3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

Jie Jing, Men Jing, Wang Chun-mei, Gao Xue-jun, and Li Qing-zhang

College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

Introduction

GHR (growth hormone receptor)is the stimulator of lactation and the member of growth hormone receptor superfamily, which is a transmembrane glycoprotein consisting of 620 amino acid residues. The research on GHR is focused on the relationship among GH (growth hormone), GHBP (growth hormone band protein),GHRH (growth hormone-releasing hormone), SS(growth hormone release-inhibiting hormone)and so on (Gonzalez, 2007). Resent discovery declares that it is the SNP of GHR that affects the characteristic of milk production (Signorelli, 2009). The disease of diabetes mellitus as well as metabolic disorder is credited to it (Strawbridge, 2007). Apart from the well-known runt disease, the abnormal expression dose between GH and GHR might result in distemper of sepsis, excessive or tardy growth of prostate cells(Strawbridge, 2008), functional disturbance of hypothalamus (Deepak, 2010). Experimental result in mice showed that the lack of GHR in nude mice led to a limited catheter branch and branch severely degraded.The same result has been found in dairy goats (Flint,1997).

CXCR4 is a molecular closely related to metastasis.MiR-139 can inhibit CXCR4 expression by binding to the 3'-UTR of CXCR4 mRNA (Fu, 2009). Another research showed that miR-139 can affect FoxO1 expression, but has no work on the mRNA (Hasseine,2009). Others say miR-139 suppresses metastasis and progression of hepatocellular carcinoma by downregulating ROCK2 (Wong, 2011). miR-139 is also downregulated in glioblastomas (Pang, 2009).

The current role of miR-139 in the mammary gland has not been reported, while miR-139 in cancer and inflammation research has been reported many times acting on the site, so miR-139 was chosen for the study.

The purpose of the experiment was to find if miR-139 was the target miRNA of GHR on DCMECs.A luciferase reporter vector of GHR 3' UTR was constructed, and the luciferase reporter vector as well as miRNA eukaryotic expression vector were transferred into DCMECs, using the dual-luciferase reporter assay system to quantitiate the reporter activity.

Materials and Methods

Main reagent and apparatus

DCMECs were obtained from the Key Laboratory Dairy Science of Education Ministry, pMIR-Report vector and dual-luciferase reporter assay system were purchased from Promega Company. pMD18-T vector T4DNA ligase, JM109 competent cell, restriction enzyme SpeⅠ/Hind Ⅲ, Taq DNA ligase, dNTP, and DNA marker were purchased from TaRaKa Company,gel recovery reagent kit and plasmid small extraction reagent kit were purchased from AXYGEN Company, lipofectamine 2000 transfection reagent was purchased from Roch Company, DMF-12 culture media, trypsin, fetal bovine serum, benzylpenicillin,and streptomycin were purchased from Gibco (Invitrogen)Company.

Cell culture

DCMECs were cultured with DMF-12 medium supplemented with 10% fetal bovine serum in the condition of 37℃, 5% carbon dioxide and saturated humidity (Pang, 2009).

Prediction of miR-139's target sequence on GHR gene

The sequence of miR-139 and the target gene of GHR were obtained from miRbase. Targetscan 5.1 provided the 3'-UTR of GHR as well as the complementary region.

Construction of luciferase vector

The Primier 5.0 was used to design the primer, and it was inserted with the SpeⅠ/Hind Ⅲ recognition site.The upstream sequence was: 5'-GACTAGTCGCTTT AACCAGGAAGACA-3' SpeⅠ; and the downstream sequence was : 5'-CCAAGCTTGGAGGTATAAT CTGGGAC-3' (Hind Ⅲ). The RNA extracted from DCMECs was used as the template for RT-PCR. The PCR product was recollected from the gel. After that, it was linked with pMD18-T vector under the condition of 16℃ overnight and was sent to it to Invitrogen Company to get the sequence result. It was designated as GHR-T-107. GHR-T-107 and the luciferase-report vector were digested with restriction enzyme SpeⅠ/Hind Ⅲ. Then, the two digested products were ligated.The new recombinant plasmid was transformated to the competent cells JM109. Then it was delivered to Invitrogen Company after the right result of dualdigestion of SpeⅠ/Hind Ⅲ. The recombinant plasmid was named GHR-LUC-107 (Wu, 2010; Lin, 2010).

