Characterization of HMGA1P6 transgenic mouse embryonic fibroblasts

ABSTRACT

Latest studies have shown that deregulated pseudogene transcripts contribute to cancer working as competing endogenous RNAs. Our research group has recently demonstrated that the overexpression of two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, has a critical role in cancer progression. These pseudogenes work sustaining the expression of HMGA1 and other cancer-related genes. We generated a mouse model overexpressing HMGA1P6 to better study the HMGA1-pseudogene function in a more physiological context. Here, we show the proliferation rate and the susceptibility to senescence of mouse embryonic fibroblasts obtained from HMGA1P6-overexpressing mice to better characterize the HMGA1-pseudogene function. Indeed, our study reports that mouse embryonic fibroblasts (MEFs) derived from HMGA1P6 mice express higher HMGA1 mRNA and protein levels. Moreover, these cells grow faster and senesce later than wild-type sustaining the oncogenic role of ceRNA crosstalk mediated by HMGA1Ps.

Background

Around 70–90% of the genome is transcribed into RNAs but just 1–2% of our genes code for functional proteins. What about the rest of these RNAs? Today, the main objective of the scientific community is to reveal the function of these “non-coding” RNAs (ncRNAs). Currently, several ncRNAs have been found, such as long non-coding RNAs (lncRNAs) [1], circular RNAs (circRNAs) [2], microRNAs (miRNAs) [3] and pseudogenes [4]. Many of these latter are considered as a class of defunct copies of protein-coding genes generated by mutations or random replications during the evolution. Pseudogenes are classified into two types: processed and non-processed considering whether they arise from gene retrotransposition or gene duplication, respectively [4]. For many years they have been considered as junk, since they derive from functional genes but they lost the ability to produce proteins. However, recent advances have underlined a key role for pseudogenes in cellular physiology and several diseases [4,5]. Two pseudogenes, HMGA1P6 and HMGA1P7 (HMGA1Ps), have been recently isolated in our laboratory [6,7]. They were generated from HMGA1 oncogene [8–10] by an ancestral retrotransposition. HMGA1-pseudogenes show only a small number of mismatches in their mRNAs with respect to HMGA1, and they have been found only in human genome [4,6,7]. Therefore, HMGA1Ps are modulated by HMGA1-targeting miRNAs since they have practically the same microRNA Responsive Elements (MREs). Consequently, an increasing number of HMGA1-pseudogene transcripts derepress HMGA1 and other miRNA-sharing mRNAs from the inhibition promoted by miRNAs, generating the so-called competitive endogenous RNA (ceRNA) mechanism [11,12]. As expected, several human cancer types show the upregulation of HMGA1-pseudogenes such as thyroid carcinoma, pituitary tumors, ovarian carcinoma, breast carcinoma, larynx carcinoma [4,6,7,11–13]. Intriguingly, in these tumors, HMGA1Ps overexpression is positively correlated with HMGA1 amounts and other cancer-associated genes (HMGA2, VEGF, EZH2, IGF2, H19, EGR1) that suffer the same microRNAs inhibiting activity. Thus, the overexpression of HMGA1Ps may switch on an oncogenic ceRNA net, where mRNAs can dialogue through the competition for microRNAs [4,6,7,11–13]. Then, to test the HMGA1-pseudogene function in a more physiological context, we produced an overexpressing mouse model for both HMGA1P6 and HMGA1P7 [7,11,12]. Considering that embryonic fibroblasts (MEFs) from HMGA1P7-overexpressing transgenic mice displayed a higher HMGA1 expression level, and consequently, a higher growth rate and a reduced susceptibility to senescence, the aim of this study is to evaluate whether the MEFs derived from HMGA1P6 mice show the same behavior of that of HMGA1P7 ones.

Methods

Cell culture

12.5-day-old embryos from HMGA1P6 and WT mice were minced and used to establish MEF single-cell suspensions. Primary MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 1% glutamine (Thermofisher, Waltham, MA, USA), 1% penicillin/streptomycin and 1% gentamicin (Thermofisher, Waltham, MA, USA). The cells (4 × 10⁵ cells/dish) were plated in a series of 6-cm culture dishes and counted daily with a Burker chamber for 8 consecutive days to extrapolate growth curves.

