ABSTRACT

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. The kinesin KIF18A is an important regulator of those processes in animal germ cells. Post-transcriptional regulation of KIF18A has not been extensively explored. Owing to the presence of PUM-binding elements (PBEs), KIF18A mRNA is a potential target of PUM proteins, where PUM refers to Pumilio proteins, RNA-binding proteins that act in post-transcriptional gene regulation. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assays, by applying an appropriate luciferase construct encoding wild-type KIF18A 3′-UTR, upon PUM overexpression or knockdown in TCam-2 cells, representing human male germ cells. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function, an MTS proliferation assay, and apoptosis and cell cycle analysis using flow cytometry, was performed upon KIF18A mRNA siRNA knockdown. KIF18A significantly influences proliferation, apoptosis and the cell cycle, with its effects being opposite to PUM effects. Repression by PUM proteins might represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells.

INTRODUCTION

Infertility is a worldwide problem which affects ∼15% of couples globally, with half of cases being a result of male infertility (for a review see Inhorn and Patrizio, 2015). Despite the availability of various medical procedures, such as artificial reproductive technology, treating infertility is not possible in many cases (Bhartiya, 2015). It is believed that dysfunction of fertility may occur during the fetal life of an individual, at the time when the germline is established, or may take place later, during the germ cell maturation. It is of note that male infertility is frequently associated with testicular germ cell tumours, and is thus considered as a risk factor for such tumours (Matzuk and Lamb, 2008).

Kinesins are enzymatic proteins playing the role of molecular motors in intracellular transportation of cargo along microtubules and in cellular movements (Sperry, 2012), these activities being catalysed by ATP hydrolysis (Vale and Milligan, 2000). Depletion of members of kinesin-8 family of proteins causes abnormalities in mitotic spindle lengthening (Goshima et al., 2005), chromosome alignment failure (Stumpff et al., 2008) and cell cycle dysfunction (Straight et al., 1998). KIF18A supports chromosome congression during mitosis by reducing the amplitude of kinetochore oscillations in pre-anaphase and slows down poleward movements in anaphase (Stumpff et al., 2008).

Although KIF18A is ubiquitously expressed, its role in mice was demonstrated in germ cell development (Czechanski et al., 2015). Namely, point mutation of the KIF18A gene region encoding the motor domain in germ cell lineage in fetal gonads leads to chromosome alignment defects and mitotic arrest (in G2/M phase), due to spindle assembly MAD2 checkpoint activation (MAD2 localization to kinetochores provides a signal that inhibits progression from metaphase to anaphase) (Li and Benezra, 1996). Those abnormalities trigger apoptosis, resulting in germ cell depletion and infertility (Czechanski et al., 2015). Interestingly, KIF18A dysregulation in somatic cells, despite chromosome alignment defects, does not cause either mitotic arrest nor apoptosis (Czechanski et al., 2015). In a more recent study, KIF18A gene disruption caused delayed postnatal growth and increased mortality of the mouse (Fonseca et al., 2019). Although the KIF18A orthologue was demonstrated to be involved in the mouse gametogenesis, its precise role in human germ cells has not been studied yet.

Notably, the expression of kinesin-8 is often dysregulated in cancer and therefore may play an important role in that process. Interestingly, overexpression of KIF18A was reported to promote tumour formation, invasion and metastasis in colorectal cancer, while its knockdown triggered proliferation decrease in cancer cell lines, altogether indicating a potential oncogenic activity (Nagahara et al., 2011). Upregulation of KIF18A in various types of human cancers indicates that accurate regulation of the KIF18A level is pivotal for preserving proper cell cycle progression. Although there is a report on transcriptional regulation of KIF18A gene expression potentially being mediated by the FOXM1 transcriptional factor (Müller et al., 2014), as well as post-transcriptional regulation in cancer via TTP and HUR RNA-binding proteins (Hitti et al., 2016), the mechanisms of KIF18A regulation were not studied extensively.

Pumilio (PUM) proteins are highly conserved RNA-binding proteins that target mRNAs by binding the 8-nt long consensus PUM-binding element (PBE) UGUAHAUW (H stands for A, G or U, and W for A or U), located mostly within the 3′UTR (Bohn et al., 2018). PBE recognition by PUMs is mediated by a highly conserved PUF domain and depends on PUM cooperation with several protein cofactors (Bohn et al., 2018; Wickens et al., 2002). Both of the mammalian paralogues, PUM1 and PUM2, are very similar in structure (Spassov and Jurecic, 2003), and PUM1 has been shown to be important for the mouse germline development (Chen et al., 2012). We sought to investigate KIF18A potential regulation by PUM proteins due to presence of two identical PBEs (UGUAAAUA) within its 3′UTR, which we selected in our recent screens for PUM1 and PUM2 targets in TCam-2 cells (Smialek et al., 2019 preprint). These cells originate from human seminoma, a type of testis germ cell tumour (TGCT) representing human male germ cells at an early stage of prenatal development (de Jong et al., 2008). Notably, PBE motifs are conserved in mammals, given that both are present in the 3′UTR KIF18A of the mouse homologue. Our results indeed show that KIF18A is regulated by PUM1 and PUM2 proteins, they both repress KIF18A expression and this repression may impact the effect of KIF18A on proliferation, apoptosis and the cell cycle progression of TCam-2 cells.

