MCPIP1 contributes to clear cell renal cell carcinomas development
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Monocyte Chemoattractant protein-induced protein 1 (MCPIP1), also known as Regnase-1, is encoded by the ZC3H12a gene, and it mediates inflammatory processes by regulating the stability of transcripts coding for proinflammatory cytokines and controlling activity of transcription factors, such as NF-κB and AP1. We found that MCPIP1 transcript and protein levels are strongly downregulated in clear cell renal cell carcinoma (ccRCC) samples, which were derived from patients surgically treated for renal cancer compared to surrounded normal tissues. Using Caki-1 cells as a model, we analyzed the role of MCPIP1 in cancer development. We showed that MCPIP1 expression depends on the proteasome activity; however, hypoxia and hypoxia inducible factor 2 alfa (HIF2α) are key factors lowering MCPIP1 expression. Furthermore, we found that MCPIP1 negatively regulates HIF1α and HIF2α levels and in the case of the last one, the mechanism is based on the regulation of the half time of transcript coding for HIF2α. Enhanced expression of MCPIP1 in Caki-1 cells results in a downregulation of transcripts encoding VEGFA, GLUT1, and IL-6. Furthermore, MCPIP1 decreases the activity of mTOR and protein kinase B (Akt) in normoxic conditions. Taken together, MCPIP1 contributes to the ccRCC development.
KeywordsClear cell renal cell carcinoma (ccRCC) MCPIP1 (Regnase-1) HIF1α HIF2α Angiogenesis
Kidney cancer is among the ten most common cancers in both men and women. More than 90% of kidney cancers originate from epithelial cells and are referred to as renal cell carcinoma (RCC). The most frequent form of this type of cancer is clear cell renal cell carcinoma (ccRCC), encompassing 75% of all renal cell carcinomas .
In more than 90% of ccRCC samples, the loss of short arm chromosome 3 is observed . This deletion includes the locus for the von Hippel–Lindau (VHL) tumor suppressor gene, responsible for regulating the stability of hypoxia inducible factors, HIF-1α and HIF-2α. The VHL protein forms a stable complex with other proteins, possessing E3 ubiquitin ligase activity. This complex is responsible for targeting HIFs for polyubiquitination and subsequent proteasomal degradation . Under hypoxic conditions, or when the VHL gene is mutated or lost, HIFs accumulate, leading to increased expression of genes encoding vascular endothelial growth factor (VEGFA), platelet derived growth factor (PDGF), transforming growth factor-α (TGFα), erythropoietin (EPO), as well as receptors potentially important in RCC oncogenesis . Further studies have shown that other genes in the short arm of chromosome 3, which are responsible for the chromatin remodeling and epigenetic modifications, are crucial for ccRCC development [5, 6, 7].
Inflammatory pathways have been extensively studied in ccRCC; however, data concerning the role of proteins that regulate stability of transcripts coding for proinflammatory mediators are limited. In our previous study, we analyzed the role of Monocyte Chemoattractant protein-induced protein 1 (MCPIP1), encoded by the ZC3H12a gene. MCPIP1 (also known as Regnase-1) possesses the N terminus of the PilT protein (PilT N terminus or PIN domain), which has RNase properties and regulates half time of transcripts coding for certain proinflammatory cytokines including: IL-1β , IL-2  or IL-6 . Moreover, MCPIP1 also suppresses microRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs, counteracting Dicer, a central ribonuclease in miRNA processing . Besides well-documented RNAse properties, MCPIP1 is considered a negative regulator of the NF-кB signaling pathway [12, 13].
In the present study, we hypothesized a role of MCPIP1 in the etiology of ccRCC. To this purpose, we analyzed ccRCC samples and adjacent normal tissues from patients surgically treated for renal cancer to estimate the level of transcripts coding for MCPIP1. Additionally, we determined correlations between MCPIP1 mRNA levels and transcripts coding for other proteins important for ccRCC development and invasiveness. To clarify the impact of MCPIP1 on ccRCC biology, we used ccRCC cell lines and assessed the potential mechanistic role of MCPIP1 in ccRCC development and examined the possible usefulness of this protein as a therapeutic target in ccRCC therapy.
