Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer

doi:10.21203/rs.3.rs-5254983/v1

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Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer

https://doi.org/10.21203/rs.3.rs-5254983/v1

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Cervical cancer (CC) remains a significant health threat to women worldwide, with a pressing need for novel therapeutic targets. Despite recent advances, the molecular mechanisms underlying CC progression are not fully understood. Nuclear transport proteins, key regulators of macromolecule movement between cellular compartments, have emerged as potential targets in cancer therapy. However, the specific role of Importin 7 (IPO7) in CC development and its clinical implications remain poorly characterized, indicating a significant gap in our current understanding.

Here, we investigated IPO7's role in CC, leveraging clinical samples, bioinformatics analyses from TCGA and GEO databases, and experimental models. We found that IPO7 is upregulated in CC and associated with poor prognosis. IPO7 knockdown in cell lines and animal models revealed decreased cell proliferation, reduced colony formation, inhibited migration and invasion, and enhanced apoptosis. To uncover IPO7's molecular mechanisms, we performed mass spectrometry analysis, identifying MSI2, an RNA-binding protein, as a potential cargo. Further validation confirmed a direct interaction between IPO7 and MSI2, with IPO7 overexpression coupled with MSI2 knockdown abrogating oncogenic effects. Sequencing analysis of IPO7-knockdown cells indicated the MYC targets pathway and co-immunoprecipitation assays confirmed a direct interaction between MYC and MSI2, suggesting that IPO7 may facilitate the nuclear transport of MSI2 and MYC, thereby promoting cancer progression. Clinically, elevated MSI2 expression in CC patients, particularly in advanced stages, correlated with poorer outcomes.

Our findings elucidate the role of IPO7 in CC, demonstrating its potential as a therapeutic target. The interaction between IPO7, MSI2, and MYC provides a novel avenue for developing targeted therapies. Importantly, our results underscore the importance of IPO7-mediated nuclear transport in CC progression, presenting a promising strategy for enhancing patient outcomes and advancing CC treatment.

Health sciences/Diseases/Cancer/Gynaecological cancer/Cervical cancer

Biological sciences/Cancer/Gynaecological cancer/Cervical cancer

Cervical cancer

nuclear transport

Importin 7

MSI2

Prognosis

Cervical cancer (CC) is the predominant malignancy affecting the female reproductive system. According to the Global Cancer Statistics 2020 report, CC is the fourth leading cause of cancer-related mortality in women, with an estimated 604,000 new cases and 342,000 deaths worldwide¹. In 2020, the World Health Organization Assembly introduced a comprehensive initiative to eradicate CC, including strategies such as HPV vaccination, CC screening, and management of precancerous lesions². The management of CC faces challenges in achieving satisfactory outcomes, largely due to the difficulty of implementing interventions on a large scale³. The development of CC associated with high-risk HPV infections is a multifaceted process involving various stages and factors, which complicates the effectiveness of vaccines in improving the prognosis of HPV-infected individuals or CC patients⁴. T Targeted therapy represents a significant advancement in treating late-stage CC patients who have undergone standard surgical procedures. Therefore, a thorough investigation into the pathogenesis of CC and the discovery of new molecular targets is essential to improve prognostic outcomes.

Nuclear transport proteins, or karyopherins, are crucial in mediating the transport of signals and substances between the nucleus and cytoplasm⁵. These proteins facilitate the translocation of macromolecular proteins through the nuclear pore complex, which is essential for the transportation process⁶. The superfamily of nuclear transport proteins is comprised of α and β families, each possessing distinct structural and functional characteristics.

The Karyopherin-β family, encompassing importins, exportins, and biportins, plays a vital role in mediating the transport of macromolecules, especially proteins, through the nuclear pore complex (NPC) by recognizing specific nuclear localization signals (NLSs) or nuclear export signals (NESs)^{5, 7}. This transport process relies on the GTPase RanGTP for cargo binding and dissociation^{8, 9}. Under normal conditions, karyopherins precisely transport specific cargo proteins to their designated locations, thus facilitating proper physiological functions. Conversely, dysfunction of karyopherins results in the mislocalization of nucleo-cytoplasmic elements, including proto-oncogenes, tumor suppressor genes, and cell cycle regulatory proteins, thus contributing to tumor development⁵. Targeting karyopherins to disrupt nuclear-cytoplasmic communication and material transport in cancer cells could be a promising strategy to enhance the survival rates of individuals with CC.