RESULTS

KIF18A expression is repressed by PUM1 and PUM2 proteins in TCam-2 cells

We have previously shown that KIF18A mRNA is a candidate target of PUM1- and PUM2-mediated post-transcriptional gene regulation in TCam-2 cells by combining CLIP-Seq and RNA-Seq (Smialek et al., 2019 preprint). The KIF18A mRNA contains two identical PBE motifs in its 3′UTR (Fig. 1A). To confirm our preliminary results, we overexpressed PUM1 or PUM2 in fusion with a DDK tag in TCam-2 cells, followed by UV crosslinking and RNA co-immunoprecipitation using anti-DDK antibody. By this approach, we found binding of KIF18A mRNA to PUM1 and PUM2 (Fig. 1B). Furthermore, we demonstrated KIF18A mRNA and protein upregulation upon an efficient PUM1, PUM2 and simultaneous PUM1 and PUM2 (PUM1/PUM2) knockdown, in comparison to cells treated with control siRNA (Fig. 1C,D). By these two approaches, we demonstrate that KIF18A is under PUM1 and PUM2 repression.

Fig. 1.

The influence of PUM1 and PUM2 proteins on KIF18A expression. (A) Diagram of the KIF18A mRNA with PBE motifs (UGUAAAUA) within 3′UTR represented as boxes. (B) RT-qPCR showing enrichment of KIF18A mRNA in PUM1 and PUM2 RNA immunoprecipitates (CoIP) obtained using anti-DDK antibody compared to pCMV6-entry empty vector as a negative control. Additionally Act5c mRNA from Drosophila melanogaster spike-RNA was measured serving as a reference gene for RT-qpCR (left panel). Representative western blot showing PUM1 and PUM2 binding to beads (right panel). 3% as Input (as well as flow-through) and 10% of the CoIP PUM (1 or 2) was loaded on the SDS-PAGE gel. This experiment was performed in three biological replicates, with five technical repeats in each. (C) RT-qPCR showing KIF18A mRNA upregulation upon PUM1, PUM2 and PUM1/PUM2 siRNA silencing, as measured 72 h post-transfection. ACTB and GAPDH mRNAs were used as reference genes for RT-qPCR (left panel). Representative western blot showing efficiency of PUM1, PUM2 and PUM1/PUM2 silencing are shown (right panel). Experiments were performed in three biological replicates and five technical repeats in each. (D) Upregulation of KIF18A protein level upon PUM1, PUM2 and PUM1/PUM2 silencing. Representative western-blots showing KIF18A protein upregulation (upper right panel), as an effect of PUM1, PUM2 and PUM1/PUM2 protein silencing (lower right panel). ACTB served as reference in western blot experiments. The experiment was performed in three biological replicates and three technical repeats. Quantitative results are mean±s.d. **P<0.005; ***P<0.0005; ****P<0.00005.

Fig. 1.

The influence of PUM1 and PUM2 proteins on KIF18A expression. (A) Diagram of the KIF18A mRNA with PBE motifs (UGUAAAUA) within 3′UTR represented as boxes. (B) RT-qPCR showing enrichment of KIF18A mRNA in PUM1 and PUM2 RNA immunoprecipitates (CoIP) obtained using anti-DDK antibody compared to pCMV6-entry empty vector as a negative control. Additionally Act5c mRNA from Drosophila melanogaster spike-RNA was measured serving as a reference gene for RT-qpCR (left panel). Representative western blot showing PUM1 and PUM2 binding to beads (right panel). 3% as Input (as well as flow-through) and 10% of the CoIP PUM (1 or 2) was loaded on the SDS-PAGE gel. This experiment was performed in three biological replicates, with five technical repeats in each. (C) RT-qPCR showing KIF18A mRNA upregulation upon PUM1, PUM2 and PUM1/PUM2 siRNA silencing, as measured 72 h post-transfection. ACTB and GAPDH mRNAs were used as reference genes for RT-qPCR (left panel). Representative western blot showing efficiency of PUM1, PUM2 and PUM1/PUM2 silencing are shown (right panel). Experiments were performed in three biological replicates and five technical repeats in each. (D) Upregulation of KIF18A protein level upon PUM1, PUM2 and PUM1/PUM2 silencing. Representative western-blots showing KIF18A protein upregulation (upper right panel), as an effect of PUM1, PUM2 and PUM1/PUM2 protein silencing (lower right panel). ACTB served as reference in western blot experiments. The experiment was performed in three biological replicates and three technical repeats. Quantitative results are mean±s.d. **P<0.005; ***P<0.0005; ****P<0.00005.