Transient cell transfection

The lipofectamine 2000 transfection reagent was used following the detail of the instruction manual. The DCMECs were vaccinated to the 6-well cell culture plate on the day before transfection, the cell concentration was 2×105and 1 000 μL DMF-12 culture media supplemented with 10% fetal bovine serum was used for each well. The fresh media after transfection was changed. The recombinant plasmid was diluted with 50 μL DMF-12 culture media free of serum. Each well was filled with 200 ng GHR-LUC-107, 800 ng miRNA (miR-139, miR-16a, and miR-30d), and 20 ng pRL-TK as the standard internal reference to calibrate the difference between different group, and the miRNA was taken as control. 4 μL lipofectamine 2000 was added to each well, mixed together and incubated for 15 min at room temperature. And was added to each well. After 48 h the cells were splitted and the luciferase activity was detected. Each group had three parallels, and all reactions were run in triplicate.

Detection of luciferase activity

The luciferase activity was detected following the details of dual-luciferase assay kit. PBS was used to wash the cell before PLB (PLB for 15 min)and the luciferase activity was detected with the Glomax20/20luminometer dual-luciferase assay system. 100 μL LARⅡ and 20 μL PLB conduction for each tube, the first luciferase activity (No.1)was detected. 100 μL Stop&Glo@ was added, and the second luciferase activity (No.2)was detected. The No.1/No.2 was taken as the final luciferase activity (Nolde, 2007; Tanaka,2009; Welch, 2007; XIA, 2009).

Statistical analysis

The data were presented as X±SD of three separate experiments, each performed in triplicate. Signif i cance levels were analyzed by one-way ANOVA, followed by Duncan' multiple range tests (STATISTICA;StatSoft, Tulsa, OK). Differences with P＜0.05 were considered statistically signif i cant.

Results

Prediction of target sequence on GHR gene

miRNA and miRNA targeting GHR3'-UTR were predicted by Targetscan 5.1 software and miRbase software.

GHR 3'-UTR –luciferase vector

PCR product was inserted into the SpeⅠ/Hind Ⅲmodified luciferase-report vector. After transient transfection to the JM109 competent cell, the recombinant plasmid was extracted from selected bacterial colonies and 1 μL of plasmid solution was subjected to agarose gel electrophoresis and both SpeⅠ/Hind Ⅲdigestion for identif i cation.

It was identif i ed that a 107 bp recombinant plasmid was ligated to the luciferase report vector (Figs. 1, 2).It was designated as GHR-Luc-107.

Fig. 1 PCR amplif i cation of part of GHR 3'-UTR sequence and restriction analysis of GHR-Luc-107 reporter plasmid

Fig. 2 Peak sequencing

miR-139 regulated GHR 3'-UTR

To prove the regulation between GHR 3'-UTR and miR-139, the recombinant plasmid and miRNA(miR-139, miR-16a, and miR-30d)were transferred into DCMECs using lipofectamine 2000 transfection reagent. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter activity. The results showed that the luciferase activity of reporter construction treated with miR-139 decreased about 20.87%±0.0370% compared with the control group (miR-16a and miR-30d)(Fig. 3).

Fig. 3 Relative luciferase activity

Discussion

MicroRNAs, an abundant class of small non-coding RNAs, are emerging as important post-transcriptional regulators of gene expression in many organs. More and more studies have shown that they regulate many genes in the transcription or translation level.Although microRNA is only 22-24nt, it can work by blocking or degradating mRNA of the target gene.People develop some Biology Software (Zhang, 2005;Lewis, 2003; 2005; Wang, 2008)according to micro-RNA mechanism, such as Targetscan 5.1, miRbase and Pictar. Compared with miRNA target gene prediction method, the method of the miRNA target genes for experimental validation is not much, there is no one quick, easy, and high-throughput. Identif i cation of the most direct way is to use fl uorescence quantitative PCR and Western blotting methods to detect mRNA levels and protein levels after the cells were transfected with miRNA overexpression or knocked down to determine the corresponding relationship between miRNA and target gene. This method can be used directly to identify the miRNA target genes accurately but can't identify the target sites. So the luciferase reporter method was used.

Based on the software forecast, we used two control plasmids to prove that miR-139 can decrease the activity of GHR-Luc-107 greatly, which might probably be reasoned as the interaction between micro-RNA and the target mRNA 3'-UTR. The luciferase activity of 30d group had no decreases suggesting it might not work with the target gene, and that of 16a group showed a little decrease which might mean the main target gene of miR-16a was not GHR-LUC-107,but was one of its target genes. Of course, the GHR protein level should be detected in the future. In short,the recombinant vector laid the foundation for the research of GHR gene expression.

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