Importin 7 (IPO7), a notable member of the karyopherin family, belongs to the β nuclear transport protein family. The sequence features two antiparallel helices, enhancing its binding affinity with associated proteins¹⁰, and thus making it a key component of the nuclear-cytoplasmic transport mechanism. Nonetheless, the specific biological functions of IPO7 in CC remain insufficiently understood and necessitate further investigation. This study aims to clarify the role of IPO7 in CC progression and, concurrently, to identify and validate potential IPO7 cargo proteins by isolating cytoplasmic and nuclear proteins and conducting mass spectrometry analysis.

2.1 Clinical samples and database analysis

The CC tissues microarray (TMA) containing 15 normal cervix tissues, 78 cervical intraepithelial neoplasia tissues, and 100 CC tissues from Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All tissue specimens were evaluated by two pathologists. This research was authorized by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital(2020-YS-075), and written informed consent was obtained from all patients before this study. CC data were acquired from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/), and normal cervix uterus data were acquired from The Cancer Genome Atlas (GTEx) (https://www.gtexportal.org/home/). The gene expression profile results were downloaded from the Gene Expression Omnibus database (GSE9750, GSE7803, GSE6791, GSE63514) at the NCBI. Database between a high-IPO7 expression group and a low-IPO7 expression group based on the median of TCGA-CESC IPO7 profiles and Kaplan–Meier analysis was conducted by the above grouping method. Gene Ontology Metascape analysis was performed using Metascape software (http://metascape.org/gp/index.html ) using default settings¹¹.

2.2 Culture of cell lines and transfection

Human CC Cell lines HeLa and SiHa were preserved in Shanghai Cancer Institute. All of the cells were cultured in a suggested medium (Invitrogen, USA) according to ATCC protocols. siRNA and plasmids transfection were performed using Lipofectamine 2000 (Invitrogen, USA). siRNAs targeting various genes were purchased from Asia-Vector Biotechnology (Shanghai, China), and the sequences are listed in Supplementary Files. To generate stable cell lines with IPO7 knockdown, Hela cells were infected with LV3-puro-shIPO7 (sh1) and cell clones were selected for puromycin resistance.

Human IPO7 coding sequence and Ubiquitin coding sequence was amplified and cloned into the expression plasmid PcDNA3.1 + for overexpressing IPO7-FLAG and UB-HA. The entire coding sequences of wild-type MSI2 and del-type MSI2 (deleted the NLS region) were subcloned into the expression plasmid PcDNA3.1+. The detailed information of plasmids and used primers is given in supplemental file.

2.3 Quantitative real-time PCR (qRT-PCR)

The total RNA was isolated from CC cell lines, quantified by NANO 2000 (Thermo Fisher Scientific, USA), subjected to reverse transcription and subsequently conducted to quantitative real-time PCR utilizing a 7500 Real-time PCR system (Applied Biosystems, USA). Relative mRNA expression was calculated by the 2−△△CT method, and was normalized by endogenous β-actin.

2.4 Western blotting analysis

For Western blotting, total protein was extracted using cell lysis buffer (Beyotime, China). Otherwise, subcellular nuclear cytoplasm protein fractionation was proceeded using NE-PER Nuclear and Cytoplasm Extraction Reagents (Thermo Fisher Scientific, USA) according to instructions. Western blotting was performed as previously describe¹². Concentrations of antibodies were used according to the manufacturer's instructions and the antibodies are listed in Supplementary File 1 (Table 4).

2.5 Co-Immunoprecipitation (Co-IP)

For Co-IP, immunoglobulin G (IgG) or specific antibody was incubated with protein G-agarose beads for 30 min at room temperature, and immunoprecipitated with protein at 4°C overnight. Then the beads were washed three times with PBS and eluted by 1x protein SDS loading buffer. Extracted proteins were separated by SDS-PAGE in a 7–15% gel, transferred to a nitrocellulose membrane (Millipore, Burlington, USA), and blocked in 5% BSA. Then, the nitrocellulose membrane was exposed to primary antibodies and species-specific secondary antibodies. Antibodies are presented in Supplementary file1.