As a next step, we sought to investigate whether regulation by PUM proteins is mediated by the KIF18A 3′UTR containing the two PBE motifs. For that purpose, we prepared a construct encoding the full-length KIF18A 3′UTR downstream of Renilla luciferase (Fig. 2A). We performed a dual-luciferase assay to measure expression of that construct 24 h post PUM1 and PUM2 overexpression, compared to empty pCMV6-entry vector as a negative control. We found that, upon PUM1 and PUM2 overexpression, KIF18A 3′UTR was downregulated (Fig. 2B, left panel), in comparison to the empty pCMV6-entry vector overexpression. A significantly weaker effect of PUM2 compared to PUM1 could be accounted for by a much lower PUM2 overexpression level compared to PUM1 (Fig. 2B, right panel). To confirm that result, we performed PUM1 and PUM2 siRNA knockdown, and compared luciferase expression in those cells to that in cells expressing control siRNA. As expected, we observed upregulation of luciferase construct expression (Fig. 2C, left panel) upon an efficient knockdown of PUM1 and PUM2 alone, as well as upon a simultaneous PUM1/PUM2 knockdown (Fig. 2C, right panels). Taken together, these results show that KIF18A mRNA is repressed by PUM1 and PUM2 in TCam-2 cells, and this effect is mediated by the 3′UTR.

Fig. 2.

The effect of PUM1 and PUM2 on the expression of the KIF18A LUC-3′UTR construct measured by dual-reporter luciferase assay. (A) Diagram of the KIF18A LUC-3′UTR construct with boxes indicating the PBE motifs (UGUGAAAUA). (B) The effect of PUM1 and PUM2 overexpression on the expression of the KIF18A LUC-3′UTR construct. Luminescence values (RLU, relative luciferase units) are expressed as a percentage of the RLU of samples transfected with reporter construct only, which was set to 100%. Renilla luciferase values were normalized using firefly luciferase measurements (left panel). Representative western blot showing PUM1 and PUM2 overexpression level (anti-DDK antibody); ACTB serves as a reference (anti-ACTB antibody) (right panel). (C) The effect of PUM1, PUM2 and PUM1/PUM2 knockdown on KIF18A LUC-3′UTR construct expression (left panel). The level of construct upregulation upon PUMs silencing is shown as the percentage RLU in comparison to control siRNA set to 100%. Representative western blot showing the level of PUM1, PUM2 and PUM1/PUM2 silencing; ACTB serves as a reference (right panel). All experiments were performed in three biological replicates with two technical repeats in each. Quantitative results are mean±s.d. **P<0.005; ****P<0.00005.

Fig. 2.

The effect of PUM1 and PUM2 on the expression of the KIF18A LUC-3′UTR construct measured by dual-reporter luciferase assay. (A) Diagram of the KIF18A LUC-3′UTR construct with boxes indicating the PBE motifs (UGUGAAAUA). (B) The effect of PUM1 and PUM2 overexpression on the expression of the KIF18A LUC-3′UTR construct. Luminescence values (RLU, relative luciferase units) are expressed as a percentage of the RLU of samples transfected with reporter construct only, which was set to 100%. Renilla luciferase values were normalized using firefly luciferase measurements (left panel). Representative western blot showing PUM1 and PUM2 overexpression level (anti-DDK antibody); ACTB serves as a reference (anti-ACTB antibody) (right panel). (C) The effect of PUM1, PUM2 and PUM1/PUM2 knockdown on KIF18A LUC-3′UTR construct expression (left panel). The level of construct upregulation upon PUMs silencing is shown as the percentage RLU in comparison to control siRNA set to 100%. Representative western blot showing the level of PUM1, PUM2 and PUM1/PUM2 silencing; ACTB serves as a reference (right panel). All experiments were performed in three biological replicates with two technical repeats in each. Quantitative results are mean±s.d. **P<0.005; ****P<0.00005.

Knockdown of KIF18A causes a decrease in proliferation of TCam-2 cells, while knockdown of PUMs induces the opposite effect

Since it was reported that KIF18A influences mouse germ cell proliferation (Czechanski et al., 2015; Luo et al., 2018; Zhong et al., 2019), we first investigated whether human KIF18A induces such an effect on TCam-2 cells, a cell line that originates from male germ cells. For that purpose, we performed an MTS proliferation assay upon KIF18A knockdown, starting at 24 h up to 120 h post transfection. We observed that proliferation of cells decreased upon KIF18A knockdown and this effect was observed during that period, in comparison to cells with control knockdown (siCTRL).

