Mol. Cells 2020; 43(7): 607-618
Published online July 10, 2020
https://doi.org/10.14348/molcells.2020.0058
© The Korean Society for Molecular and Cellular Biology
Correspondence to : khs307@pusan.ac.kr
microRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. miRNAs inhibit gene expression by binding to the 3′ untranslated region (UTR) of their target gene. miRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p share the binding sites in the 3’ UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p to inhibit the activity of VNTR. In conclusion, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p acts as a repressor of VNTR activity in the 3′ UTR of SHISA7.
Keywords long terminal repeat, miR-3681-5p, SHISA7, transposable elements, variable number tandem repeat
Transposable elements (TEs) comprise approximately half of the eukaryotic genome and are known to insert into the genome as stable genomic components (Bourque et al., 2018; Wicker et al., 2007). TEs are divided into two classes—retrotransposons (class I) and DNA transposons (class II). Retrotransposons use the ‘copy and paste’ mechanism. The retrotransposons are first transcribed into an RNA intermediate, which is reverse transcribed into a DNA intermediate (Agren and Clark, 2018; Bourque et al., 2018; Lander et al., 2001; Wicker et al., 2007). Long terminal repeat (LTR) is a member of the retrotransposon family and the composition of LTR in humans is approximately 9%. An LTR consists of innate enhancer activity, however, transcription factor (TF), nucleotide substitutions, transcription start sites, and epigenetics may provide alternative enhancer activity of LTR elements (Thompson et al., 2016). LTRs recruit TF and enhance the transcription of host genes (Gonzalez-Cao et al., 2016; Hu et al., 2017). TEs can generate regulators of genes, such as microRNAs (miRNAs), TFs, and variable number tandem repeats (VNTRs) (Feschotte, 2008; Lee et al., 2019; Piriyapongsa et al., 2007; Qin et al., 2015; Roberts et al., 2014; Sabino et al., 2014).
miRNAs are small non-coding RNA molecules, approximately 19 to 24 nucleotides long, which are essential for gene regulation (Ambros et al., 2003; Bartel, 2004; Kim, 2005; O'Brien et al., 2018). miRNAs have a unique seed region, approximately six nucleotides long, that binds to the 3′ untranslated region (UTR) of the target mRNA. In most of cases, miRNAs regulate gene expression by inhibiting gene expression; however, recent study has reported the presence of enhancer miRNAs (Lee et al., 2019). In addition, some miRNAs bind to the 5′ UTR of the target transcripts and to coding sequences, to regulate target gene expression (Brummer and Hausser, 2014; Gu et al., 2014; Hausser et al., 2013; Lee et al., 2009; Li et al., 2016; Liu et al., 2015). In studies on miRNA, the number of miRNA binding sites in the target gene was not an important consideration in selecting the best target genes. Previous studies have suggested that miRNAs derived from transposable element (MDTE) and MDTEs share sequences (Lee et al., 2019; Petri et al., 2019; Piriyapongsa and Jordan, 2007; Piriyapongsa et al., 2007; Qin et al., 2015; Roberts et al., 2014). Hsa-miR-3681 is derived from an LTR element and has not been studied much. A few studies on hsa-miR-3681 have reported on its dysregulation in human disease and cancer and downregulation in cervical cancer (Shi et al., 2019; Vaira et al., 2014; Vaz et al., 2010).
One of target genes for hsa-miR-3681-5p,
In this study, the relative expression of the MDTEs, hsa-miR-3681-5p and its target
Experiments with the Western chimpanzee were carried out in accordance with the guidelines and regulations approved by the Animal Experimentation Committees of Kyoto University (2018-004). Experiments with Crab-eating monkey were carried out in accordance with guidelines and regulation approved by Korea Research Institute of Bioscience & Biotechnology (KRIBB-AEC-15046).
The sequence of hsa-miR-3681-5p was downloaded from miRbase v22.1 (http://www.mirbase.org) (Kozomara et al., 2019), and then the sequence was used to localized in human genome (GRCh38) and to download sequence of LTR16D1 by UCSC Genome Browser (http://genome.ucsc.edu) (Agarwal et al., 2015). The sequences of hsa-miR-3681-5p and LTR16D1 were aligned using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999) to compare the analogy. TargetScanHuman (http://www.targetscan.org/vert_72/) (Agarwal et al., 2015) was used to list out the target gene candidates of hsa-miR-3681-5p (Table 1).