2.6 Immunohistochemistry (IHC) analysis

Briefly, the primary antibodies anti-IPO7 (1:200, Abcam), anti-MSI2 (1:100, Abcam) were incubated with the slides first. Each tissue sample was scored according to the proportion of stained cells (0–5% scored “–”, 5–25% scored “+”, 25–50% scored “++”, 50–75% scored “++”, and 75–100% scored “+++”). The scoring was judged independently by two pathologists in a blinded manner. We designated the final score as a high or low expression group as follows: a score of “-” and “+” were defined low expression of IPO7 (or MSI2) and a score of “++” and “+++” were defined high expression IPO7 (or MSI2).

2.7 Immunofluorescence (IF) assay

Cells were cultured on coverslips in 8-well plates, and were fixed and permeabilized with 4% paraformaldehyde and 0.05% Triton X-10. BSA (1%) was used for blocking. Then cells were probed with the anti-IPO7 antibody (1:200, Abcam) and anti-MSI2 antibody (1:50, Abcam) overnight at 4°C, followed by an Alexa Fluor 594 conjugated anti-rabbit antibody (1:200 Jackson) and Fluor 488–conjugated anti-mouse antibody (1:200, Jackson). DAPI (4′,6-diamidine-2′-phenylindole, 1:1000, Invitrogen) was used for nuclear staining. IF images were captured by confocal laser scanning microscope (Carl Zeiss).

2.8 Cell proliferation assay

Cell proliferation was measured by using a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Japan). The cells were cultured on 96-well plates, and OD450 was measured 1 h after addition of CCK-8 at 0, 24, 48, 72, and 96 h.

2.9 Colony formation assay

The CC cells (2 × 10³) were cultured on per well of a 6-well plate for 2 weeks. Then the cell culture plates were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Photographs were acquired and the cell numbers were counted.

2.10 Cell migration and invasion assay

The Transwell migration assay was performed using Corning chambers (Corning, USA), featuring 8-µm pores. A total of 1 × 105 cells were resuspended in serum-free culture medium and seeded in the upper chamber, conversely complete medium containing 10% FBS was placed in the lower chamber. After 24-hour incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet, and then observed and quantified under an inverted microscope. Transwell invasion assay were performed as previously described except the chambers were coated with Matrigel (Corning, USA).

2.11 Cell apoptosis assay

Target cells were cultured under serum deprivation for 24 hours to assess apoptosis. Subsequently, the cells were detached with 0.25% trypsin and resuspended for propidium iodide (PI) and Annexin V-FITC staining. The samples were processed to FACS Canto-Plus flow cytometer (BD Biosciences).

2.12 Cell cycle assay

Cell cycle distribution was assessed by flow cytometry using the cell cycle/apoptosis analysis kit. The target cells were fixed in pre-cooling 75% ethanol for 12h at 4°C, and incubated in PI for 30 min at 37°C. The cells were harvested for flow cytometry analysis (BD Biosciences) and analysed with the Modfit LT 5.1 software.

2.13 Mouse xenograft model

a total of 1×106 IPO7-shRNA or control shRNA Hela cells were injected subcutaneously in the axilla of BALB/c female mice (5–6 weeks of age, n = 5 per group). The tumor diameters were measured by Vernier caliper every 7 days. The tumor volume was estimated based on the equation V = 1/2 (a x b x b). After 5 weeks, the mice were killed to dissect and weight xenograft tumors. The xenograft tumor samples were fixed and used for subsequent IHC analysis.

2.14 Ubiquitination assay

Cells were transfected with the HA-Ubiquitin plasmid, treated with the proteasome inhibitor MG132 (25 mM) (Sigma, M7449) for 6 h, and subsequently lysed in ubiquitination assay buffer. The cell lysis was co-immunoprecipitated with protein G-agarose beads conjugated with MSI2 antibody, and Western blotting was performed with an anti-HA antibody (Proteintech, China) to detect ubiquitinated MSI2.

