1 regulated cholesterol efflux. Recent studies indicated that PPARI3 enhances cholesterol efflux by inducing the transcription of LXR[alpha] (Soumian et al., 2005). PPARI3 also induces the expression of ABCA1 and ABCG1, and promotes cholesterol efflux from macrophages through a transcriptional cascade mediated by LXR[alpha] (Chawla et al., 2001; Wong et al., 2008; Hu et al., 2013). LXR[alpha] can also activate LPL. Some studies have revealed that cholesterol-induced LPL gene expression in the liver is directly regulated by RXR/LXR heterodimers in a tissue-specific manner, which is mediated predominantly by LXR[alpha] in vivo (Zhang et al., 2001).

LXR[alpha] is important for cholesterol metabolism in human and mice. Bovine LXR[alpha] gene also controls the cholesterol level and the outflow of cholesterol efflux in muscle. Given that the importance of LXR[alpha] gene, regulating cholesterol in metabolism, exploring LXR[alpha] gene function in cholesterol metabolism in muscle cells to balance the cholesterol content and improving the quality of beef are of great significance. Packaging and proliferating lentivirus with RNA interference (RNAi) technique is a conserved biological response to double-stranded RNA (Hannon, 2002), RNAi is a sequence-specific process that regulates genetic functions and provides defense against virus at the post-transcriptional level in mammalian cells and animals (Song et al., 2003; Jacque et al., 2002). Lentivirus vector is a kind of inactivated HIV virus with many advantages, such as high transfection rate, stable expression in target cells and good security, so it has been wildly used in transgenic research as a crucial tool.

Lentiviral expression vector is a kind of long-acting system which can infect host cells with high-efficiency. Thus, we use this mediation system to achieve functional gene research on bovine muscle satellite primary cells. Considering the importance of LXR[alpha] gene and related study on bovine has not been reported yet.

We hypothesized that LXR[alpha] might influence some cholesterol metabolism regulating genes. To address this hypothesis, we successfully construct virus vector in order to transfect bovine cells and detect some cholesterol regulation genes. To our knowledge, this study first demonstrated the interference of LXR[alpha] on bovine muscle satellite cells in vitro to investigate LXR[alpha] gene's function and its target genes. Furthermore, it can provide us with valuable information for further studying of bovine LXR[alpha] gene mechanism.

MATERIALS AND METHODS

Unless stated otherwise, all chemicals and bio-chemicals used in this study are tested for cell culture and are of molecular biology grade.

Cell culture: HEK 293T Cells, the bovine muscle satellite cells were given by NBCIC (National Beef Cattle Improvement Center, Northwest AandF University, Yangling, China), and identified with cell markers by Immunofluoresence staining, cultured in Dulbecco's modified Eagle's medium (DMEM, gibco) with stable L-glutamine supplemented with 20% fetal bovine serum (FBS, gibco), 10% horse serum (HS, gibco), 100U/ml penicillin (sigma), 100ug/ml streptomycin (sigma) (complete growth medium), incubated at 37 AdegC under 5% CO2 atmosphere (Thermo).

Design of shRNA sequence and system: The sequence of bovine LXR[alpha] (NM_001014861.1) in GenBank. Interference shRNA sequences were designed on online software. Four shRNA oligos and the shRNA-NC sequence oligo were shown in table 1. Synthetic single-stranded DNA oligo was diluted with ddH2O. Then, we mixed 0.5ul of each of top strand oligo and bottom strand oligo and 2ul 10xoligo annealing buffer, adding 8ul ddH2O up to a final volume of 20ul to anneal. Oligo mixture was heated at 95AdegC for 5 min, and then placed at room temperature for 20 min forming double-stranded DNA. Synthetic double-stranded DNA was diluted to10nM with ddH2O. The reaction mixture containing 4ul 5x ligation, 2ul pcDNA 6.2-GW/Em GFP-miR, 4ul 10nM ds oligo, 1ul 1U/ul T4 DNA ligase and 9ul ddH2O was placed for 30min at room temperature. The products were then transformed into E.coli DH5[alpha] competent cells. Monoclonal colony was selected. Next, plasmids were extracted and sequenced.

The four plasmids (14MR0054-01~14MR0054-04) were respectively recombined into pDONR221 vector by Invitrogen BP carrier recombination system. 100ul of DH5[alpha] competent cells was transformed by 5ul recombination reaction solution. Positive clones were screened and sequenced. Purpose sequence was further restructured to pLenti6.3/V5-DEST vector by LP restructuring system. DH5[alpha] competent cells were transformed by recombination reaction solution again; positive clones were selected and sequenced. Our results proved that lentiviral vector carrying interference sequence has been successfully achieved. The names of the four lentivirus vectors were shown in table 2.