The structural interaction between hsa-miR-3681-5p and the 3′ UTR of
The putative TF binding sites near the 9.5 VNTRs of the 3′ UTR of
Total RNAs from 13 human tissues (brain, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, testis, prostate, and uterus) and 10 mouse tissues (brain, heart, lung, liver, kidney, spleen, stomach, smooth muscle, testis, and spinal cord) were purchased from Clontech (USA).
A total of 14 female crab-eating monkey tissue samples (cerebellum, cerebrum, heart, lung, liver, kidney, spleen, stomach, small intestine, large intestine, uterus, pancreas, bladder, and spinal cord) were provided by the National Primate Research Center (Korea). Total RNA was extracted from 14 tissue samples of the female crab-eating monkey by using Hybrid-RTM (GeneAll, Korea), according to the manufacturer’s instructions.
Tissue samples from the female Western chimpanzee were provided from the Kumamoto sanctuary, Kyoto University, to Primate Research Institute in Inuyama, Japan, via GAIN and the male Western chimpanzee samples were provided by the Primate Research Institute. All experiments using samples from the Western chimpanzee were done at the Primate Research Institute. Total RNA was extracted from 11 tissues of healthy male Western chimpanzees (cerebellum, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, and testis) and female Western chimpanzees (cerebellum, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, and ovary) by using Hybrid-RTM, according to the manufacturer’s instructions.
The RNA samples were quantitated using the ND-1000 UV-Vis spectrophotometer (NanoDrop, USA). A total of 500 ng of quantified RNA was reverse transcribed from mRNA by using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) and from miRNA by using HB miR Multi Assay KitTM system Ⅰ (HeimBiotek, Korea).
The quantitative polymerase chain reaction (qPCR) primer information for
The miRNA level cDNA samples in human, male and female Western chimpanzee, female crab-eating monkey, and male mouse were used with the HB_I Real-Time PCR Master mix kit (HeimBiotek) to perform qPCR, according to the manufacturer’s protocol in a Rotor-Gene Q system, under the following qPCR conditions; initialization step at 95°C for 2 min, followed by 45 thermal cycles of 95°C for 5 s, 55°C for 10 s, and 72°C for 15 s; standard melting conditions of the ramp ranged from 55°C to 99°C, with a 1°C rise at each step. Small nuclear RNA (snRNA) U6 was used as the reference gene for normalization of relative expression analysis for miR-3681-5p (5′-UAGUGGAUGAUGCACUCUGUGC-3′). All samples were amplified in triplicates, and the relative expression data was analyzed according to the 2-ΔC t method, where ΔCt = Ct(hsa-miR-3681-5p) – Ct(U6). All the bar in the graphs were plotted as the mean ± SD.
Genomic DNA was extracted from a human cell line HEK293A using DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer’s protocol. Genomic DNA was then used for PCR amplification using the primer list shown in Table 2. The 20 µl PCR mix contained 10 µl of 2× TOPsimple DyeMix (aliquot)-HOT premix (Enzynomics, Korea), 7 µl of distilled water, 1 µl of gDNA template, and 1 µl of forward and reverse primers (10 pmole/µl) each. PCR conditions were as follows: initialization step at 95°C for 5 min, followed by 35 thermal cycles of 94°C for 40 s, annealing temperatures listed in Table 2 for 40 s, 72°C 40 s, and the final elongation step at 72°C 5 min. The PCR products were separated on a 1.5% agarose gel, and purified with ExpinTM Gel SV (GeneAll), according to the manufacturer’s protocol. The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) using the LigaFastTM Rapid DNA Ligation System (Promega), as per the manufacturer’s protocol. The cloned plasmid DNA was isolated by using ExprepTM Plasmid SV, mini (GeneAll).