To investigate the influence of PUM proteins on proliferation, we also performed an MTS proliferation assay upon PUM1 and PUM2 siRNA silencing. We found that knockdowns of both PUMs resulted in the upregulation of cell proliferation in comparison to cells with control knockdown (siCTRL). Moreover, the positive effect of PUM1 knockdown on cell proliferation was more pronounced than that of PUM2. This effect of knockdown of the PUMs was opposite to that of KIF18A knockdown (Fig. 3A). The expression of PUM1, PUM2 and KIF18A genes was efficiently silenced as shown in Fig. 3B, starting from the 48 h post-transfection time point.

Fig. 3.

Effect of KIF18A, PUM1 and PUM2 knockdown on cells proliferation. (A) The MTS proliferation assay was performed on cells at 24, 48, 72, 96 and 120 h post-transfection with KIF18A, PUM1 or PUM2 siRNA (each point represents an average of three biological replicates with seven technical repeats in each); absorption measurements were performed at 450 nm (Formazan) minus 750 nm (background) at 4 h after addition of MTS reagent. (B) Representative western blots showing efficiency of KIF18A siRNA or PUMs silencing starting from 24 h post-transfection in comparison to ACTB serving as reference. Quantitative results are mean±s.d. **P<0.005; ***P<0.0005.

Fig. 3.

Effect of KIF18A, PUM1 and PUM2 knockdown on cells proliferation. (A) The MTS proliferation assay was performed on cells at 24, 48, 72, 96 and 120 h post-transfection with KIF18A, PUM1 or PUM2 siRNA (each point represents an average of three biological replicates with seven technical repeats in each); absorption measurements were performed at 450 nm (Formazan) minus 750 nm (background) at 4 h after addition of MTS reagent. (B) Representative western blots showing efficiency of KIF18A siRNA or PUMs silencing starting from 24 h post-transfection in comparison to ACTB serving as reference. Quantitative results are mean±s.d. **P<0.005; ***P<0.0005.

KIF18A knockdown causes significant upregulation of apoptosis in TCam-2 cells, which is opposite to the effect of PUM proteins

It was previously shown that depletion or a single amino acid substitution in KIF18A causes elevated apoptosis in germ cells in mice (Czechanski et al., 2015). In addition, KIF18A gene knockout stimulated apoptosis, which resulted in the absence of germ cells in seminiferous tubules and infertility (Czechanski et al., 2015). Therefore, we investigated potential influence of human KIF18A on apoptosis in TCam-2 cells. We observed that, upon KIF18A knockdown, the population of TCam-2 cells was reduced by half (data not shown). For the half of cells that survived, an almost 2.5 times increase in apoptosis ensued in comparison to control cells (Fig. 4A, upper panel). The knockdown efficiency of KIF18A is shown in Fig. 4A (lower panel). KIF18A depletion caused significant increase in apoptosis in the TCam-2 human germ cell model. The effect on apoptosis is visible on representative dot-plots (Fig. 4B).

Fig. 4.

The effect of KIF18A siRNA silencing on apoptosis, as measured using imaging flow cytometry and Annexin V staining assay. (A) Graph showing the effect of KIF18A knockdown on apoptosis in comparison to control apoptosis level set as 1 (upper panel). Representative western blot using anti-KIF18A antibody showing the efficiency of KIF18A silencing 48 h post-transfection in comparison to control siRNA (siCTRL). ACTB detection using anti-ACTB antibody served as a reference (lower panel). (B) Representative Amnis Ideas graphs showing living, early apoptotic, late apoptotic and necrotic cells at 48 h post-transfection with control (left panel) and KIF18A siRNA (right panel). All experiments were performed in three biological replicates with two technical repeats in each. Quantitative results are mean±s.d. *P<0.05.

Fig. 4.

The effect of KIF18A siRNA silencing on apoptosis, as measured using imaging flow cytometry and Annexin V staining assay. (A) Graph showing the effect of KIF18A knockdown on apoptosis in comparison to control apoptosis level set as 1 (upper panel). Representative western blot using anti-KIF18A antibody showing the efficiency of KIF18A silencing 48 h post-transfection in comparison to control siRNA (siCTRL). ACTB detection using anti-ACTB antibody served as a reference (lower panel). (B) Representative Amnis Ideas graphs showing living, early apoptotic, late apoptotic and necrotic cells at 48 h post-transfection with control (left panel) and KIF18A siRNA (right panel). All experiments were performed in three biological replicates with two technical repeats in each. Quantitative results are mean±s.d. *P<0.05.