Packaging lentivirus: When the 293T cells reached 70%-80% confluence, cells were plated at 1.0x106 cells per 10cm cell culture dish and cultured overnight. The media canbe removed directly before transfection. 9ug of Packaging Mix and 3ug of the lentiviral expression plasmids were added to 1.5mlOpti-MEM medium that was then preheated at 37AdegC. 36ul lipofectamine 2000 was added to 1.5ml Opti-MEM medium and mixed gently; then, the mix was kept at room temperature for 5min.

Next, we mixed plasmid solution and lipofectamine 2000 diluent and put them at room temperature for 20min.

Then, 3ml of Plasmid liposome complex was added into cell dish and incubated at 37AdegC in 5% CO2 for 6h, after which we removed the primary medium and replaced it with fresh DMEM with 10% FBS. The supernatant was collected at 48h by centrifugation at 3000 rpm/min for 10min and filtrated by 0.45um membrane filter unit.

The virus stock solution was ultra-centrifuged at 50000g for 2h; the supernatant was removed and suspended in opti-MEM culture medium to determine the titer. Lentiviral stocks should be aliquoted and stored at -80AdegC.

Determination of viral titers: HEK 293T cells were seeded into 96-well plate at a density of 8x10 cells per well. Lentivirus was diluted with DMEM supplemented with 2% FBS and 8ug/ml Polybrene (DFP medium). The details of the viral stock dilution were shown in table 3.

Then, we carefully removed the culture medium from 96-well plate and replaced it with 100ul diluted virus solution. The cells were incubated in at 37AdegC with 5% CO2. After transfection for 24 hours, GFP expression was observed under fluorescence microscope, virus titer was calculated after transfection for 96 hours. Lentivirus vectors of pLenti-01~pLenti-04 were transduced into HEK293T cells, which were screened under the same view in fluorescent and bright fields of microscope (Fig. 1).

Determination of MOI value: The bovine muscle satellite cells were maintained in DMEM supplemented with 20% of FBS and 10% HS. After the confluence, the cells were trypsinized and counted, 1x104 cells were seeded in 96-well plates with 2ml of medium, which were incubated overnight. According to MOI value of 2, 5, 10, 20, 50, 100, 200 and 300, we diluted lenti 6.3-RNAi with DMEM supplemented with 2% FBS and 8ug/ml polybrene. Cells were lightly washed once with Phosphate Buffered Saline (PBS) and lentivirus liquid with different MOI values was added to the corresponding wells respectively. After 72 hours, the expression of GFP was observed.

RNA extraction: Total RNA was extracted from bovine muscle satellite cell samples using Trizol buffer, and the following reverse was performed using a High-capacity cDNA Reverse Transcription Kit.

qRT-PCR: The qPCR reaction system was 17.3ul ultrapure water, 2.5ul 10xPCR buffer, 2ul magnesium ions, 0.2ul dNTPs, 0.5ul primers, 0.5ul 50xsybr, 1.2ul cDNA and 0.3ul of Taq DNA polymerase. The PCR amplification conditions were:2min at 95AdegC; 40cycles of 95AdegC for 10s, 30s at 60AdegC and 45s at 70AdegC.The expression level of mRNA was determined by cycle threshold (Ct) normalized with that of PPP1R11 using the 2-IICt formula. Samples transfected with negative interference vector were used as a control.

Data Analysis: SYBR Green and cDNA were used for Quantitative Real-time PCR(RT-PCR).The expression level was quantified via 2-IICt. Analytical data was normalized to the mRNA expression level of endogenous control [beta]-actin. Primer sequences were described in table 4.

RESULTS

Lentiviral packaging, the titer determination results: 293T cells were first transfected with lentivirus vectors for 24 hours, and then observed under fluorescent (Fig. 1A-D) and light (Fig. 1 a-d) microscopes.

Significant GFP could be found after transfection with virus in 100uL at 2x10-8 ml. The virus titers for Lenti-14MR0054-01, -02, -03 and -04 were 3x108, 2x108, 2.5x108 and 3.5x108 TU/ml, respectively.

LXR[alpha] mRNA expression: Bovine muscle satellite cells were infected with pLenti-01, -02, -03 and -04 lentiviruses for 48 h, and then these cells were collected qRT-PCR analysis. We found that the LXR[alpha] mRNA expression levels were decreased to 0.46, 0.23, 0.12 and 0.29 as compared to negative control lentivirus. The silencing efficiencies of the four viruses were 54%, 77%, 88% and 71%, respectively (Fig. 2).