Characteristics of ccRCC and non-tumor tissues from patients
Histologic grade (Fuhrmann) N (%)
Limited to the kidney
Invasion into major veins
Tumor size (cm)
Proliferation index (S + G2/M) [%]
Caki-1 as a model to study ccRCC
To further assess the role of VHL in MCPIP1 regulation, we utilized A498 cell line, which possess completely disrupted VHL function. First, we compared both cell lines on the molecular level. As shown in Suppl. Fig. 1a, the VHL transcript level is much higher in Caki-1 cells than in A498, but protein is only detected in Caki-1. Both cell lines express similar levels of mRNA coding for HIF1α, but HIF1α protein was visualized by western blot only in Caki-1 cells. HIF2α expression was detectable in both cell lines at the mRNA and protein levels; however, the level of HIF2α was higher in A498 cells, probably due to inactive VHL  (Suppl. Fig. 1). Both cell lines differed in the level of MCPIP1 transcript and protein. In Caki-1 cells, twofold higher transcript levels were observed in comparison with A498 cells, but the reverse situation was seen in case of protein. Caki-1 cells expressed significantly lower levels of protein than did A498 cells (Suppl. Fig. 1). Moreover, the level of the transcript coding for glucose transporter (GLUT1) and VEGFA was lower in Caki-1 than in A498 cells (Suppl. Fig. 1).
Overexpression of either wild-type VHL or its mutant form in the A498 cell line did not influence the level of MCPIP1; thus, the regulation of MCPIP1 is VHL independent. As a positive control for VHL activity, a diminished level of transcripts coding for VEGFA and GLUT1 was observed. In the mutant form of VHL, the decrease was not observed for either transcript. Therefore, in active form of VHL, HIFs are degraded, and there is no expression of transcripts coding for VEGFA and GLUT1 (Suppl. Fig. 2).
Hypoxia decreases the level of MCPIP1
Increased MCPIP1 level following MG132 treatment is the result of de novo expression
As MCPIP1 levels are dependent on proteasome activity in normoxia, it was also interesting to see whether such phenomena takes place in cells cultured under low oxygen conditions. Caki-1 cells were treated with MG132 and cultured in both normoxia and hypoxia for 4, 8, and 24 h. Compared to control cells (DMSO treated cells), we observed an accumulation of MCPIP1 after MG132 stimulation, in both normoxia, and hypoxia; however, the effect was not so prominent in cells kept 24 h in low oxygen concentration (Fig. 4b, c).
To determine, whether accumulation of MCPIP1, after inhibition of proteasome, is the result of de novo synthesis, cycloheximide (CHX, 5 µg/ml) was added to cells treated with MG132 for 4, 8 and 24 h. The addition of CHX to cells treated with MG132 resulted in a strong decrease of MCPIP1 protein level in cells cultured in normoxia after 8 h, and even more prominently after 24 h. The effect was especially strong in cells cultured in hypoxic conditions for 24 h in comparison with cells treated only with MG132. This experiment showed that accumulation of MCPIP1 in Caki-1 cells depends on de novo synthesis as inhibition of translation with CHX inhibited MCPIP1 protein increase following MG132 treatment and the effect is especially meaningful in hypoxic conditions (Fig. 4b, c).
HIF2α negatively regulates MCPIP1 levels
MCPIP1 overexpression in Caki-1 cells affects cell viability
As shown in Fig. 1, most human ccRCC samples have very low levels of MCPIP1 when compared to control, non-tumor tissues. Therefore, we hypothesized that diminished levels of MCPIP1 in human ccRCC samples may be a factor that promotes tumor development. To determine whether our hypothesis was correct, Caki-1 cells were transduced with a lentiviral vector expressing MCPIP1 in a doxycycline (DOX)-dependent manner (Tet-ON system; v-MCPIP1). In each experiment, Caki-1 cells transduced with an empty lentiviral vector (v-PURO) and treated with DOX served as a control. After transduction, cells were under puromycin selection (2 μg/ml) for 10 days in order to enrich the amount of cells containing vector (empty or expressing MCPIP1). Cells were then seeded in appropriate numbers on a plate in normoxic conditions, stimulated with DOX for 24 h, and finally cultured for another 24 h, in hypoxia or normoxia.
