The role of CSE1L silencing in the regulation of proliferation and apoptosis via the AMPK/mTOR signaling pathway in chronic myeloid leukemia

ABSTRACT Objectives Chromosome segregation 1-like (CSE1L) is abundant and strongly expressed in solid tumors. However, the expression and role of CSE1L in chronic myeloid leukemia(CML) remain largely unknown. Materials and methods The relative expression levels of CSE1L in bone marrow granulocytes from patients with primary CML and non-hematologic controls were measured by flow cytometry. Cell counting kit-8 analysis, DNA Content Quantitation Assay, and Annexin V-PE/7-AAD staining were applied to assess the effects of CSE1L knockdown on cell proliferation, cell cycle progression, and apoptosis. Results Elevated expression of CSE1L was detected in bone marrow granulocytes of patients with primary CML. In the CML cell line K562 cells, CSE1L knockdown impaired cell proliferation blocked the cell cycle shift from G0/G1 phase to the S phase, and promoted apoptosis. Knockdown of CSE1L reduced Bcl-2 protein expression and increased Bax protein expression. Meanwhile, knockdown of CSE1L enhanced the expression of phospho-AMPK protein and decreased the expression of phospho-mTOR protein. The expression of total AMPK and mTOR proteins was not affected. In addition, CSE1L expression levels were decreased in imatinib-treated K562 cells. Conclusions CSE1L plays a pivotal role in K562 cell survival and growth. These functions may be partially dependent on the AMPK/mTOR signaling pathway to achieve. In addition, CSE1L may have had a future impact on the treatment of CML patients.


Introduction
Chronic myeloid leukemia (CML) is a bone marrow hyperplastic malignant tumor that accounts for approximately 15% of all adult leukemia [1,2].The BCR/ABL oncoprotein encoded by the BCR/ABL oncogene maintains constitutive tyrosine kinase activity, induces downstream signaling pathway activation, promotes CML cell proliferation, and inhibits cell apoptosis [3].Imatinib targeting BCR/ABL has emerged as a firstline drug for CML treatment [4].However, modern drugs have significant side effects and adverse reactions, often leading to chemotherapy discontinuation [5,6] and remission in only 20-30% of patients [7].Moreover, most people with CML cannot be cured with drugs and must continue to take them for the rest of their lives, and once they stop, they quickly relapse [8].Therefore, it is essential to profoundly investigate the regulatory mechanisms of CML cell biological properties and discover BCR/ABL-related molecules to treat CML.
Chromosome segregation 1-like(CSE1L, also named CAS/XPO2) is a multifunctional protein with a molecular weight of 110 kDa found on human chromosome 20q13 [9].CSE1L is associated with microtubules and mitotic spindles and is thought to play a role in tumor growth and is a potential oncogene [10,11].CSE1L is involved in cell apoptosis, embryo development, microvesicle formation, cell survival, chromosomal installation, and cytoplasmic transport [12][13][14][15] and is favorably linked with tumor grade, metastasis, and prognosis [16,17].It is highly expressed in numerous forms of solid tumors, including gastric, oral, lung, and others [18][19][20].However, the expression status of CSE1L in CML, its carcinogenic effect, and the underlying molecular mechanism are still unclear.
Here, we systematically evaluated CSE1L expression using bone marrow specimens from CML patients and controls.we found that CSE1L was highly enriched in bone marrow granulocytes from CML patients.Next, we performed functional experiments using K562 cells as a CML model to explore the role of CSE1L in CML.We demonstrated that CSE1L was closely associated with the proliferation, apoptosis, and cycle of K562 cells.Excitingly, we confirmed that CSE1L expression was reduced in K562 cells treated with imatinib.In addition, we found that CSE1L was involved in the AMPK/mTOR signaling pathway.
Taken together, our findings suggest that CSE1L offers significant clues for further study of the mechanism and clinical therapy of CML.

Clinical sample information
From January 2021 to January 2022, all 68 hematology inpatients at the Second Hospital of Shanxi Medical University were screened.Finally, 47 patients with a primary diagnosis of CML and 21 controls(no hematologic disease or other tumors) were included in this study.All leukemia patients were diagnosed based on morphology, immunology, cytogenetics, molecular biology, and biochemistry [21].Specimens were stored in EDTA anticoagulant tubes at 4°C and tested within three days.All patients knew the study's purpose and completed written informed consent forms.All procedures were conducted in line with the principles of the Declaration of Helsinki and the relevant guidelines.
The expression level of CSE1L in CML patients 2 ul of PC7-labeled CD45 antibody and 30 ul of EDTA anticoagulant were added to the flow cytometry, respectively.50 ul of Buffer 1(Fixative Reagent) was then injected.10 min later Reagent 2(Permeabilization) was added in 50 ul increments.10 min later 1 ul of anti-CSE1L was used.30 min later the second antibody was protected from light.25 min later the relative expression level of CSE1L was detected by flow cytometry.
Cell line and cell culture CML K562 cells were donated by the Basic Laboratory of Hematology Diseases, the Second Hospital of Shanxi Medical University.K562 cells are lymphoblasts isolated from the bone marrow of a chronic myeloid leukemia patient.The K562 cells were incubated in RPMI-1640 medium (Gibco Company Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Punosai, Wuhan, China).K562 cells were incubated at 37°C in humidified air with 5% CO 2 .