The lung cancer derived cell line A549 was grown at 37°C in a 5% (v/v) CO2 incubator at Rosewell Park Memorial Institute (RPMI) (Gibco, USA), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco), and 1% (v/v) antibiotic-antimycotic (Gibco). The cells were plated in 24-well plates at a cell-density of 1 × 105 cells/well and grown to 80% confluence. After 24 h of incubation at 37°C in a 5% (v/v) CO2 incubator, the cells were transfected with psi-CHECK2 vector cloned with different
The sequence of hsa-miR-3681-5p (5′-UAGUGGAUGAUGCACUCUGUGC-3′) was downloaded from miRbase v22.1 and used for blast search using the UCSC genome browser (GRCh38), to confirm the location and the nearest gene in human chromosomes. The seed region of hsa-miR-3681-5p is 5′-AGUGGAU-3′ and hsa-miR-3681 is present in the p arm of human chromosome 2. Moreover, hsa-miR-3681 overlaps with LTR16D1. The sequence of LTR16D1 was downloaded from the UCSC genome browser to determine the analogy between target miRNA and its derived gene (Fig. 1). The alignment result shows that hsa-miR-3681-5p is derived from LTR16D1, as seen from the perfectly matched sequence.
The target gene list of hsa-miR-3681-5p was downloaded from Target Scan Human and the candidates were selected based on the highest number of total seed sites that are complementary to the 3′ UTR of the target gene (Table 1). Out of 2,424 target genes, 13 were selected from the list and
The schematic structure of
Three primers were designed from the 3′ UTR of
Relative expression analyses of hsa-miR-3681-5p and
Except for humans, the relative expression results for the female and male Western chimpanzees, crab-eating monkeys, and mice showed similar patterns for 9-mer hsa-miR-3681-5p binding sites. The male Western chimpanzees had the highest relative expression in the testis and the stomach for both one 9-mer hsa-miR-3681-5p binding primers (Fig. 3B). The female Western chimpanzees had the highest relative expression in the cerebellum for both 9-mer hsa-miR-3681-5p binding primers (Fig. 3C). The female crab-eating monkeys had the highest relative expression in the bladder for both primers (Fig. 3D). The male mice had the highest relative expression in the spleen and the lowest relative expression in the kidney and the brain, for both primers (Fig. 3E). The relative expression data in graph of all hsa-miR-3681-5p and primers are shown in Supplementary Figs. S2-S6.
To analyze the correlation between the 3′ UTR of
The 3′ UTR of
The sequence and name of the TFs near hsa-miR-3681-5p 9-mer seed regions are indicated within the box in Fig. 5. The prediction of TF provided the possibility of VNTR in the 3′ UTR of
The schematic structure of
This study focused on the idea that the number of miRNA binding sites in the 3′ UTR of a target gene will affect its expression. For evolutionary profiling, examination of the relative expression analysis on MDTE hsa-miR-3681-5p was performed by qPCR in the target gene of hsa-miR-3681-5p
We hypothesized that the number of 9-mer hsa-miR-3681-5p binding sites affects the relative expression data and the luciferase assay. Previously, a miRNA study reported that miR-185 downregulates the expression of
Another diagnosis of enhancer activity shown in both hsa-miR-3681 mimic and hsa-miR-3681-5p inhibitor is VNTRs. VNTR and miRNA studies are scarce; however, a few studies have been done on brain disease related genes. A study analyzed autism and neurotransmitter related genes and miRNAs; MAOA, MAOB, DRD2, miRNA-431, and -21 for genomic sequence variations (Salem et al., 2013). Another study revealed that VNTRs regulate miRNA-137 by alternative splicing and this event increases the risk of schizophrenia (Pacheco et al., 2019). The deletion form of miRNA-137 transcripts is more frequent when an increased number of VNTRs were detected. Similar to our report, one of the reports used the luciferase assay to check VNTR and miRNA-491 expression (Jia et al., 2016). The result shows that when miRNA-491 mimic treatment was done with different number of VNTR copies, the expression was downregulated when more copies of VNTRs were included. In contrast, the expression was upregulated when anti-miRNA-491 was treated to different quantities of VNTR copies. In our report, the both hsa-miR-3681 mimic and hsa-miR-3681-5p inhibitor treatment result shows upregulation in all luciferase assays. The promoter activity is different within the length of VNTRs in the LTR12C element and it affects the expression of
The illustration of the summary of this study can be seen in Fig. 7. miRNA derived from LTR or any of the TEs, called MDTEs, binds to the 3′ UTR of the target gene. In some cases, the 3′ UTR of target gene contains VNTRs such as
This research was supported by the Cooperative Research Program of Primate Research Institute, Kyoto University (2019-B-7) and the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07049460). We thank Kumamoto sanctuary and Primate Research Institute in Inuyama, Japan for providing the samples of Western chimpanzee male and female samples.