To determine whether the expression of exogenous MCPIP1 changes the biology of Caki-1, we performed additional assays. The impact of MCPIP1 on cell viability was measured using an MTT assay. As shown in Fig. 6b (upper panel), cells cultured under hypoxic conditions were less viable than cells in normoxia; however, cells overexpressing MCPIP1 displayed a further significant decrease in growth under hypoxic conditions as compared to cells cultured in normoxia.
Furthermore, we observed a prominent decrease in proliferation of cells overexpressing MCPIP1, as measured by the BrdU incorporation assay. In normoxia, the proliferation rate of control cells (v-PURO) was higher than in cells overexpressing MCPIP1, while in hypoxia, the proliferation rate was generally lower than in normoxia. Control cells proliferated more efficiently than those, expressing MCPIP1(Fig. 6b, lower panel). Decreased growth was correlated with the level of ATP content which was significantly lower for cells with enhanced expression of MCPIP1. Additionally, cells with induced expression of MCPIP1 displayed activation of caspase 3/7. Cells overexpressing MCPIP1 and cultured in normoxia had 3.5 times higher caspase 3/7 activity than did control cells. In hypoxia, cells overexpressing MCPIP1 displayed stronger activation of caspase 3/7, but this difference was not statistically significant (Fig. 6b, lower panel).
MCPIP1 strongly decreases the levels of HIFs, GLUT1, VEGFA, and IL-6 and influences the activity of signaling pathways
In the next step, we determined the activation of selected signaling proteins that are important in tumor growth. We examined how enhanced expression of MCPIP1 influences the activation status of HIFs, NF-κB, p38, ERK1/2, Akt, and mTOR. Although the level of endogenous (v-PURO) and exogenous MCPIP1 (v-MCPIP1) was generally lower in cells cultured in hypoxia than in normoxia, the changes in activity of the mentioned proteins were observed after MCPIP1 overexpression.
The most prominent changes were observed for HIFs. First of all, hypoxia strongly activates accumulation of HIFs as both proteins were not detectable in cells cultured in normoxic conditions. Interestingly, the cells overexpressing MCPIP1 and cultured in hypoxia exhibit a significantly lower level of HIF1α and HIF2α in comparison with the cells transduced with an empty vector. Furthermore, overexpression of MCPIP1 (v-MCPIP1) in normoxic conditions results in the lower activation of mTOR and protein kinase B (Akt) and higher activation of ERK1/2 and p-38, and p-65 (subunit of NF-κB), although in case Akt, ERK1/2, and p-65 changes are not statistically significant. Moreover, in hypoxic conditions, cells overexpressing MCPIP1 display slightly increased phosphorylation of mTOR and ERK1/2 although changes were not statistically significant. In case of Akt and p-65 molecules, a decreasing trend was observed, but these differences were also not statistically significant (Fig. 7b, c).
MCPIP1 acts as an endonuclease controlling stability of many transcripts; therefore, we ask the question whether diminished level of HIF2α might be the result of regulation of HIF2α transcript stability by MCPIP1. We carried out experiment where cells overexpressing MCPIP1 were treated with actinomycin D and then, total RNA was isolated in selected time points, and Q-RT-PCR was done. We found that cells overexpressing MCPIP1 display shorter half time of transcript coding for HIF2, thus MCPIP1 regulates HIF2α on transcript level (Fig. 7d).
In this study, we found that MCPIP1 levels are substantially lower in ccRCC samples than in the surrounding tissues. Using Caki-1 cell line as a model, we found that proteasome regulates MCPIP1 expression; however, in the cells cultured under hypoxic conditions, the accumulation of MCPIP1 is not so efficient as it is in the case of cells kept under normal oxygen supply. We showed that one of the factors negatively regulating MCPIP1 during hypoxic conditions is HIF2α; however, the exact mechanism is still unknown. Furthermore, we also found that MCPIP1 negatively regulates the levels of HIFs and that in the case of HIF2α, the mechanism is based on the shortening of the half-life of its transcript coding for this protein. Negative regulation of HIF2α by MCPIP1 may explain, at least partially, some features of ccRCC development, including proinflammatory and proangiogenic changes. MCPIP1 overexpression strongly reduces transcripts coding for VEGFA, IL-6, and GLUT1, especially in hypoxic conditions. Additionally, MCPIP1 decreases phosphorylation of mTOR and Akt in normoxic conditions and, finally, decreases proliferation rate of Caki-1 cells and activates caspases 3/7.