Given the effect of KIF18A on apoptosis, as the next step, we sought to identify apoptotic genes, the level of which is changed by KIF18A depletion in TCam-2 cells. To this aim, upon KIF18A siRNA knockdown in TCam-2 cells, at 48 and 72 h post-transfection, we performed real-time quantitative PCR (RT-qPCR) measurement of expression of five anti-apoptotic genes: AKT1, BCL2, CREB1, CCND1 and PIK3CA and one pro-apoptotic BCL2L1 (shorter isoform). All of them are highly expressed in TCam-2 in our previous RNA-Seq screens (Smialek et al., 2019 preprint). Our analysis showed that KIF18A knockdown caused a significant downregulation of the mRNA level of BCL2, an anti-apoptotic gene (Petros et al., 2001), and upregulation of anti-apoptotic (Chen et al., 2019; Singh et al., 2019) CCND1 and PIK3CA. Upregulation of the pro-apoptotic shorter isoforms of BCL2L1 mRNA occurring upon KIF18A knockdown was in line with the pro-apoptotic effect of KIF18A knockdown, as with downregulation of anti-apoptotic BCL2 (Fig. S1A). Finally, KIF18A had no effect either on AKT1 or CREB1 mRNA expression (Fig. S1A). We have previously shown that overexpression of PUM1 strongly and PUM2 slightly upregulates apoptosis, and that overexpression of both caused downregulation of TCam-2 cell cycle (Janecki et al., 2018).

Knockdown of KIF18A causes TCam-2 cell cycle arrest in G2/M phase

Since depletion or mutation of the KIF18A orthologue in mice caused germ cell cycle arrest (Czechanski et al., 2015), we sought to assess whether such an effect also takes place in the TCam-2 human germ cell model. To this end, upon KIF18A siRNA knockdown, we performed cell cycle analysis at 48 h post-transfection using propidium iodide (PI) staining and flow cytometry. In these conditions, we observed differences in the distribution of cell cycle phases. Namely, we found a lower number of cells in G0/G1 and S phases, in favour of cells representing G2/M phase (Fig. 5A, upper panel). The efficient knockdown of KIF18A in cells compared to control cells transfected with control siRNA is illustrated on Fig. 5A (lower panel). The quality of the separation of cells being at different cell cycle phases is shown by ModFit LT Software (Verity Software House) (Fig. 5B).

Fig. 5.

The effect of KIF18A siRNA silencing on the cell cycle, as measured by flow cytometry using PI. (A) The cell cycle changes are represented as the percentage of cells in each phase (G0/G1;S; G2/M) upon KIF18A knockdown in comparison to the baseline (0%) of siRNA control (upper panel). Representative western blot using anti-KIF18A antibody showing the level of KIF18A knockdown at 48 h post-transfection, compared to ACTB (anti-ACTB antibody), serving as the reference (lower panel). (B) Representative ModFit graphs showing separation quality of particular cell cycle phases 48 h post-transfection upon control (left panel) and KIF18A siRNA silencing (right panel). All experiments were performed in three biological replicates with two technical in each. Quantitative results are mean±s.d. *P<0.05; **P<0.005.

Fig. 5.

The effect of KIF18A siRNA silencing on the cell cycle, as measured by flow cytometry using PI. (A) The cell cycle changes are represented as the percentage of cells in each phase (G0/G1;S; G2/M) upon KIF18A knockdown in comparison to the baseline (0%) of siRNA control (upper panel). Representative western blot using anti-KIF18A antibody showing the level of KIF18A knockdown at 48 h post-transfection, compared to ACTB (anti-ACTB antibody), serving as the reference (lower panel). (B) Representative ModFit graphs showing separation quality of particular cell cycle phases 48 h post-transfection upon control (left panel) and KIF18A siRNA silencing (right panel). All experiments were performed in three biological replicates with two technical in each. Quantitative results are mean±s.d. *P<0.05; **P<0.005.

DISCUSSION

The mechanisms responsible for the regulation of expression of KIF18A, one of the kinesins playing specific roles in germ cell development, have not been investigated extensively. The presence of PBE motifs in 3′UTR suggested regulation by PUM proteins. Likewise, KIF18A mRNA was one of the candidates selected as a PUM-regulated mRNA in our recent global screens for PUM1 and PUM2 targets in TCam-2 cells (Smialek et al., 2019 preprint). Here, we propose that KIF18A expression is regulated by PUM proteins for the following reasons. First, in co-immunoprecipitation assays, we demonstrated enrichment of KIF18A mRNA in anti-PUM immunoprecipitates, indicating interaction with PUM proteins. Second, upon PUM siRNA silencing, we observed KIF18A upregulation at mRNA and protein level. Third, the luciferase reporter assay upon PUM protein overexpression or silencing, shows that repression by PUM proteins is mediated by 3′UTR of KIF18A. Fourth, KIF18A mRNA in mice contains two PBE motifs in 3′UTR, indicating that this type of KIF18A post-transcriptional regulation could be evolutionarily conserved in mammals. Altogether, our results clearly show that KIF18A mRNA is repressed by PUMs in TCam-2 cells and that this regulation is mediated by the 3′UTR. The repression by PUMs may be part of the post-transcriptional regulatory mechanism of KIF18A gene expression.