H.E.L. designed and performed all the experiments including writing the manuscript, J.W.H. and S.J.P. provided the crab-eating monkey samples, H.I. provided the Western chimpanzee samples, and H.S.K. commented and revised the manuscript.
The authors have no potential conflicts of interest to disclose.
The list of target gene candidates of hsa-miR-3681-5p
Gene name | Abbreviation | Coordinates | Transcript ID | Total sites | 8mer | 7mer-m8 | 7mer-A1 | 6mer |
---|---|---|---|---|---|---|---|---|
ADP-ribosylation factor-like 10 | ARL10 | chr5:176,365,373-176,381,963 | ENST00000310389.5 | 4 | 0 | 2 | 2 | 2 |
Solute carrier family 8 (sodium/calcium exchanger), member 2 | SLC8A2 | chr19:47,428,017-47,471,893 | ENST00000236877.6 | 7 | 0 | 0 | 7 | 4 |
Myopalladin | MYPN | chr10:68,105,215-68,211,985 | ENST00000358913.5 | 4 | 1 | 1 | 2 | 1 |
Zinc finger and BTB domain containing 37 | ZBTB37 | chr1:173,868,082-173,903,547 | ENST00000367701.5 | 4 | 0 | 4 | 0 | 2 |
K(lysine) acetyltransferase 7 | KAT7 | chr17:49,788,681-49,835,026 | ENST00000259021.4 | 4 | 0 | 2 | 2 | 5 |
Glutamate receptor, ionotropic, N-methyl D-aspartate 2B | GRIN2B | chr12:13,537,337-13,980,356 | ENST00000609686.1 | 5 | 2 | 1 | 2 | 5 |
Lysine (K)-specific demethylase 5A | KDM5A | chr12:280,057-389,320 | ENST00000399788.2 | 5 | 0 | 3 | 2 | 4 |
RNA binding motif protein 28 | RBM28 | chr7:128,297,685-128,343,908 | ENST00000223073.2 | 4 | 1 | 1 | 2 | 4 |
Neuronal growth regulator 1 | NEGR1 | chr1:71,395,943-72,282,539 | ENST00000357731.5 | 4 | 1 | 2 | 1 | 4 |
Zinc finger and BTB domain containing 20 | ZBTB20 | chr3:114,304,949-115,155,228 | ENST00000462705.1 | 4 | 0 | 3 | 1 | 3 |
Peroxisomal biogenesis factor 26 | PEX26 | chr22:18,077,990-18,105,396 | ENST00000329627.7 | 4 | 1 | 3 | 0 | 0 |
Peptidylprolyl isomerase (cyclophilin)-like 4 | PPIL4 | chr6:149,504,495-149,546,043 | ENST00000340881.2 | 4 | 0 | 3 | 1 | 3 |
The list of primer information on SHISA7 and the reference gene
Primer | Sequences | Temperature (°C) | Details |
---|---|---|---|
9mer1 | F: CATCTCCAGGGATCCACTTC | 55 | One 9mer hsa-miR-3681-5p in 3’ UTR of |
R: GACACCAGGGTTATGGTGGA | |||
9mer3 | F: AGGATCCACGAGAGCCAAT | 59 | Three 9mer hsa-miR-3681-5p in 3’ UTR of |
R: ATGGTGACAGACAGCACTGG | |||
9mer6 | F: CACTACCTCCCAGTATCCA | 55 | Six 9mer hsa-miR-3681-5p in 3’ UTR of |
R: ATGGTTGCAGTGGACTCT | |||
F: GAA ATC CCA TCA CCA TCT TCC AGG | 55 | The reference gene; Glyceraldehyde 3-phosphate dehydrogenase ( | |
R: GAG CCC CAG CCT TCT CCA TG |
Each primer was designed based on the quantity of 9mer hsa-miR-3681-5p binding sites. The name of the primer, sequence of each forward (F) and reverse (R) primer, annealing temperature and details about the primer is shown in the table.
Mol. Cells 2020; 43(7): 607-618
Published online July 31, 2020 https://doi.org/10.14348/molcells.2020.0058
Copyright © The Korean Society for Molecular and Cellular Biology.