Current treatment for ccRCC is based on nephron-sparing and minimally invasive surgery, which is usually considered for tumors smaller than 4 cm in diameter (stage pT1a), while radical nephrectomy is used for tumors larger than 4 cm. Despite increased detection of early cases, approximately 50% of patients display metastatic disease [21, 22]. Current therapies are based on the treatment with immunomodulatory drugs , multiple tyrosine kinase receptor inhibitors [24, 25, 26], inhibitors of mammalian target of rapamycin , or dendritic cell (DC)-based vaccines [28, 29]. Targeted therapies have markedly improved patient outcomes, but they rarely induce complete responses, and the major problem of such therapies is resistance to treatment. This work provides a strong rationale for testing novel agents to improve ccRCC therapy.
MCPIP1 plays a key role in the regulation of inflammatory processes via controlling the levels of transcripts coding for inflammation-related modulators but also proteins involved in many other biological processes [8, 10, 12, 13, 30]. The impact of inflammation on cancer development was shown as early as 1863 by Virchow . Many subsequent studies confirmed that tumor microenvironment is often infiltrated by innate and adaptive immune system cells, releasing growth factors, proangiogenic factors and extracellular matrix (ECM)-modifying enzymes. All these factors facilitate angiogenesis while promote growth, invasion, and metastasis of tumor cells.
We observed that the levels of MCPIP1 transcript and protein were diminished in the vast majority of ccRCC tissues. For subsequent studies, to investigate the mechanism of MCPIP1 downregulation in ccRCC samples, we used Caki-1 cell line as a model. These cells possess an active form of VHL and are characterized by significantly lower level of MCPIP1 in comparison with the A498 cell line, which features an inactive form of VHL. This protein is responsible for assigning other proteins for proteasomal degradation and has a well-documented role in ccRCC development [32, 33]. Furthermore, somatic VHL mutations are detected in 60–90% of patients with ccRCC. Aberrations in VHL gene function either through mutation or promoter hypermethylation [34, 35]. Our experiments performed in both Caki-1 and A498 cells transfected with wild-type or mutated pVHL indicated that this protein is not involved in MCPIP1 regulation. The fact that MCPIP1 is diminished on both transcript and protein levels in most ccRCC cases is additional evidence that in these cells a VHL-independent mechanism that is responsible for downregulation of this RNase exists.
Our previous studies on HepG2 and HeLa cells indicated that inhibition of proteasome resulted in the accumulation of MCPIP1 and that this upregulation of MCPIP1 in MG132 treated cells is a result of de novo synthesis [12, 19]. Similar phenomenon was observed in Caki-1 cells after treatment with MG132, in both hypoxic and normoxic conditions. Administration of proteasome inhibitor to Caki-1 cell culture medium led to the increase of MCPIP1 in normal and low oxygen supply conditions, although, in hypoxia, the effect was less prominent than in normoxia. The fact that MCPIP1 is increased in MG132-treated cells may be explained by enhanced activation of NF-κB and p38 MAP kinase. Both proteins were shown to be important in proteasome dependent MCPIP1 gene upregulation [12, 19]. A strong accumulation of MCPIP1 after proteasome inhibition in Caki-1 cells is the result of de novo synthesis, as the experiment with addition of cycloheximide and MG132 indicated. The fact that this synthesis was not so efficient in hypoxic conditions means that hypoxia activates factor(s) that negatively regulate MCPIP1 expression.
Under hypoxic conditions, MCPIP1 is strongly downregulated on transcript and protein levels. This phenomenon is observed for ccRCC cell line with active (Caki-1) and inactive (A498) VHL form. Furthermore, the same effects were observed for non-cancer cells: human embryonic kidney cells (HEK 293) and an immortalized proximal tubule epithelial cell line from normal adult human kidney (HK-2). The fact that MCPIP1 is downregulated under hypoxic conditions has an important impact on many cellular processes. MCPIP1 influences the levels of many transcripts coding for proinflammatory cytokines (IL-6, IL-1, and many others), transcription factors (NFkappaB, C/EBPbeta), signaling proteins (JNK, Akt/mTor) [8, 9, 10, 11, 12, 13, 36, 37, 38]. The whole list of targets is not known yet; thus, it is difficult to predict which other cellular processes are altered by diminished level of MCPIP1.