Mouse embryo samples

The use of mouse embryos experiments performed in this study were approved by the Ministero della Salute (project “Ruolo degli pseudogeni di HMGA1 nel cancro” Cod. 893/2013). The methods and experiments were carried out in accordance with the approved guidelines by the Ministero della Salute in complaints with the European Communities Council Directive (63/2010/EEC).

RNA extraction and quantitative reverse transcription PCR

Total RNA was extracted from tissues and cell cultures with Trizol (Thermofisher, Waltham, MA, USA) according to the manufacturer’s instructions [14]. For mRNA detection, we reverse-transcribed total RNA from cell lines by using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany), and then Real-time PCR was performed by using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the following primers:

HMGA1P6-Fw 5ʹ-gcagacccacaaaactgga-3ʹ HMGA1P6-Rev 5ʹ-gagcaaagctgtcccatcc-3ʹ

Hmga1-Fw 5ʹ-ggcagacccaagaaactgg-3ʹ Hmga1-Rev 5ʹ-ggcactgcgagtggtgat-3ʹ

G6pd-Fw 5ʹ-cagcggcaactaaactcaga-3ʹ G6pd-Rev 5ʹ-ttccctcaggatcccacac-3ʹ

The 2^−ΔΔCt formula was used to calculate the differential gene expression.

Protein extraction, western blotting and antibodies

Protein extraction and Western blotting were performed as previously reported [15,16]. Briefly, for total cell extracts, MEFs were lysed in RIPA buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P40, and a mix of protease inhibitors), and centrifugated at 13,000 rpm at 4°C for 30 min. Proteins were run on 12.5% denaturing condition polyacrylamide gels and transferred to nitrocellulose filters. Membranes were incubated with 5% BSA in TTBS and then with the specific primary antibodies. The primary antibodies used were anti-γ-Tubulin (sc-17787) and anti-HMGA1, this latter has been described elsewhere [17]. Blots were visualized by using the Western blotting detection reagents (GE Healthcare, Chicago, IL, USA).

SA-β-gal assay

Briefly, 4 × 10⁴ MEFs were plated 24 hours before the assay. The day after, MEFs were washed with PBS and incubated with a fixation buffer (2% [w/v] formaldehyde, 0.2% [w/v] glutaraldehyde in PBS) for 10 minutes. Then, MEFs were washed 3 times with PBS and stained overnight in the staining solution (40 mM citric acid/sodium phosphate, pH 6.0; 150 mM NaCl; 2.0 mM MgCl2; 1 mg/ml X-gal) at 37°C. The following day, the staining solution was substituted by PBS, and, by light microscopy (at least 24 fields), the stained and unstained MEFs were counted.

Statistical analysis

Data were analyzed using a two-sided unpaired Mann–Whitney test (GraphPad Prism, GraphPad Software, Inc.). Values of P < 0.05 were considered statistically significant. The mean +/− s.d. of three or more independent experiments is reported.

Results

The expression of HMGA1P6 in MEFs explanted from HMGA1P6 transgenic mice was verified by qRT-PCR. HMGA1P6 mRNA was present in HMGA1P6 MEFs and absent in the WT counterpart (Figure 1), since HMGA1P6 has been found only in human genome. Interestingly, both HMGA1 mRNA and protein levels were upregulated (~1.4-Fold Change) in HMGA1P6-overexpressing MEFs compared with their WT control (Figure 1). These data are in agreement with our previous results obtained on HMGA1P7 MEFs. Moreover, the growth rate of these HMGA1P6-overexpressing MEFs was significantly higher than that of the WT controls (~50% less) (Figure 2). Then, by measuring senescence-associated β-gal (SA-β-gal) activity, we tested the susceptibility of MEFs to senescence. Indeed, at culture passage 6, SA-β-gal activity was found in WT MEFs, whereas it was strongly reduced in the HMGA1P6-overexpressing counterparts (Figure 2). These results indicate that HMGA1P6 overexpression decreased the predisposition to cellular senescence.

Figure 1.

HMGA1 is upregulated in HMGA1P6-overexpressing MEFs. (a) Relative levels of HMGA1P6 mRNA from WT and three different HMGA1P6 transgenic MEF preparations by qRT-PCR analysis. (b) (upper panel) Relative levels of HMGA1 mRNA in WT and three different HMGA1P6 transgenic MEF preparations by qRT-PCR analysis. (c) Western blot analysis and relative densitometric values of HMGA1 protein expression in the same samples of (B). The results are reported as the mean of values with error bars indicating SD (mean ± SD).