Hee-Eun Lee1,2,3 , Sang-Je Park3
, Jae-Won Huh3,4
, Hiroo Imai5
, and Heui-Soo Kim2,6, *
1Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea, 2Institute of Systems Biology, Pusan National University, Busan 46241, Korea, 3National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Korea, 4Department of Functional Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB) School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Korea, 5Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan, 6Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 46241, Korea
Correspondence to:khs307@pusan.ac.kr
microRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. miRNAs inhibit gene expression by binding to the 3′ untranslated region (UTR) of their target gene. miRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p share the binding sites in the 3’ UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p to inhibit the activity of VNTR. In conclusion, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p acts as a repressor of VNTR activity in the 3′ UTR of SHISA7.
Keywords: long terminal repeat, miR-3681-5p, SHISA7, transposable elements, variable number tandem repeat
Transposable elements (TEs) comprise approximately half of the eukaryotic genome and are known to insert into the genome as stable genomic components (Bourque et al., 2018; Wicker et al., 2007). TEs are divided into two classes—retrotransposons (class I) and DNA transposons (class II). Retrotransposons use the ‘copy and paste’ mechanism. The retrotransposons are first transcribed into an RNA intermediate, which is reverse transcribed into a DNA intermediate (Agren and Clark, 2018; Bourque et al., 2018; Lander et al., 2001; Wicker et al., 2007). Long terminal repeat (LTR) is a member of the retrotransposon family and the composition of LTR in humans is approximately 9%. An LTR consists of innate enhancer activity, however, transcription factor (TF), nucleotide substitutions, transcription start sites, and epigenetics may provide alternative enhancer activity of LTR elements (Thompson et al., 2016). LTRs recruit TF and enhance the transcription of host genes (Gonzalez-Cao et al., 2016; Hu et al., 2017). TEs can generate regulators of genes, such as microRNAs (miRNAs), TFs, and variable number tandem repeats (VNTRs) (Feschotte, 2008; Lee et al., 2019; Piriyapongsa et al., 2007; Qin et al., 2015; Roberts et al., 2014; Sabino et al., 2014).
miRNAs are small non-coding RNA molecules, approximately 19 to 24 nucleotides long, which are essential for gene regulation (Ambros et al., 2003; Bartel, 2004; Kim, 2005; O'Brien et al., 2018). miRNAs have a unique seed region, approximately six nucleotides long, that binds to the 3′ untranslated region (UTR) of the target mRNA. In most of cases, miRNAs regulate gene expression by inhibiting gene expression; however, recent study has reported the presence of enhancer miRNAs (Lee et al., 2019). In addition, some miRNAs bind to the 5′ UTR of the target transcripts and to coding sequences, to regulate target gene expression (Brummer and Hausser, 2014; Gu et al., 2014; Hausser et al., 2013; Lee et al., 2009; Li et al., 2016; Liu et al., 2015). In studies on miRNA, the number of miRNA binding sites in the target gene was not an important consideration in selecting the best target genes. Previous studies have suggested that miRNAs derived from transposable element (MDTE) and MDTEs share sequences (Lee et al., 2019; Petri et al., 2019; Piriyapongsa and Jordan, 2007; Piriyapongsa et al., 2007; Qin et al., 2015; Roberts et al., 2014). Hsa-miR-3681 is derived from an LTR element and has not been studied much. A few studies on hsa-miR-3681 have reported on its dysregulation in human disease and cancer and downregulation in cervical cancer (Shi et al., 2019; Vaira et al., 2014; Vaz et al., 2010).
One of target genes for hsa-miR-3681-5p,
In this study, the relative expression of the MDTEs, hsa-miR-3681-5p and its target
Experiments with the Western chimpanzee were carried out in accordance with the guidelines and regulations approved by the Animal Experimentation Committees of Kyoto University (2018-004). Experiments with Crab-eating monkey were carried out in accordance with guidelines and regulation approved by Korea Research Institute of Bioscience & Biotechnology (KRIBB-AEC-15046).
The sequence of hsa-miR-3681-5p was downloaded from miRbase v22.1 (http://www.mirbase.org) (Kozomara et al., 2019), and then the sequence was used to localized in human genome (GRCh38) and to download sequence of LTR16D1 by UCSC Genome Browser (http://genome.ucsc.edu) (Agarwal et al., 2015). The sequences of hsa-miR-3681-5p and LTR16D1 were aligned using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999) to compare the analogy. TargetScanHuman (http://www.targetscan.org/vert_72/) (Agarwal et al., 2015) was used to list out the target gene candidates of hsa-miR-3681-5p (Table 1).