To investigate the mechanism involved in a negative regulation of MCPIP1 in hypoxic conditions in both control cells and those treated with MG132, we tested the role of HIFs in this process. Numerous studies have shown that HIFs are crucial in tumor development [39, 40, 41, 42, 43]. Importantly, both HIF isoforms may independently regulate gene expression, e.g., in human endothelial cells, IL-8 expression is decreased by HIF-1α, whereas HIF-2α overexpression leads to the induction of IL-8 mRNA and protein level; however, of the HIF subunits, HIF-2α appears to be more oncogenic than HIF-1α activating pro-tumorigenic target genes. In addition, recent studies indicated that HIF-1α, more than HIF-2α, can undergo proteasomal degradation in VHL -/- RCC cells, thus HIF-2α is an important player in ccRCC development [44, 45]. It is also worth noting here that using MDR and logistic regression methods, we discovered a complex network of interactions involving six genes implicated in RCC (SCARB1, GNAS1, BIRC5, EPAS1, VDR and MC1R), of which the EPAS1 gene encodes HIF2α . Using Caki -1 cells as a model, for the first time we have shown that HIF2α, but not HIF1α, is a key player in the downregulation of MCPIP1 transcript and protein, particularly in hypoxia. HIF-1 and HIF-2, despite some overlapping effects, can uniquely regulate distinct genes. HIF2α may regulate gene expression contributing to various stages of MCPIP1 downregulation by: binding directly to specific binding sites present in the regulatory regions of target genes and activating gene expression or stimulating miRNA expression that will affect target gene expression. The evidence for the latter is derived from the studies of the influence of MCPIP1 overexpression on Caki-1 cell viability. In spite of MCPIP1 expression from exogenous template (a vector overexpressing MCPIP1), the level of this protein was diminished in hypoxia. Vector regulatory sequences do not contain sequences recognized by HIF2α; thus, it is possible that regulation of MCPIP1 expression is triggered by some miRNAs regulated by HIF2α activated in hypoxia. It can be with an agreement what we observe in the experiment where MCPIP1 transcript level is analyzed in Caki-1 cells kept in hypoxic conditions for 12 and 24 h. The level of MCPIP1 mRNA is higher after 12 h than after 24 h. Following miRNA-dependent hypothesis of MCPIP1 regulation, one can imagine that after induction of hypoxia HIF2α is activated. Then, this transcription factor induces expression of specific miRNA that regulates MCPIP1 transcript. As a result, MCPIP1 transcript level is lower after 24 h than after 12 h, because miRNA continues degradation in cell cultured upon hypoxic conditions. However, the exact mechanism of this regulation deserves further investigation.
Under normoxic conditions, overexpressed MCPIP1 significantly reduces mTOR and Akt activity, while changes in hypoxia are almost not detectable. In case of mTOR, there is a delicate tendency to upregulation, although the difference is not statistically significant. From the other hand, phosphorylation of both proteins in ccRCC samples is enhanced; therefore, this phenomenon might be at least partially the result of diminished level of MCPIP1 in this tumor. At the end, it must enhance proliferation rate of tumor cells. Besides having an influence on mTOR and Akt, overexpressed MCPIP1 displays a tendency to a higher levels of the phosphorylated forms of MAP kinases (p38 and ERK1/2) in the same conditions, while in hypoxia only slightly stronger phosphorylation of ERK1/2 was observed, without influence on p38 kinase. It is well known that both kinases are activated by stress. In our experimental conditions, cells were exposed to stress induced by hypoxia and by overexpression of MCPIP1. Both kinases are important in response to hypoxic shift and cells adaptation to oxygen deprivation . In the case of MCPIP1, there are evidences that it induces production of reactive oxygen species (ROS), what results in the generation of cellular stress . MCPIP1 does not change phosphorylation status of NF-κB subunit, p65. It is possible that the effect of MCPIP1 on selected signaling pathways would be more meaningful under hypoxic conditions in case of higher level of this protein. Unfortunately, MCPIP1 is diminished in hypoxia, even after overexpression, and further studies are essential to understand this mechanism.