The next important question we addressed was whether KIF18A, acting as a post-transcriptional PUM target, impacts processes crucial for germ cell development, such as proliferation, the cell cycle and apoptosis of TCam-2 cells, and if so, whether those effects are in line with KIF18A repression by PUM proteins. Our results showed that indeed this was the case.

First, the anti-proliferative effect of KIF18A depletion in TCam-2 cells was in line with the effect of PUM proteins. Therefore, the activity of PUM in downregulating proliferation may be partially accounted for by repression of KIF18A. Given that PUM proteins regulate numerous RNA targets (Galgano et al., 2008; Smialek et al., 2019 preprint), there are probably other genes with similar properties to KIF18A which are worth further investigation.

Second, we found that KIF18A depletion caused an increase of TCam-2 cell number in G2/M phase and a decrease in G1 and S phase. We postulate that the increased TCam-2 cell number in the G2/M phase indicates an inhibition of cell cycle progression. Such an effect would be consistent with previous reports showing that KIF18A depletion caused mitotic arrest accompanied by the presence of unaligned chromosomes in HeLa cells, as well as mitotic arrest of mouse male germ cells, resulting in infertility (Huang et al., 2009; Liu et al., 2010). Moreover, according to a recent study on human somatic HeLa and retinal pigment epithelial cell line (RPE1), loss of chromosomal alignment upon KIF18A depletion gives rise to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit and the formation of micronuclei (Fonseca et al., 2019). Such abnormalities could potentially also arise in TCam-2 cells upon KIF18A depletion and cause mitotic arrest. The effect of KIF18A depletion on TCam-2 cell cycle is also consistent with our recent report documenting that PUM proteins inhibit TCam-2 cell cycle progression (Janecki et al., 2018). Therefore, we propose that post-transcriptional modulation of KIF18A level represents a mechanism by which PUM proteins influence the TCam-2 cell cycle.

Third, we have observed a significant pro-apoptotic effect of KIF18A depletion on TCam-2 cells, which was in line with the recently reported repressive effect of PUM proteins (Janecki et al., 2018). This pro-apoptotic effect is in line with that seen in mouse germ cells, which upon chromosome alignment defects caused by KIF18A depletion, undergo mitotic arrest and apoptosis (Czechanski et al., 2015). We show that this KIF18A-mediated effect in TCam-2 cells might be accounted for by impacting a group of genes that are related to apoptosis. Namely, a positive influence of KIF18A depletion on pro-apoptotic BCL2L1 gene expression as well as its negative effect on expression on anti-apoptotic BCL2 gene expression may account for its anti-apoptotic activity in TCam-2 cells. Interestingly, we have previously reported that PUM overexpression causes upregulation of apoptosis (Janecki et al., 2018), the same effect as KIF18A knockdown. This suggests that KIF18A is among the targets of PUM proteins influencing apoptosis. Our analysis showed that KIF18A knockdown was associated with downregulation of the mRNA level of the anti-apoptotic BCL2, which is consistent with the described anti-apoptotic role of KIF18A. However, KIF18A knockdown also was associated with upregulation of anti-apoptotic CCND1 and PIK3CA genes, which is opposite to what one could expect and therefore these processes require further studies.

The precise regulation of proliferation, apoptosis and cell cycle is crucial for germ cell development, and dysregulation of these processes may lead to infertility or cancer. TCam-2 cells, while representing human male germ cells, originate from a type of the most frequent type of human TGCT, the seminoma (for a review, see Batool et al., 2019). TGCTs are the most common solid tumours in young men, and their incidence is on the rise (Rosen et al., 2011). Our finding that KIF18A depletion inhibits proliferation of TCam-2 cells, is consistent with previous reports showing KIF18A-stimulated proliferation of other cancer cells in vitro and in vivo, such as clear cell renal carcinoma, breast cancer or lung adenocarcinoma (Chen et al., 2016; Zhang et al., 2010; Zhong et al., 2019). In addition, the effect of KIF18A depletion, associated with upregulation of apoptosis and cell cycle arrest represent features promoting cancer.

Altogether, this study broadens the current knowledge concerning regulation of KIF18A in germ cells, further studies of KIF18A regulation by PUM proteins in the context of pathways governing processes such as cell proliferation, cell cycle and apoptosis is of high priority. Considering that these three processes are crucial for germ cell development and cancer, such further studies may shed light on the molecular basis of the functional relationships between these two processes.