The structural interaction between hsa-miR-3681-5p and the 3′ UTR of
The putative TF binding sites near the 9.5 VNTRs of the 3′ UTR of
Total RNAs from 13 human tissues (brain, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, testis, prostate, and uterus) and 10 mouse tissues (brain, heart, lung, liver, kidney, spleen, stomach, smooth muscle, testis, and spinal cord) were purchased from Clontech (USA).
A total of 14 female crab-eating monkey tissue samples (cerebellum, cerebrum, heart, lung, liver, kidney, spleen, stomach, small intestine, large intestine, uterus, pancreas, bladder, and spinal cord) were provided by the National Primate Research Center (Korea). Total RNA was extracted from 14 tissue samples of the female crab-eating monkey by using Hybrid-RTM (GeneAll, Korea), according to the manufacturer’s instructions.
Tissue samples from the female Western chimpanzee were provided from the Kumamoto sanctuary, Kyoto University, to Primate Research Institute in Inuyama, Japan, via GAIN and the male Western chimpanzee samples were provided by the Primate Research Institute. All experiments using samples from the Western chimpanzee were done at the Primate Research Institute. Total RNA was extracted from 11 tissues of healthy male Western chimpanzees (cerebellum, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, and testis) and female Western chimpanzees (cerebellum, heart, lung, liver, kidney, spleen, stomach, small intestine, colon, muscle, and ovary) by using Hybrid-RTM, according to the manufacturer’s instructions.
The RNA samples were quantitated using the ND-1000 UV-Vis spectrophotometer (NanoDrop, USA). A total of 500 ng of quantified RNA was reverse transcribed from mRNA by using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) and from miRNA by using HB miR Multi Assay KitTM system Ⅰ (HeimBiotek, Korea).
The quantitative polymerase chain reaction (qPCR) primer information for
The miRNA level cDNA samples in human, male and female Western chimpanzee, female crab-eating monkey, and male mouse were used with the HB_I Real-Time PCR Master mix kit (HeimBiotek) to perform qPCR, according to the manufacturer’s protocol in a Rotor-Gene Q system, under the following qPCR conditions; initialization step at 95°C for 2 min, followed by 45 thermal cycles of 95°C for 5 s, 55°C for 10 s, and 72°C for 15 s; standard melting conditions of the ramp ranged from 55°C to 99°C, with a 1°C rise at each step. Small nuclear RNA (snRNA) U6 was used as the reference gene for normalization of relative expression analysis for miR-3681-5p (5′-UAGUGGAUGAUGCACUCUGUGC-3′). All samples were amplified in triplicates, and the relative expression data was analyzed according to the 2-ΔC t method, where ΔCt = Ct(hsa-miR-3681-5p) – Ct(U6). All the bar in the graphs were plotted as the mean ± SD.
Genomic DNA was extracted from a human cell line HEK293A using DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer’s protocol. Genomic DNA was then used for PCR amplification using the primer list shown in Table 2. The 20 µl PCR mix contained 10 µl of 2× TOPsimple DyeMix (aliquot)-HOT premix (Enzynomics, Korea), 7 µl of distilled water, 1 µl of gDNA template, and 1 µl of forward and reverse primers (10 pmole/µl) each. PCR conditions were as follows: initialization step at 95°C for 5 min, followed by 35 thermal cycles of 94°C for 40 s, annealing temperatures listed in Table 2 for 40 s, 72°C 40 s, and the final elongation step at 72°C 5 min. The PCR products were separated on a 1.5% agarose gel, and purified with ExpinTM Gel SV (GeneAll), according to the manufacturer’s protocol. The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) using the LigaFastTM Rapid DNA Ligation System (Promega), as per the manufacturer’s protocol. The cloned plasmid DNA was isolated by using ExprepTM Plasmid SV, mini (GeneAll).
