Expression and subcellular localization of HSPC117 in min pig tissues and the PK15 cell line

2.1Animal

Three female min pigs were studied. The min pigs were slaughtered at 8 months of age and samples of back muscle, leg muscle, kidney, spleen, fat, uterine, small intestine, lung, and oviduct were collected.

2.2Reverse transcription and real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from each sample using a RNA extraction kit. The purity and integrity of the total RNA were assessed. The qualified sample total RNAs were used for reverse transcription. The first-strand cDNA was synthesized using an M-MLV First Strand Kit (Invitrogen) with oligo-dT as a primer according to the manufacturer’s protocol.

Gene expression was quantified using the Stratagene Mx3000P qPCR system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured in all of the tissue samples to normalize all of the mRNA quantifications. The specific primers used in these tests included GAPDH (F, 5′ACACTCACTCTTCTACCTTTG3′; R, 5′ CAAATTCATTGTCGTACCAG3′), HSPC117 (F, 5′TCCCATAGTAACGCCAATA 3′; R, 5′CTGGGTGTCCACAATCAA3′). All of the PCR amplifications were performed in triplicate independent biological replicates (3 samples of each kind of organ); no template controls were included. The relative amount of mRNA to GAPDH in each sample was calculated using a relative quantification standard curve.

2.3Construction of EGFP-C1-HSPC117 expression plasmid

The porcine HSPC117 coding sequence (GenBank: DQ 508263.1) was amplified by PCR from PK15 cell RT-PCR products using specific primers containing restriction sites at 5′ ends. F (5′AAGCTTATGA GTCGCAGCTATAATGATGAG3′) and R (5′GGATCCCTATCCTTTGATCACAGCAATTGGTC3′).

The PCR product was cloned into pEGFP-C1 vector at HindIII/BamHI sites to construct pEGFP-C1-HSPC117 (EGFP-HSPC117) and fully sequenced.

2.4Cell lines, cultures, and transfections

The PK15 cells were cultured in DMEM supplemented with 10% fetal calf serum at 38∘C in a humidified incubator (5% CO2 and 95% air). Prior to the day of transfection, the fusion rate of PK15 cells had reached approximately 80–90% and the PK15 cells were plated into 35-mm plates. EGFP-HSPC117 was transiently transfected in PK15 cells using 10 μL Lipofectamine 2000 reagent and 7 μg EGFP-HSPC117 plasmid, according to the reagent’s protocol. The PK15 cells were cultured in the transfection medium for 6 h and then incubated in DMEM medium with 10% serum.

2.5Analysis of fluorescence images

The transfected PK15 cells were cultured for 24 hours, 48 hours and 72 hours. Prior to observation, the cells were stained with Hoechst 33342 for 10 min. An Axioimager fluorescence microscope (Zeiss) with a 20× or a 40× objective and Axiovision imaging software (Zeiss) was used for imaging. GFP-HSPC117 and Hoechst 33342 fluorescence analyses were conducted using the GFP (488-nm) and DAPI (340-nm) channels, respectively.

2.6Statistical analysis

Data assays were performed using one-way ANOVA using SPSS 15.0 software. All of the data are presented as mean ± SEM. Moreover, P< 0.05 was considered to be statistically significant. Three replicates were analyzed for each experiment.

3.1Relative expression of HSPC117 mRNA in tissues

To determine the relative expression of HSPC117 mRNA in tissues from the min pig, qPCR was used to analyze mRNA levels in samples of 10 types of tissue. As shown in Fig. 1, the HSPC117 mRNA expression levels in most of the tissues (back muscle, colon, leg muscle, liver, kidney, uterus, spleen, and oviduct) were similar (P> 0.05). However, the HSPC117 mRNA expression levels in fat and lung tissues were a significant 0.12 and 0.19 times lower, respectively, compared than in back muscle (P< 0.01).

Figure 1.

The HSPC117 mRNA expression level in samples of 10 types of tissue (back muscle, colon, fat, leg muscle, liver, kidney, uterus, spleen, lung, and oviduct) GAPDH was used as the control.