MATERIALS AND METHODS

Cell culture and transfection

TCam-2 cells were cultured in RPMI with GlutaMAX medium (Gibco, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (HyClone) and 1% (v/v) antibiotic–antimycotic solution (Lonza). The cells were transfected with plasmid (pCMV6-entry from Origene) and siRNA constructs (Santa Cruz Biotechnology) using the Neon Transfection System (Life Technologies) according to the manufacturer's protocol.

Luciferase assays

For luciferase assays, 2×105 TCam-2 cells were transfected with 1.5 μg of plasmid DNA encoding PUM1, PUM2 or an empty plasmid in the pCMV6-entry vector system (OriGene Technologies), plus the full-length 3′UTR of KIF18A in the psiCheck2 dual luciferase vector system (Promega, Germany) at a 10:1 plasmid DNA:luciferase vector ratio. Transfected cells were cultured in 12-well plates for 24 h in standard medium without antibiotic antimycotic solution. For PUM transient knockdown experiments, 1.5×105 TCam-2 cells were transfected with 40 nM siRNA and 150 ng of psiCheck2 vector constructs as described above, and then cultured in 12-well plates for 48 h to achieve effective PUM knockdown. Transfections were performed in three biological repetitions. Cells were lysed and luminescence from each well was measured twice using a Glomax-Multi Detection System luminometer (Promega) and the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer's protocol. The mean Renilla to firefly luciferase luminescence ratios and standard deviations were calculated from three experiments. Luminescence ratios for each combination of constructs and/or siRNA are presented as a percentage of relative luciferase units (RLU). The sample transfected with empty pCMV6-entry or control siRNA plus the reporter construct was considered as 100%.

MTS proliferation assay

TCam-2 cells were transfected with 40 nM siRNA mixture of three different KIF18A sequences (Santa Cruz Biotechnology, sc-96629), PUM1 (Santa Cruz Biotechnology, sc-62912), PUM2 (Santa Cruz Biotechnology, sc-44773) and control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) (sequences in Table S1). These were performed in three biological replicates (three independent transfections; seven technical repeats/wells on a 96-well plate) and were cultured for 24, 48, 72, 96 and 120 h after transfection. To obtain a similar number of viable cells per well, the same (5000 cells per well) number of cells were seeded to 96-well plates directly after transfection. The cell viability was measured using GLOMAX (Promega) at 450 nm (and 750 nm for cell background) wavelength at 24, 48, 72, 96 and 120 h after transfection and 4 h after adding 20 µl of CellTiter 96® AQ One Solution Reagent (Promega) to each well containing transfected TCam-2 cells.

The expression of KIF18A, PUM1, PUM2 and ACTB proteins was measured 24, 48, 72, 96 and 120 h after transfection by western blotting using anti-KIF18A, -PUM1, -PUM2 and -ACTB antibodies. ACTB expression was used as a reference to estimate the protein content in each blot.

Western blotting

Overexpression and silencing efficiency was measured by western blot analysis under standard conditions, using a nitrocellulose membrane, horseradish peroxidase (HRP)-conjugated secondary antibodies, and semi-quantitative measurement of protein levels was performed using ImageLab 5.1 software (Bio-Rad). The chemiluminescent signal was detected using Clarity™ Western ECL Substrate for HRP (Bio-Rad).

Antibodies

For the measurements of knockdown efficiency, the primary antibody anti-KIF18A (Thermo Fisher Scientific, PA5-31477, 1:5000), anti-PUM1 (Santa Cruz Biotechnology, sc-65188, 1:1000) and anti-PUM2 (Santa Cruz Biotechnology, sc-31535, 1:250) were used. The primary antibody anti-DDK (FLAG) (OriGene Technologies, TA50011, 1:1000) was used for detection of proteins overexpressed from the pCMV6-entry vector. Anti-ACTB reference protein (Sigma-Aldrich, A2066, 1:10,000) antibody was also used. Goat anti-rabbit IgG-HRP (Sigma-Aldrich, A6154, 1:25,000), mouse anti-goat IgG-HRP (Santa Cruz Biotechnology, sc-2354, 1:50,000) and mouse IgGκ-HRP (Santa Cruz Biotechnology, sc-516102, 1:10,000) were used in the study as a secondary antibodies.

Flow cytometry

Cell cycle analysis was performed 48 h after TCam-2 transfection with 40 nM control and KIF18A siRNAs. For this purpose, TCam-2 cells were washed with PBS and fixed in cold 100% methanol on ice for 10 min. After incubation at 37°C for 15 min in 50 µg/ml propidium iodide (PI) (Sigma-Aldrich) containing 330 µg/ml RNase A (Sigma-Aldrich), cells were incubated for 1 h on ice and finally measured using the S3e™ Cell Sorter (Bio-Rad) apparatus. Data files were analysed using ModFit LT (Verity Software House).