For the first time, we report that MCPIP1 negatively regulates HIF1α and HIF2α level under hypoxic conditions. Conversely, one potential reason is that cells overexpressing MCPIP1 display lower expression of transcripts coding for GLUT1 and VEGFA, which is the result of HIFs negative regulation by MCPIP1. As already mentioned, there are many evidences that MCPIP1 acts as an RNase controlling stability of transcripts. Direct regulation of IL-6 by MCPIP1 is already well described . Here we show for the first time that MCPIP1 controls half-life of transcript coding for HIF2α but not for VEGFA. It is possible that downregulation of VEGFA transcript might be the effect of negative regulation of HIF2α by MCPIP1. On the basis of these observations, we can assume that the prominent vascularization observed in ccRCC samples might be explained by diminished level of MCPIP1 in this tumor. Besides HIFs, also IL-6 exhibits proangiogenic properties acting as an inductor of VEGFA in a STAT3 and HIF-dependent manner [49, 50, 51, 52]. IL-6 may also directly influence blood vessel formation by promoting endothelial progenitor cell migration and proliferation [53, 54], and/or the stimulation of vascular smooth muscle cell (VSMC) migration . Interestingly, Baldewijns and coauthors found no significant association between VHL mutation or methylation and angiogenesis/tumor parameters . It is possible that MCPIP1 is a missing link, because its expression and stability is not dependent on VHL but has a significant influence on the level of proangiogenic factors.
Consequently, decreased MCPIP1 levels in ccRCC may promote tumor development and metastasis. Additionally, our results clearly indicate that MCPIP1 also influences cell metabolism, by negative regulation of GLUT1 transcript. Strong, MCPIP1-dependent downregulation of HIFs correlates with worsened cell survival, proliferation rate, ATP content and activation of caspases 3/7. All these changes triggered by MCPIP1 in Caki-1 cells are potentially important in tumor biology, making it plausible that this protein is important for consideration as a therapeutic target in ccRCC.
Negative roles of MCPIP1 in cell proliferation and viability were already described in different cancer cell types. Overexpression of MCPIP1 modulates transcriptome including miRNAs, in human neuroblastoma cells . Furthermore, it was also shown that MCPIP1 functions as a potent tumor suppressor and induces apoptosis of breast tumor cells by selectively enhancing mRNA decay of antiapoptotic gene transcripts, including Bcl2L1, Bcl2A1, RelB, Birc3, and Bcl3 . It is possible that MCPIP1 influences several cellular processes acting as an RNase and regulating stability of different important RNA molecules. However, regardless of the type of cellular process in which MCPIP1 is involved, its significance in antitumor response seems to be undisputed.
Materials and methods
Patient tissue samples
Normal and tumor renal tissues pairs were obtained from patients surgically treated for renal cancer in Oncology Center in Krakow under the supervision of the local ethical committee (approval no 68/KBL/OIL/2011). Samples were subjected to histological evaluation by pathologists and diagnosed according to criteria set forth by World Health Organization classification. In each case, the sample was divided into two pieces, with one frozen at −80 °C (for protein isolation), and the other was incubated overnight in RNA-later (Invitrogen) at 4 °C and then stored in −80 C to be used for RNA isolation.
Human clear cell renal cell carcinoma cell lines, Caki-1 and A498, were cultured in Eagle’s minimal essential medium (EMEM) (Lonza) containing 10% fetal bovine serum (FBS), (BioWest SAS, Nuaillé, France) at 37 °C in a 5% CO2 atmosphere. Cells were passaged three times a week, at 80–90% confluence.
Transfection/transduction of Caki-1
For stable expression of MCPIP1, doxycycline-dependent overexpression Tet-ON system was used. Caki-1 cells were seeded on 6-well plates at 50–70% confluency (3 × 105 cells per well). Lentivirus was added at MOI-50. Cells were incubated with viruses for 24 h, and then medium was replaced. After an additional 24 h, puromycin was added at concentration 2 µg/ml and selection of stably transduced cells was carried out for next 10 days. Stimulation of MCPIP1 expression was performed in FBS-free medium (DMEM/F12 supplemented with BSA (0.5 mg/ml), transferrin (5 μg/ml) and Na2SeO3 (2 ng/ml)) to decrease Tet-ON system leakage.
