Abstract

   MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate
   gene or protein expression by targeting mRNAs and triggering either
   translational repression or mRNA degradation. Distinct expression
   levels of miRNAs, including miR-29b, have been detected in various
   biological fluids and tissues from a large variety of disease models.
   However, how miRNAs “react” and function in different cellular
   environments is still largely unknown. In this study, the regulation
   patterns of miR-29b between human and mouse cell lines were compared
   for the first time. CRISPR/Cas9 gene editing was used to stably
   knockdown miR-29b in human cancer HeLa cells and mouse fibroblast
   NIH/3T3 cells with minimum off-targets. Genome editing revealed
   mir-29b-1, other than mir-29b-2, to be the main source of generating
   mature miR-29b. The editing of miR-29b decreased expression levels of
   its family members miR-29a/c via changing the tertiary structures of
   surrounding nucleotides. Comparing transcriptome profiles of human and
   mouse cell lines, miR-29b displayed common regulation pathways
   involving distinct downstream targets in macromolecular complex
   assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling
   pathways; miR-29b also demonstrated specific functions reflecting cell
   characteristics, including fibrosis and neuronal regulations in NIH/3T3
   cells and tumorigenesis and cellular senescence in HeLa cells.

   Subject terms: Gene regulation, RNA metabolism

Introduction

   MicroRNAs are a class of 18–24 nucleotides long small non-coding RNAs
   that affect cellular gene and protein expression by modulating the
   stability and translational efficiency of their target messenger RNAs
   (mRNAs)^[32]1. miRNAs have been discovered in various organisms, and
   exhibited their critical roles in diverse pathological processes and as
   potential therapeutic targets for treating diseases^[33]2,[34]3. The
   biogenesis of miRNAs starts with RNA polymerase II or III dependent
   transcription of miRNA gene in the nucleus, which generates a long
   primary miRNA (pri-miRNA) that can be several hundred nucleotides to
   kilobases in length^[35]1,[36]4. Pri-miRNAs are then cleaved into
   approximately 70 nucleotides hairpin structured precursor miRNAs
   (pre-miRNAs), which are transported to cytoplasm and undergo further
   cleavage to form highly unstable miRNA duplexes^[37]5,[38]6. The
   passenger strand of the duplex usually forms miRNA* which usually goes
   through rapid degradation, whereas the guide strand steers miRNA
   induced silencing complex (miRISC) to target mRNA transcripts^[39]6.
   The nucleotide 2–8 from the 5′ end of mature miRNA is called the seed
   sequence, which can bind to the 3′ UTR of mRNA through Watson-Crick
   base pairing and induce translational repression and/or mRNA
   degradation of target mRNA^[40]1. Nearly half of miRNA genes are found
   in tandem within clusters and share the same promoters^[41]1,[42]7,
   underpinning the similar regulatory features of these miRNAs.

   miR-29b belongs to the miR-29 family, which is consisted of miR-29a,
   miR-29b-1, miR-29b-2 and miR-29c^[43]8–[44]10. miR-29b-1 and miR-29b-2
   are transcribed from different genome loci but have identical mature
   sequences, thus they are both termed miR-29b^[45]8–[46]10. In humans,
   miR-29a and miR-29b-1 are located at chromosome 7q32, separated by 652
   bases, and have the same pri-miRNA transcripts, whereas miR-29b-2 and
   miR-29c are separated by 507 bases at chromosome 1q32, and are
   transcribed into the same primary miRNAs^[47]11,[48]12. They are called
   miR-29a/b-1 cluster and miR-29b-2/c cluster respectively, due to the
   close localizations and that they share the same promoters^[49]12. For
   mir-29b genes, nucleotide 11–32 forms miR-29b*, and nucleotides 52–74
   forms mature functional miR-29b^[50]13.

   miR-29b has been associated with various disorders including fibrotic
   diseases, cancers, and neurodegenerative diseases^[51]14–[52]16. miR-29
   family members are critical regulators of extracellular matrix (ECM)
   proteins and signalling pathways associated with fibrosis via targeting
   collagens, fibrillins, and elastin^[53]16. miR-29b can support
   osteoblast differentiation either by inhibiting the accumulation of
   extracellular matrix proteins COL1A1, COL5A3 and COL4A2, or by directly
   downregulating inhibitors of osteoblast differentiation, such as HDAC4,
   TGFβ3, ACVR2A, CTNNBIP1 and DUSP2^[54]17. Also, miR-29b directly
   regulates CDK6 (cell cycle dependent kinase 6), which is responsible
   for retinoblastoma (Rb) protein phosphorylation, in acute myeloid
   leukemia (AML)^[55]11, mantel cell lymphoma (MCL)^[56]18 and in
   cervical carcinogenesis^[57]19. In addition, miR-29 family expression
   is markedly upregulated in normal aging mice and in response to DNA
   damage, involving a potential miR-29-Ppm1d phosphatase-p53 regulatory
   feedback loop^[58]20. miR-29b is highly expressed in brains and has
   shown dysregulated expression levels in neurodegenerative
   disorders^[59]21,[60]22. miR-29b can target BACE1 in sporadic AD
   patients^[61]21, in cases of spinocerebellar ataxia 17^[62]23, in brain
   development of mice and in primary neuronal cultures^[63]21. miR-29b
   was reported to regulated human secreted glycoprotein – progranulin,
   which is involved in frontotemporal dementia^[64]24. miR-29b is also
   among a list of miRNAs that were upregulated in exosomes released from
   prion disease cell model^[65]25.