Detection of apoptotic TCam-2 cells was performed 48 h after transfection of TCam-2 cells with control and KIF18A siRNA constructs using an Annexin V–FITC Apoptosis Detection Kit (Beckman Coulter), according to the manufacturer's protocol, followed by flow cytometry using a FlowSight instrument (Amnis). The results were analysed using Image Data Exploration and Analysis Software version 6.0 (IDEAS® v6.0, Amnis). For all flow cytometry experiments, three biological repetitions with two technical repetitions in each were performed.

RNA co-immunoprecipitation

For RNA co-immunoprecipitation (RIP) experiments, 2×106 TCam-2 cells were transfected with 15 µg of pCMV6-entry empty (only DDK), and PUM1–DDK and PUM2–DDK coding sequences. At 24 h post-transfection TCam-2 cells were washed twice with ice-cold PBS and subjected to UV cross-linking at 254 nm on a HEROLAB CL-1 Cross-linker for 60 s (0.03 J). For one RIP reaction, 2×106–3×106 cells were lysed in 1 ml of RIPA Lysis Buffer (with 1% Triton X-100) for 30 min with rotation at 4°C. Lysates were centrifuged (104g for 10 min), and the supernatant was mixed with anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823, 50 µl) with protease and RNase inhibitors. The RIP reaction was held for 3 h at 4°C on a rotator in a final volume of 1 ml. Then, magnetic beads were washed three times with RIPA buffer (without Triton X-100) followed by treatment with proteinase K at 55°C for 30 min. Total RNA was isolated from 80% magnetic beads using a TRIzol® Reagent (Life Technologies) according to the manufacturer's protocol. 20% of the magnetic beads, 3% of input and flow-through were loaded on 8% PAGE gels for western blot analysis.

Real-time quantitative PCR

To measure KIF18A mRNA enrichment in anti-PUM1 and PUM2 RIPs, we added 200 ng of total D. melanogaster RNA (treated as spike-in RNA) to both of the two samples of total RNA isolated from the RIP (∼50-100 ng) for later reverse transcription of both RNA pools together and RT-qPCR experiments (in which Act5c mRNA from the D. melanogaster spike-in RNA was treated as reference).

To measure KIF18A, PUM1, PUM2, AKT1, BCL2, BCL2L1, CREB1, CCND1 and PIK3CA mRNA levels, total RNA from cell cultures was isolated using TRIzol® Reagent (Life Technologies) according to the manufacturer's protocol. RNA was treated with DNase I (Sigma-Aldrich) and reverse-transcribed using the Maxima First Strand cDNA Synthesis Kit (Life Technologies) according to the manufacturer's protocol. Total cDNA was used as a template for qPCR amplification. The reaction was carried out using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) in 10 μl volumes containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 3.5 mM MgCl2, 0.2× Sybr Green, 0.2 mM dNTP mix (dATP, dCTP, dGTP, dTTP), 0.2 μM forward and reverse primers and 0.5 U JumpStart™ Taq DNA polymerase (Sigma-Aldrich, D4184). Specific primers are listed in Table S2. Amplification parameters were as follows: initial denaturation 95°C, 2.5 min and 40 cycles of denaturation 95°C, 15 s, annealing 10 s (annealing temperatures for each primer pair are given in Table S2), extension 72°C, 15 s. ACTB and GAPDH were used for normalization.

Statistical analysis

A one-way unpaired t-test was used to estimate statistical significance. P<0.05 (*) was considered statistically significant.

Acknowledgements

We thank Anna Spik for excellent technical assistance, Jaruzelska lab and Simon Anthony Patterson for reading and correcting the manuscript.

Footnotes

Author contributions

Conceptualization: M.J.S., E.I., J.J.; Methodology: M.J.S., B.K., E.I., D.M.J., M.P.S.; Software: M.J.S., E.I.; Validation: M.J.S., B.K., J.J.; Formal analysis: M.J.S., B.K., E.I., D.M.J., M.P.S., J.J.; Investigation: M.J.S., B.K., E.I., M.P.S.; Resources: K.K.-Z., J.J.; Data curation: M.J.S., J.J.; Writing - original draft: M.J.S., B.K., J.J.; Writing - review & editing: M.J.S., B.K., E.I., D.M.J., M.P.S., K.K.-Z., J.J.; Visualization: M.J.S.; Supervision: M.J.S., J.J.; Project administration: M.J.S., J.J.; Funding acquisition: M.J.S., J.J.

Funding

This study was supported by grants from the National Science Centre Poland (Narodowe Centrum Nauki) no 2013/09/B/NZ1/01878 to J.J. and ETIUDA6 scholarship no 2018/28/T/NZ1/00015 to M.J.S.

Peer review history

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Competing interests

The authors declare no competing or financial interests.

Supplementary information