Caki-1 cells at 50–70% confluence were transfected with wild-type VHL or its mutant form del-VHL (a deletion of C-terminal domain) (both vectors were kind gift of Professor Sánchez-Prieto R ) using Lipofectamine 2000. Procedure was performed according to manufacturer’s recommendation. For 6-well plate, 3 × 105 cells were seeded in 2 ml and the following day, the transfection was performed. Briefly 7.5 µl of Lipofectamine 2000 was diluted in 100 μl of EMEM without FBS and 2.5 µg DNA (p-VHL vector, p-mVHL or empty vector) was diluted in 100 µl EMEM without FBS. After 5 min, diluted plasmid DNA and Lipofectamine 2000 were combined and incubated 15 min at room temperature. DNA-lipofectamine complexes were then added to cells and mixed gently by rocking. To reduce toxicity, cell medium was changed 5 h after transfection and the cells were incubated for 24–72 h for further experiments.
Adenoviral vectors containing HIF1α or HIF2α cDNA (Ad-HIF1α, Ad-HIF2α) were a kind gift from Prof. Seppo Yla-Herttuala (Kuopio, Finland) and Prof. Lorenz Poellinger (Stockholm, Sweden). The pAdHIF-1α was generated as described previously . Briefly, the construct was stabilized against prolyl hydroxylation and subsequent ubiquitin-mediated proteolytic degradation in normoxic conditions by point mutations (P402A/P563A). A control vector harboring green fluorescent protein (GFP) cDNA (AdGFP) was produced using the AdenoX system as described previously .
Transfection with small interfering RNA (siRNA)
Cells were transfected with 25 nM of chemically synthesized siRNA targeted against human HIF1α or HIF2α mRNA. As a control scrambled siRNA was used. Briefly, cells were plated into 6-well plates 1 day prior to transfection to obtain 50–70% confluency. siRNA and Lipofectamine 2000 were separately diluted in Opti-MEM without serum, incubated 5 min at room temperature, combined, and then incubated for 20 min at room temperature. Forty-eight hours post-transfection protein cell lysates were prepared and used for further analysis.
Caki-1 cells were seeded on 12-well plate (1.2 × 105 cells/well) at approximate 70% confluency. Following 24-h incubation, plates were placed in cell culture incubators with hypoxic condition (1% O2) or in atmospheric conditions (21% O2). Protein and RNA were isolated after 12 and 24 h of incubation.
Total RNA was isolated using the guanidium isothiocyanate (GTC) method. The integrity of ribosomal RNA and DNA contamination was examined using denaturing formaldehyde gel electrophoresis (l%). Protein and phenol contamination and concentration of total RNA was assessed by determining the ratio A260/280 and A260/230, respectively (NanoDrop).
For the real-time PCR, 1 μg of total RNA was reverse-transcribed using the oligo(dT) primer (Promega), random hexamers (EURx), and M-MLV reverse transcriptase (Promega). Following synthesis, cDNA was diluted fivefold and real-time PCR was carried out using the Eco Real-Time PCR System (Illumina) and a Sybr Green-based master mix (A&A Biotechnology). For tissue samples peptidylprolyl isomerase A (PPIA) was used as a reference gene. For cell cultures, each sample was normalized to reference gene ribosomal protein S13 (RPS13). For mRNA half-life analysis as a reference gene 28S ribosomal RNA was used (28S rRNA). The relative level of transcripts was quantified by the ΔΔCT method.