The lung cancer derived cell line A549 was grown at 37°C in a 5% (v/v) CO2 incubator at Rosewell Park Memorial Institute (RPMI) (Gibco, USA), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco), and 1% (v/v) antibiotic-antimycotic (Gibco). The cells were plated in 24-well plates at a cell-density of 1 × 105 cells/well and grown to 80% confluence. After 24 h of incubation at 37°C in a 5% (v/v) CO2 incubator, the cells were transfected with psi-CHECK2 vector cloned with different
The sequence of hsa-miR-3681-5p (5′-UAGUGGAUGAUGCACUCUGUGC-3′) was downloaded from miRbase v22.1 and used for blast search using the UCSC genome browser (GRCh38), to confirm the location and the nearest gene in human chromosomes. The seed region of hsa-miR-3681-5p is 5′-AGUGGAU-3′ and hsa-miR-3681 is present in the p arm of human chromosome 2. Moreover, hsa-miR-3681 overlaps with LTR16D1. The sequence of LTR16D1 was downloaded from the UCSC genome browser to determine the analogy between target miRNA and its derived gene (Fig. 1). The alignment result shows that hsa-miR-3681-5p is derived from LTR16D1, as seen from the perfectly matched sequence.
The target gene list of hsa-miR-3681-5p was downloaded from Target Scan Human and the candidates were selected based on the highest number of total seed sites that are complementary to the 3′ UTR of the target gene (Table 1). Out of 2,424 target genes, 13 were selected from the list and
The schematic structure of
Three primers were designed from the 3′ UTR of
Relative expression analyses of hsa-miR-3681-5p and
Except for humans, the relative expression results for the female and male Western chimpanzees, crab-eating monkeys, and mice showed similar patterns for 9-mer hsa-miR-3681-5p binding sites. The male Western chimpanzees had the highest relative expression in the testis and the stomach for both one 9-mer hsa-miR-3681-5p binding primers (Fig. 3B). The female Western chimpanzees had the highest relative expression in the cerebellum for both 9-mer hsa-miR-3681-5p binding primers (Fig. 3C). The female crab-eating monkeys had the highest relative expression in the bladder for both primers (Fig. 3D). The male mice had the highest relative expression in the spleen and the lowest relative expression in the kidney and the brain, for both primers (Fig. 3E). The relative expression data in graph of all hsa-miR-3681-5p and primers are shown in Supplementary Figs. S2-S6.
To analyze the correlation between the 3′ UTR of
The 3′ UTR of
The sequence and name of the TFs near hsa-miR-3681-5p 9-mer seed regions are indicated within the box in Fig. 5. The prediction of TF provided the possibility of VNTR in the 3′ UTR of
The schematic structure of
This study focused on the idea that the number of miRNA binding sites in the 3′ UTR of a target gene will affect its expression. For evolutionary profiling, examination of the relative expression analysis on MDTE hsa-miR-3681-5p was performed by qPCR in the target gene of hsa-miR-3681-5p
We hypothesized that the number of 9-mer hsa-miR-3681-5p binding sites affects the relative expression data and the luciferase assay. Previously, a miRNA study reported that miR-185 downregulates the expression of
Another diagnosis of enhancer activity shown in both hsa-miR-3681 mimic and hsa-miR-3681-5p inhibitor is VNTRs. VNTR and miRNA studies are scarce; however, a few studies have been done on brain disease related genes. A study analyzed autism and neurotransmitter related genes and miRNAs; MAOA, MAOB, DRD2, miRNA-431, and -21 for genomic sequence variations (Salem et al., 2013). Another study revealed that VNTRs regulate miRNA-137 by alternative splicing and this event increases the risk of schizophrenia (Pacheco et al., 2019). The deletion form of miRNA-137 transcripts is more frequent when an increased number of VNTRs were detected. Similar to our report, one of the reports used the luciferase assay to check VNTR and miRNA-491 expression (Jia et al., 2016). The result shows that when miRNA-491 mimic treatment was done with different number of VNTR copies, the expression was downregulated when more copies of VNTRs were included. In contrast, the expression was upregulated when anti-miRNA-491 was treated to different quantities of VNTR copies. In our report, the both hsa-miR-3681 mimic and hsa-miR-3681-5p inhibitor treatment result shows upregulation in all luciferase assays. The promoter activity is different within the length of VNTRs in the LTR12C element and it affects the expression of
The illustration of the summary of this study can be seen in Fig. 7. miRNA derived from LTR or any of the TEs, called MDTEs, binds to the 3′ UTR of the target gene. In some cases, the 3′ UTR of target gene contains VNTRs such as
This research was supported by the Cooperative Research Program of Primate Research Institute, Kyoto University (2019-B-7) and the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07049460). We thank Kumamoto sanctuary and Primate Research Institute in Inuyama, Japan for providing the samples of Western chimpanzee male and female samples.