Lentivirus transduction
The shRNA-CSE1L lentivirus was designed and synthesized by Han Hang Seng (Shanghai, China).The lentiviral vector of RNA interference-mediated gene silencing is HBLV-h-shRNA-CSE1L1-ZsGreen-PURO.The empty vector of lentivirus in negative control is HBLV-h-ZsGreen-PURO NC.2 × 10 5 K562 cells in the logarithmic growth phase were seeded into six-well plates with 2 ml fresh medium well.According to the titer of K562 cells (1 × 10 8 TU/ ml) and multiplicity of infection (MOI, 30), 60 µl of lentivirus and 3 ul of Polybrene (Han Hang Seng, Shanghai, China) were added to each well.The cells were screened with puromycin (Solarbio, Beijing, China).The infected cells that had been stabilized were used in future studies.

RNA isolation and quantitative PCR (qRT-PCR)
Total RNA was isolated from 1 × 10 6 cells using a TRIzol kit (TaKaRa, Dalian, China) and amplified using qRT-PCR.The cDNA (from total RNA; 1 μ g/reaction mixture) was synthesized with the Reverse Transcription Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions.For qRT-PCR analysis, Applied Biosystems 7300 Fast Real-Time PCR System (ABI, USA) and SYBR Green real-time PCR Master Mix were used.The reverse transcriptional PCR reaction parameters were 37°C for 15 min and 85°C for 5 s.The cDNA is kept at 4°C.For a total of 40 cycles, the Real-Time PCR conditions were as follows: initial denaturation for 30 s at 95°C, denaturation for 5 s at 90°C, annealing for 34 s at 60°C, and extension for 15 s at 95°C.All qPCR experiments were conducted with RNA extracts from three independent batches of cells, and each reaction was run in triplicate.The β-actin was used as an endogen gene.The 2 delta Ct method was used to determine the relative expression of CSE1L mRNA.The following are the PCR primers(oligo-dT): CSE1L-F:

Cell cycle and apoptosis assay
Cell Cycle was detected using the DNA Content Quantitation Assay (Solarbio, Beijing, China). 1 × 10 6 of K562 cells were collected according to the manufacturer's instructions.The cells were fixed overnight at 4°C using 70% alcohol.The next day, the cells were added RNaseA at 37°C for 30 min.The cells were then stained with propidium iodide staining solution (PI) and incubated for 30 min at 4°C protected from light.Samples were analyzed using flow cytometry at 488 nm.
Apoptosis was detected using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Solarbio, Beijing, China).A certain number of cells were collected and stained with 5 ul Annexin V-PE, and 10 ul 7-AAD, according to the manufacturer's instructions.After incubation, the stained cells were analyzed by flow cytometry.

Statistical analysis
Statistical analysis was performed using SPSS 25.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.3.1 (Graph-Pad inc., san Diego, USA).The comparison between the groups was analyzed via Student's t-test and Mann-Whitney U test.All Results were shown as mean ± standard deviation (SD) in our study.A statistically significant difference was considered to be p < 0.05.Statistical significance was set at *p < 0.05; **p < 0.01; ***p < 0.001.

CSE1L is upregulated in primary CML patients
To verify the expression level of CSE1L in primary CML patients, we examined the relative expression level of CSE1L in bone marrow granulocytes by flow cytometry.The CSE1L expression level in patients with primary CML patients was (72.20 ± 3.03)% compared with (34.23 ± 4.74)% in the control group (Figure 1).CSE1L expression was significantly higher in the primary CML patients than in the controls (p < 0.001).

Knockdown of CSE1L inhibits the proliferation of K562 cells
K562 cells were used as a CML model to create stable cell lines with CSE1L knockdown to better understand the mechanism of CSE1L in CML.Under a fluorescent microscope, the shRNA-NC and shRNA-CSE1L groups showed a more robust green light signal than the control group (Figure 2A).The infectious efficiency of shRNA-NC and shRNA-CSE1L was 99.7% and 99.6%, respectively (Figure 2B).qRT-PCR showed that CSE1L knockdown was reduced to 40% (Figure 2C).Western blot also verified the expression of CSE1L was successfully reduced to 57% (Figure 2D).Within 24 h, CCK8 data showed no significant difference in K562 cell proliferation between the shRNA-NC and shRNA-CSE1L groups.However, after 48 h, the proliferation of CSE1L knockdown group was significantly inhibited (p < 0.001) (Figure 2E).Therefore, the knockdown of CSE1L could inhibit the proliferation of K562 cells.