Figure 2.

HMGA1P6-overexpressing MEFs show a higher growth rate, and lower susceptibility to senescence. (a) MEFs were prepared from WT and HMGA1P6-overexpressing embryos at 12.5 dpc. At culture passage 3, they were plated and counted daily for 8 days. The results are reported as the mean of values with error bars indicating SD (mean ± SD). **, P < 0.01 (Mann-Whitney test). (b) Light microscopy of representative WT and HMGA1P6-overexpressing MEFs stained for β-galactosidase activity at culture passages 6. (c) Percentage of stained cells obtained from galactosidase activity at culture passages 6 in WT and HMGA1P6-overexpressing MEFs, the results are reported as the mean of values with error bars indicating SD (mean ± SD). *, P < 0.05 (Mann-Whitney test).

Discussion

It has been demonstrated that HMGA1P6 and HMGA1P7 protect HMGA1 mRNA from miRNAs able to target this gene [6]. Therefore, as well as miRNAs, HMGA1Ps are currently considered an epigenetic event that regulate HMGA1 expression, and then regulators of cellular processes where HMGA1 is involved such as cancer progression, senescence, development, metabolism and many other biological functions [7]. The participation of HMGA1 in all these fundamental cellular processes explains the reason for its fine regulation through different molecular mechanisms. Interestingly, several oncogenes seem to be protected by HMGA1P6 and HMGA1P7 from miRNA downregulation action. Indeed, the levels of HMGA2, VEGF and EZH2, which are coded for by genes targeted by HMGA1-targeting miRNAs, were enhanced by HMGA1Ps overexpression [7,11,12]. More recently, Tian et al reported that the upregulation of HMGA1P6 is directly correlated with a shorter overall survival in ovarian cancer patients [18]. Intriguingly, in the same study, it has been described the MYC regulation of HMGA1P6 expression by binding to its promoter, reporting also that HMGA1P6 is able to positively regulate both HMGA1 and HMGA2 through a ceRNA mechanism, increasing the sphere-forming efficiency and invasiveness of ovarian cancer cells [18]. Results achieved with HMGA1P6 and HMGA1P7 transgenic mice and with the relative MEFs sustain the oncogenic role of HMGA1Ps in vivo. In fact, as showed for HMGA1P7, a higher growth rate and a lower predisposition to senescence was found in MEFs deriving from transgenic mice overexpressing HMGA1P6 in comparison with the WT counterpart. For sure one of the limitations of this study is that it relies upon a single embryonic cellular model; however, we speculate that it would not be surprising to find out that HMGA1P6 contributes by the same ceRNA mechanism to the deregulation of many oncogenic pathways that exploit different genes. In the light of our result, it would be interesting to further investigate other cancer-related genes that are modulated by HMGA1P6.

Consistently, a recent paper from our group reports that a lymphoid pathology has been found in about 50% of HMGA1Ps overexpressing mice. In particular, the spleen size was strongly increased in the HMGA1Ps transgenic mice (splenomegaly) due to a lymphoid hyperplasia [19]. Furthermore, several of these mice developed carcinomas in other organs such as kidney and liver. Taken together these results strongly endorse that mRNAs may influence each other through the ceRNA connection, playing key functions both in physiology and in pathology. In conclusion, the ceRNA research topic will shed new light on the complexity and dynamics of the noncoding RNA regulatory networks involved in the induction of senescence and in pathology of several diseases.

Funding Statement

This work was supported by grants from: National Research Council of Italy (CNR) Research Project “Aging: molecular and technological innovations for improving the health of the elderly” (Prot. MIUR 2867 25.11.2011)”, CNR Flagship Projects (Epigenomics-EPIGEN), Associazione Italiana per la Ricerca sul Cancro (AIRC IG 11477). M.D.M is recipient of a Fellowship from “Programma Valere Plus”, University of Campania “L. Vanvitelli”, Caserta, Italy.

Author contributions

M.D.M., C.A., A.F. and F.E. made substantial contributions to conception and design of the study, as well as analysis and interpretation of data and drafting of the article. G.P and P.C. made substantial contributions to conception and interpretation of data and drafting of the article.

Disclosure statement

The authors declare that they have no conflicts of interest.

Ethical approval

The study was conducted in accordance with the ethical principles of the Helsinki declaration.

Informed consent

Informed consent from all participants was obtained.