   With the function diversity of miR-29b, it is speculated that miR-29b
   may exhibit cellular environment specific regulation patterns. Studies
   have shown the cell type/disease specific miRNA signatures^[66]26.
   However, there is a lack of studies that examine the specificity of
   miRNA regulation between two cellular environments. In particular
   miR-29b, a miRNA that has been implicated in various disease disorders,
   whereby identifying the regulation patterns of miR-29b would provide
   reference and guidance for its potential therapeutic usage. In this
   study we systematically designed and revealed details of the
   specificity and consistency in miR-29b regulations using the same
   editing method and experimental approaches. Using this system, we
   investigated gene regulation via miRNA clusters, mature miRNA
   generation, and differential gene expression (DEG) profiles induced by
   miR-29b stable knockdown between two cell lines. This study provides a
   comprehensive analysis into understanding the regulatory patterns of
   miR-29b in different cellular environments and species.

Results

CRISPR/Cas9 mediated stable knockdown of miR-29b in NIH/3T3 and HeLa cells

   Five different human and mouse cell lines were screened for the
   expression levels of miR-29b (Supplementary [67]1). The human
   epithelial cervix adenocarcinoma cells - HeLa cells^[68]27, and the
   mouse embryo fibroblast cells - NIH/3T3 cells^[69]28 were selected to
   establish miR-29b knockdown clones using CRISPR/Cas9 engineering, due
   to their robust expression levels of miR-29b and distinct features of
   these two cell lines (Supplementary [70]1).

   miR-29b gRNAs were designed by submitting the whole length of miR-29b
   gene sequences, including hsa-mir-29b-1, hsa-mir-29b-2, mmu-mir-29b-1
   and mmu-mir-29b-2, to [71]http://crispr.mit.edu tool. According to gRNA
   design principles, gRNAs with quality score over 55 have higher
   specificity and less predicted off-targets when applied for gene
   editing, and were thus selected for miR-29b editing. The gRNAs, their
   nucleotide sequences, the targeting localizations on the gene loci,
   quality scores, numbers of predicted off-targets and ‘in-gene’
   off-targets are illustrated (Fig. [72]1a). Mouse gRNA m-cas1 was
   designed to target the 5′ end of mmu-mir-29b-1 gene, with 23 potential
   ‘in-gene’ off-targets out of 307 predicted off-targets; m-cas2 and
   m-cas3 were used to target mmu-mir-29b-2 at the 5′ end of mmu-mir-29b-2
   in NIH/3T3 cells, with 31 and 34 potential ‘in-gene’ off-targets
   respectively (Fig. [73]1a,b). H-cas1 has the same nucleotide sequences
   with m-cas1, and is the only gRNA with quality score over 55 for
   targeting gene hsa-mir-29b-1, thus is the only gRNA used for target
   hsa-mir-29b-1 in HeLa cells (Fig. [74]1a). H-cas1 is predicted to have
   322 off-targets, with 32 of them coding for genes (Fig. [75]1b). No
   gRNAs with high quality were available for has-mir-29b-2 due to the
   short length of primary miRNAs.

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   CRISPR/Cas9 mediated miR-29b knockdown in NIH/3T3 and HeLa cells. (a)
   The gRNAs were designed by submitting the whole length of
   hsa-mir-29b-1, mmu-mir-29b-1, mmu-mir-29b-2 onto gRNA design website
   [78]http://crispr.mit.edu. The gRNA quality scores were calculated
   based on the number of potential off-targets and the number of in genes
   off-targets. (b) The gRNAs h-cas1, m-cas1, m-cas2 and m-cas3 locations,
   sequences and their PAMs were shown on the whole length sequences of
   hsa-mir-29b-1, mmu-mir-29b-1, mmu-mir-29b-2, respectively. (c) The
   expression levels of miR-29b in HeLa cell clones detected by qRT-PCR.
   (d) The expression levels of miR-29b in NIH/3T3 cells detected by
   qRT-PCR. Data shown represent the mean values of three independent
   experiments. Statistical analysis was performed using paired Student’s
   t-tests. *p < 0.05, ***p < 0.001, ****p < 0.0001.

   These four gRNAs were inserted into CRISPR plasmid px458 respectively;
   the reconstructed plasmids were termed h-cas1, m-cas1, m-cas2 and
   m-cas3 for further references. The blank vector px458 was used as the