CSE1L knockdown hinders the K562 cell cycle at G0/G1 phase
To evaluate the effect of CSE1L on the K562 cell cycle, we used flow cytometry.MODFIT software was used for cell cycle result visualization.The cell cycle analysis demonstrated that compared to the shRNA-NC group, the shRNA-CSE1L group had a more significant proportion of cells in G0/G1 and a lower proportion in the S phase (p < 0.001), with no significant difference in G2/M (Figure 3).

Knockdown of CSE1L promotes apoptosis of K562 cells
To understand the potential oncogenic mechanism of knockdown CSE1L in CML, we performed CSE1L knockdown using shRNA treatment in K562 cells.Figure 4A showed that imatinib caused a decrease in cell viability in a dose-dependent manner after treatment for 24 h.K562 cells treated with 4 μM Imatinib were used as the positive control group.Compared to the shRNA-NC group (1.29 ± 0.59)%, the shRNA-CSE1L group (6.48 ± 1.78)% showed significantly higher apoptosis rates (Figure 4B-C) (p < 0.001).Next, we observed that the important apoptotic protein Bcl-2 expression was significantly reduced after CSE1L silencing, while that of Bax was significantly increased (Figure 5D-E).These results suggested that CSE1L is involved in cell apoptosis.

Imatinib treatment inhibits the expression level of CSE1L in CML cells
To demonstrate whether CSE1L is involved in imatinibinduced apoptosis, we observed the expression level of CSE1L in imatinib-treated K562 cells using Western blotting.We treated K562 cells with 4 μM imatinib and collected the cells at 24 h to extract protein to detect CSE1L expression.Figure 5 showed that the CSE1L expression level was reduced in K562 cells treated with imatinib.These data strongly suggest that CSE1L could be an effector in imatinib-induced apoptosis in CML cells.

Knockdown of CSE1L alters the AMPK and mTOR expression in K562 cells
As AMPK has been reported to play a role in early apoptosis after Bcl-2 inhibition [22], we tested whether AMPK protein was also affected by CSE1L.We found that the phosphorylation level of AMPK was increased after the knockdown of CSE1L, while the total AMPK protein level was not significantly altered (Figure 6A).In addition, a previous study proposed that AMPK activation could lead to the downregulation of mTOR [23].In our study, knockdown of CSE1L reduced mTOR protein phosphorylation levels, while no significant alterations were seen in total mTOR protein (Figure 6B).Taken together, our results indicate that CSE1L mediates CML progression may be via AMPK/mTOR signaling.