Q-RT-PCR was carried out for MCPIP1 transcript with primers: ggaagcagccgtgtccctatg and tccaggctgcactgctcactc (NM_025079.2); for IL-6 transcript with primers: ggtacatcctcgacggcatct and gtgcctctttgctgctttcac; for RPS13 with primers: tcggctttaccctatcgacgcag and acgtacttgtgcaacaccatgtga; for VEGFA transcript with primers: agaaaatccctgtgggccttgctc and gcctcggcttgtcacatctgcaa; for SLC2A1(GLUT1) transcript with primers: tccctgcagtttggctacaa and gcaggatgctctccccatag; for CXCL8 (IL-8) with primers: atgacttccaagctggccgtggct and atgacttccaagctggccgtggct, for HIF1α with primers: caagaacctactgctaatgc and ttatgtatgtgggtaggagatg; for EPAS1(HIF2α) with primers: gcgctagactccgagaacat and tggccacttactacctgaccctt, for 28S rRNA with primers: cacccactaatagggaacgtg and ctgacttagaggcgttcagtc.
HIF2α mRNA half-life analysis
Caki-1 cells with a stable expression of MCPIP1 (Tet-ON system vectors) were seeded on 12-well plate (1.2 × 105 cells/well). After 24-h incubation, actinomycin D (BioShop) was added at a final concentration of 5 mg/ml. Total RNA was collected at the following time points: 1, 3, 6, and 8 h. After cDNA synthesis, Q-RT-PCR was carried out as described above.
After washing with ice-cold PBS, cells were harvested and lysed on ice in RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (Sigma) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). The protein concentration in cell lysates was measured with the bicinchoninic acid assay.
Cell lysates were separated by SDS/PAGE (8, 10 or 12% polyacrylamide gel), electrotransfered to PVDF membrane (Millipore,) and blocked for 1 h (at room temperature) in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (BioShop, Burlington, Canada). Membranes were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h, room temperature, chemiluminescence was detected using Immobilon Western HRP substrate (Millipore) with ChemiDoc system (BioRad). The following antibodies and dilutions were used: rabbit anti-MCPIP1 (1:1000, GeneTex), HIF1α (1:1000), HIF2α (1:1000), VHL (1:500), Akt (1:1000), Phospho-Akt (1:2000), SAPK/JNK (1:500), Phospho-SAPK/JNK (1:1000), p38 (1:1000), Phospho-p38 (1:1000), ERK1/2 (1:1000), Phospho-ERK1/2 (1:1000). All mentioned antibodies were from Cell Signaling Technology. Tubulin (1:4000, Calbiochem; Merck Millipore, Billerica, MA, USA) or in case tissue samples, GAPDH (1:30,000 Sigma-Aldrich) antibodies were used as a loading control. The following secondary antibodies were used: peroxidase-conjugated anti-rabbit IgG (1:30,000; Sigma) and peroxidase-conjugated anti-mouse IgG (1:10,000, Sigma).
ATP content assay
Cell viability was analyzed with ATPLite—Luminescence ATP Detection Assay System (PerkinElmer). Cells were plated on 96-well white plates (5 × 103 per well), and after 24 h, MCPIP1 expression was stimulated with doxycycline. Quantification of intracellular ATP content was carried out according to the manufacturer’s instructions after 48 h of exposure. The luminescence was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland).
Cell proliferation was measured with BrdU incorporation assay using the chemiluminescent BrdU Cell Proliferation Assay Kit (Roche) as it was performed . Chemiluminescence was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.) in three independent experiments, each performed in triplicate.
Cell viability was measured using the colorimetric MTT assay as described . Absorbance was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.) at 570 nm with the reference wavelength of 500 nm. Three independent experiments were performed, each in quintuplicate.
All statistical analysis was performed using Statistica 10 (Statsoft). The statistical significance for differences between normal and tumor tissue samples was assessed by the Mann–Whitney U test. Experiments were conducted in three independent repetitions. The standard error of the mean (SEM) is presented on the graphs. Testing of multiple samples was performed with ANOVA followed by Tukey’s HSD (honest significant difference) test. The p values were marked with the asterisks on the charts (*p < 0.05).
The authors wish to thank the study participants. We would like to thank Monika Dziendziel and Tomasz Uspienski for technical support.
This research was supported by the grant from National Science Centre no. 2011/03/B/NZ1/00023 (to J. Jura). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficiary of the structural funds from the European Union and the Polish Ministry of Science and Higher Education (Grants No: POIG.02.01.00-12-064/08 and 02.02.00-00-014/08) and is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.
Compliance with ethical standards
Conflicts of interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
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