LXR[alpha] related genes mRNA expression: To explore the effects of LXR[alpha] knockdown on LXR[alpha] related genes, the expression of PPAR[alpha], PPARI3, ABCA1, LPL, RXR[alpha] and apoE were also determined with by qRT-PCR analysis, and the results were summarized in fig. 3. As compared with negative control group, the mRNA expression levels of PPAR[alpha], PPARI3, ABCA1, LPL and apoE were decreased in bovine muscle satellite cells when LXR[alpha] was inhibited, whereas that of RXR[alpha] was increased (Fig. 3).

LXR[alpha] played a negative role in regulating. The results showed that up-regulation of RXR[alpha] expression was involved in LXR[alpha] suppression; therefore, the expression of LXR[alpha] and RXR[alpha] were negatively correlated.

The interference efficiency of pLenti-03 vector was 88%, which was the best among all the vectors. The relative expression of the six genes regulated by pLenti-03 was shown in fig. 4. Compared with the negative control group, except RXR[alpha], the expression level of the other five genes decreased less than one. The related genes decreased by LXR[alpha] were in the order of PPARI3>PPAR[alpha]>LPL>apoE>ABCA1. The relative expression of RXR[alpha] gene was higher than the negative control and was up-regulated. Hence, the silencing effects of bovine LXR[alpha] gene on PPAR[alpha], PPARI3, ABCA1, LPL, RXR[alpha] and apoE were significant.

DISCUSSION

Bovine muscle satellite cells were used in our experiment. The positive rate of GFP was very high when cell was infected by adenovirus with MOI value 3000, but the positive of cellular fluorescence intensity was very low. The reason for this was that adenovirus cannot be applied to infect bovine muscle satellite cells. However, the positive rate of the cells was very high when lentivirus infecting bovine muscle satellite cells with MOI 300. Although the positive rate was low and cellular fluorescence intensity was weak, which also have an obvious effect on the cellular shape. Lentiviral vector was developed basis on the gene therapy of the human immunodeficiency virus and have the high efficiency for infection and stable silencing target gene, therefore, it was used widely (Wang et al., 2012; Zhao et al., 2013).

Lentivirus as gene transfer vector was characterized by high transfection efficiency, high titer, good stability in target cells and little immunoreactivity, and has been widely used in transforming into vector of genetic engineering (Koh et al., 2009). Compared to another carrier lentiviral vector, it has its unique advantages: for some difficult to transfect cells which has a high infection rate for primary cells, stem cells and undifferentiated cells, and the genetic recombination could not occur and the vector could be stably expressed.

In this study, we found that LXR[alpha] genes were down-regulated by 88% after lentivirus infecting bovine muscle satellite cells for 48h, the expression of ABCA1 and ApoE were also decreased. Meanwhile, LXR[alpha] could increase the expression of ATP-binding cassette transporter protein and promote cholesterol efflux. Additionally, after activating LXR[alpha] in macrophages, the expression of ABCA1, ABCG1 and ApoE also increased. LXR and PPAR pathway coupling increased expression of ABCA1and regulated lipid intake and reversed transport (Tobin et al., 2000). LXR[alpha] genes might decrease the expression of ApoE. Compared with the control group, the expression of PPARI3 was decreased in bovine muscle satellite cells. When the expression of LXR[alpha] gene is increased, more heterodimers could be formed by PPARI3 and RXR, inhibiting PPARI3 and RXR forming heterodimers and decreasing the expression of PPARI3 (Yoshikawa et al., 2003).

Therefore, when LXR[alpha] was down-regulated, LXR[alpha] and PPARI3 could form a competitive mechanism, which caused decreased expression of PPARI3. However, the mechanism of up-regulated expression of RXR[alpha] has not been identified and the further study should be remained and investigated. It is of great significance to study intracellular lipid metabolic through interfering LXR[alpha] gene. Lentivirus interfering vector lays the foundation for further studying LXR[alpha] gene function in lipid metabolism in bovine muscle satellite cells.

Conclusion: lentiviral vector carrying the shRNA targeting LXR[alpha] gene was successfully constructed, and the lentiviral vector can be efficiently expressed in bovine muscle satellite cells. We also explored the effects of LXR[alpha] gene silencing on the metabolic associated genes. This study provides new insights into the regulation of bovine LXR[alpha] in cholesterol metabolism.

Acknowledgments: This work was supported by the national natural science foundation of China (31372288), the research fund for the doctoral of higher education of China (20130202120008), the China postdoctoral science foundation (2015M570811), and the fundamental research funds for the central universities of China (GK201502008).

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