Discussion
Tyrosine kinase inhibitors (imatinib, nilotinib, and dasatinib) are now widely used in the treatment of CML patients.However, these TKIs produced only transient anti-leukemic effects [24], and only half of the patients who achieved DMR maintained molecular remission after discontinuation of the TKI [25].Long-term medicines, meanwhile, cause many adverse responses and consequences in patients, such as cardiovascular toxicity, thrombotic vascular occlusion, and muscular spasm, all of which harm patients' quality of life and life [26,27].Therefore, it is urgent to explore new therapeutic targets for CML.
CSE1L has been reported to play an important role in maintaining the balance between cell proliferation and apoptosis [6,28].Cancer development and survival need CSE1L [29].Inhibition of CSE1L expression has been shown to slow the growth and spread of triplenegative breast cancer [30].Silencing CSE1L inhibits the viability of lung cancer cells and reduces malignant transformation of cancer cells [31].However, no studies on the expression of CSE1L in CML have been done.To investigate the possible molecular mechanism of CSE1L expression in CML patients, we detected the relative expression level of CSE1L in bone marrow granulocytes by flow cytometry.The results showed that strong CSE1L signals were detected in bone marrow granulocytes from patients with primary CML.In a K562 cell model,we showed that knockdown of CSE1L shifts K562 cells from low Annexin to medium Annexin levels.This result confirmed that knockdown of CSE1L can reduce the growth of K562 cells and induce apoptosis.Disruption of cell cycle checkpoints has been identified as a hallmark of cancer, with the G1-S phase being the most important regulatory point in cell cycle regulation [32,33].In our study, knockdown of CSE1L also hindered the cell cycle transition from G0/G1 phase to the S phase, without significant effect on the G2/M phase.Interestingly, our study showed that imatinib triggered a downregulation of CSE1L in K562 cells.The results suggest that CSE1L is involved in imatinib-induced killing of K562 cells.The findings suggest that CSE1L may play a significant role in the onset and progression of CML.
Since we have depicted the high expression of CSE1L in patients with CML and the cellular events involved in K562 cells, we performed further studies to explore the potential molecular pathway of silencing CSE1L-induced apoptosis.The mitochondrial pathway is one of the most significant apoptotic signaling pathways, and the Bcl-2 family plays a key role in its regulation.In a normal physiological state, apoptosis and proliferation are in dynamic balance.Excess exposure to the anti-apoptotic gene Bcl-2 can inhibit  apoptosis, whereas extra exposure to the pro-apoptotic gene Bax can reverse Bcl-2 ′ s biological activity and induce apoptosis [34].Our results showed that Bax protein expression was increased and Bcl-2 protein expression was decreased in K562 cells with knockdown of CSE1L.These results further confirmed that CSE1L promotes apoptosis of K562 cells through the mitochondrial pathway.
AMPK is expressed in various metabolic organs and can be triggered by various events in the body.AMPK activity is controlled by phosphorylation [35].AMPK negatively regulates mTOR, a serine/threonine-protein kinase [36,37].Emerging evidence has previously indicated a strong link between the AMPK/mTOR cascade and apoptosis [38].Activation of the AMPK/mTOR pathway promotes proliferation and invasion of colorectal cancer cells [39].Among these reports, activation of the AMPK/mTOR pathway was shown to induce apoptosis in pancreatic cancer cells [40].In our study, we found an increase in the expression of p-AMPK protein and a decrease in the expression of p-mTOR after silencing CSE1L in K562 cells, while total AMPK and mTOR protein expression were not significantly altered.The results suggest that silencing CSE1L may regulate cell proliferation and apoptosis through the AMPK/mTOR pathway.Further in vitro and in vivo investigation of the detailed mechanism is necessary and planned in our future work.

Conclusion
In summary, we demonstrated in the present study that CSE1L downregulation is involved in imatinibinduced CML cytotoxicity.The underlying mechanism may be that the knockdown of CSE1L activates the AMPK/mTOR signaling pathway and simultaneously inhibits Bcl-2 protein expression and increases Bax protein expression, leading to cell cycle arrest, growth inhibition, and apoptosis.Most importantly, our results indicate that CSE1L is a viable therapeutic target for CML.

Disclosure statement
No potential conflict of interest was reported by the author (s).

Figure 1 .
Figure 1.CSE1L is upregulated in primary CML patients.(A)the positive signal of CSE1L was in the second quadrant.(B)Comparison of CSE1L-positive signals in bone marrow granulocytes of primary CML patients and non-hematologic controls.

Figure 2 .
Figure 2. Knockdown of CSE1L inhibits K562 cell growth.(A-B)The transfection efficiency of CSE1L was examined in K562 cells.Green fluorescence(A) and GFP protein(B) expression were significantly increased in the K562 cells.(C-D) The CSE1L knockdown efficiency was examined in K562 cells.The mRNA (C) and protein (D) levels of CSE1L decreased in the K562 cells transfected with CSE1L-KD shRNA.(E)The CCK8 assay was used to analyze the proliferation of K562 cells at 0, 24, 48, 72, and 96 h.All Experiments were performed three times.

Figure 3 .
Figure 3. Cycle distribution of K562 cells.(A) Flow cytometry was used to determine the proportion of K562 cells in the G0/G1, S, and G2/M phases.(B) The chart shows the cell cycle data obtained by flow cytometry analysis in the shRNA-NC and shRNA-CSE1L groups.All Experiments were performed three times.

Figure 4 .
Figure 4. CSE1L knockdown promotes the apoptosis of K562 cells.(A) The viability of K562 cells after treatment with different concentrations of imatinib (0, 0.5, 1, 2, 4, 8, or 16 μM) for 24 h.(B) Flow cytometry was used to detect the apoptosis rate of cells.(C) The chart shows the cell apoptosis data obtained by flow cytometry analysis in the shRNA-NC and shRNA-CSE1L groups.(D-E) Western blot was used to analyze the relative expression level of Bcl-2 and Bax protein.β-actin was used as an internal reference.All Experiments were performed three times.

Figure 5 .
Figure 5. Imatinib alters the expression of CSE1L in K562 cells.Western blot was used to determine the relative expression levels of CSE1L protein.β-actin was used as an internal reference.All experiments were performed three times.

Figure 6 .
Figure 6.CSE1L knockdown affects AMPK/mTOR signaling pathway protein expression in K562 cells.(A-B) Western blot was used to determine the relative expression levels of t-AMPK, p-AMPK, mTOR, and p-mTOR proteins.β-actinwas used as an internal reference.All Experiments were performed three times.