The HSPC117 protein is involved in many important cell functions, such as tRNA splicing, RNA transportation, cell migration/invasion, and many other essential functions, which occur during cell growth and division [8]. Since the HSPC117 protein plays so many basic and important roles in the biological processes of cells, it is widely distributed in both cells and tissues. Previous research has demonstrated that the HSPC117 protein is expressed in tissues from humans and in some types of pigs [8, 9]. The D10Wsu52e protein (an HSPC117 homolog) has been found in mouse embryonic forebrain, midbrain, hindbrain, optic vesicles, somites, stomach, and duodenum, and the expression level of this protein changes depending on the development stage [10]. In the current study, HSPC117 mRNA expression was found in back muscle, colon, fat, leg muscle, liver, kidney, uterus, spleen, lung, and oviduct tissues from the min pig. However, the level of HSPC117 mRNA expression differed among these tissues, suggesting that HSPC117 might have different functions or be regulated differently in different tissues, although it performs some basic and important biological functions.

Figure 2.

EGFP-HSPC117 localization in PK15 cells during interphase. A: 24 h nucleus. a: 24 h EGFP-HSPC117. a’: 24 h contrast image. B: 48 h nucleus. b: 48 h EGFP-HSPC117. b’: 48 h contrast image. C: 72 h nucleus. c: 72 h EGFP-HSPC117 fluorescence. c’: 72 h contrast image.

3.2Subcellular distribution of the EGFP-HSPC117 protein in PK15 cells

Transgenic PK15 cells, which express an EGFP-HSPC117 fusion protein, were produced. EGFP-HSPC117 expression in the transgenic PK15 cells was driven by the SV40 CMVie promoter. We examined the distribution of the fusion protein in PK15 cells using fluorescence microscopy. EGFP fluorescence was visible in some PK15 cells 12 h after induction and was clearly seen in most cells 24 h after induction. Fluorescent fusion proteins were detected throughout the cytoplasm as shown by indirect fluorescence images, although EGFP expression was slightly stronger in cell nuclei than in the cytoplasm (Fig. 2). There were no marked changes in green fluorescence during the interphase after the transgenic cells, expressing EGFP-HSPC117, were cultured for 48 h or 72 h.

To elucidate the localization of EGFP-HSPC117 in PK15 cells during mitosis, transgenic PK15 cells were cultured for 3–4 days. Some cells were then observed during the mitotic phase. As shown in Fig. 3, fluorescence was detected in the condensed chromosomes of the mitotic PK15 cells, whereas much less fluorescence was seen in the cytoplasm. These observations indicate that the localization of EGFP-HSPC117 in PK15 cells changes during the mitotic phase.

Figure 3.

EGFP-HSPC117 localization in PK15 cells during mitosis. A: The apparent EGFP fluorescence was detected in the dividing daughter nucleolus during mitosis. A’ contrast image of PK15 cells during mitosis. B: fluorescent fusion proteins were detected throughout the cytoplasm during interphase. B’ contrast image of PK15 cells during interphase.

HSPC117 is an important protein complex and it participates in various functions that occur in different parts of the cell. In the current study, EGFP-HSPC117 fusion protein fluorescence was detected throughout the cytoplasm and nuclei during interphase and the distribution did not change significantly over culture time. Perez-Gonzalez et al. found that HSPC117 protein molecules combined with DDX1, FAM98B, and hCLE proteins to form a compound, which is present in both the nucleus and cytoplasm and participates in both nuclear and cytoplasmic RNA metabolism [9]. Previous studies have shown that HSPC117 is present in the cytoplasm and is involved in both RNA maturation and transportation [11, 12, 13]. However, in this study, fusion protein fluorescence was visibly concentrated in the nucleolus area of PK15 cells during the mitotic phase. To the best of our knowledge, no previous research has indicated that HSPC117 assembles in the nucleolus area during the mitotic phase. This phenomenon might be related to chromosome condensation, although there is no direct evidence for this as of yet.