H.E.L. designed and performed all the experiments including writing the manuscript, J.W.H. and S.J.P. provided the crab-eating monkey samples, H.I. provided the Western chimpanzee samples, and H.S.K. commented and revised the manuscript.
The authors have no potential conflicts of interest to disclose.
. The list of target gene candidates of hsa-miR-3681-5p.
Gene name | Abbreviation | Coordinates | Transcript ID | Total sites | 8mer | 7mer-m8 | 7mer-A1 | 6mer |
---|---|---|---|---|---|---|---|---|
ADP-ribosylation factor-like 10 | ARL10 | chr5:176,365,373-176,381,963 | ENST00000310389.5 | 4 | 0 | 2 | 2 | 2 |
Solute carrier family 8 (sodium/calcium exchanger), member 2 | SLC8A2 | chr19:47,428,017-47,471,893 | ENST00000236877.6 | 7 | 0 | 0 | 7 | 4 |
Myopalladin | MYPN | chr10:68,105,215-68,211,985 | ENST00000358913.5 | 4 | 1 | 1 | 2 | 1 |
Zinc finger and BTB domain containing 37 | ZBTB37 | chr1:173,868,082-173,903,547 | ENST00000367701.5 | 4 | 0 | 4 | 0 | 2 |
K(lysine) acetyltransferase 7 | KAT7 | chr17:49,788,681-49,835,026 | ENST00000259021.4 | 4 | 0 | 2 | 2 | 5 |
Glutamate receptor, ionotropic, N-methyl D-aspartate 2B | GRIN2B | chr12:13,537,337-13,980,356 | ENST00000609686.1 | 5 | 2 | 1 | 2 | 5 |
Lysine (K)-specific demethylase 5A | KDM5A | chr12:280,057-389,320 | ENST00000399788.2 | 5 | 0 | 3 | 2 | 4 |
RNA binding motif protein 28 | RBM28 | chr7:128,297,685-128,343,908 | ENST00000223073.2 | 4 | 1 | 1 | 2 | 4 |
Neuronal growth regulator 1 | NEGR1 | chr1:71,395,943-72,282,539 | ENST00000357731.5 | 4 | 1 | 2 | 1 | 4 |
Zinc finger and BTB domain containing 20 | ZBTB20 | chr3:114,304,949-115,155,228 | ENST00000462705.1 | 4 | 0 | 3 | 1 | 3 |
Peroxisomal biogenesis factor 26 | PEX26 | chr22:18,077,990-18,105,396 | ENST00000329627.7 | 4 | 1 | 3 | 0 | 0 |
Peptidylprolyl isomerase (cyclophilin)-like 4 | PPIL4 | chr6:149,504,495-149,546,043 | ENST00000340881.2 | 4 | 0 | 3 | 1 | 3 |
. The list of primer information on SHISA7 and the reference gene.
Primer | Sequences | Temperature (°C) | Details |
---|---|---|---|
9mer1 | F: CATCTCCAGGGATCCACTTC | 55 | One 9mer hsa-miR-3681-5p in 3’ UTR of |
R: GACACCAGGGTTATGGTGGA | |||
9mer3 | F: AGGATCCACGAGAGCCAAT | 59 | Three 9mer hsa-miR-3681-5p in 3’ UTR of |
R: ATGGTGACAGACAGCACTGG | |||
9mer6 | F: CACTACCTCCCAGTATCCA | 55 | Six 9mer hsa-miR-3681-5p in 3’ UTR of |
R: ATGGTTGCAGTGGACTCT | |||
F: GAA ATC CCA TCA CCA TCT TCC AGG | 55 | The reference gene; Glyceraldehyde 3-phosphate dehydrogenase ( | |
R: GAG CCC CAG CCT TCT CCA TG |
Each primer was designed based on the quantity of 9mer hsa-miR-3681-5p binding sites. The name of the primer, sequence of each forward (F) and reverse (R) primer, annealing temperature and details about the primer is shown in the table..
Kyung Won Kim
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