2.15 Liquid Chromatography–mass spectrometry

Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and separated utilizing the EASY-nLC 1000 ultra-high performance liquid system. Subsequently, the peptides undergo separation by the ultra-high performance liquid system, followed by injection into the NSI ion source for ionization and analysis using Orbitrap Fusion mass spectrometry. The ion source operates at 2.2 kV and facilitates the detection and analysis of peptide precursor ions and their secondary fragments utilizing high-resolution Orbitrap.

2.16 RNA-seq

Hela cells (with IPO7-shRNA and control RNA) were seeded into a 6 cm plate. Cell lysates were prepared using Trizol (TaKaRa) and then stored at − 80◦C. The following steps were performed by Newcore Biotech. Cell lysate was sequentially treated with chloroform, isopropanol, and ethanol to extract total RNA. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, Los Angeles, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, Carlsbad, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, USA). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Ipswich, USA) following manufacturer’s recommendations. The gene set enrichment analysis (GSEA) analysis was performed using GSEA software following the manufacturer’s introduction.

2.17 mRNA stability assay

Cells (2 × 10^5 per well) were seeded into a 12-well plate. Transcriptional inhibition was achieved using Actinomycin D at a final concentration of 2 µg/mL (Leagene, Beijing, China). Total RNA was isolated at 0-, 3-, and 6-hours post-treatment using Trizol reagent. Subsequently, RNA was extracted, reverse transcribed into cDNA, and subjected to quantitative real-time PCR (qRT-PCR) as previously described.

2.16 Statistical analysis

The statistical analyses were performed using SPSS Statistics software (SPSS 20, USA) and GraphPad Prism 8.0 software. The data are presented as means ± SD and were calculated using the two-tailed Student’s t-test. The relationship between IPO7/MSI2 expression level and clinical parameters was determined by the χ2 test and Fisher’s exact test. Survival analysis was conducted using the Kaplan-Meier method. The survival curves were analyzed using the log-rank test. p < 0.05 was considered significant statistically for all tests.

3.1 IPO7 is highly expressed in CC and is associated with unfavorable prognosis

This study aims to investigate the role of nuclear transport proteins in the development of CC. We analyzed the expression profiles of 27 known nuclear transport proteins in the TCGA-CESC database to evaluate their potential as prognostic indicators. Furthermore, comparative analysis of expression levels in control and tumor samples from the GEO database identified IPO7 and RanBP17 as genes with increased expression and significant prognostic value in CC. Thus, the present study concentrated on elucidating the role of IPO7 in CC progression (Supplementary Fig. 1 and Supplementary Table 1).

Through the integration of TCGA and GEO databases, a substantial increase in IPO7 expression was observed in CC tissues relative to normal controls. Additionally, an escalation in IPO7 expression was noted in tandem with the advancement of cervical lesions (Supplementary Fig. 1B, Fig. 1A, and 1B). High IPO7 expression was associated with a poor prognosis for patients (Fig. 1C). However, no correlation was detected between elevated IPO7 expression and patient age, BMI, histopathological type, or clinical stage when analyzing the TCGA database. Moreover, individuals with high IPO7 expression had decreased overall survival (OS) and disease-specific survival (DSS) rates (Supplementary Table 2).

Additionally, we obtained clinical specimens from patients diagnosed with CC who underwent surgical treatment at our hospital over the past five years. IPO7 protein expression was evaluated via immunohistochemical staining (IHC) (Fig. 1D). Table 1 presents the statistical analysis associated with the clinical data. Our results demonstrate an incremental rise in IPO7 protein expression in tandem with the severity and stage of cervical lesions (Figs. 1F-1H).

Table 1

Correlation between IPO7 expression and clinicopathological features in cervical cancer (n = 100)
		Expression of IPO7
	total	Low (%)	High (%)	P value
age
< 55 years old	72	30 (41.7%)	42 (58.3)	0.121
≥ 55 years old	28	7 (25.0)	21 (75.0)
Menopause
Yes	82	32 (39.0)	50 (61.0)	0.532
No	18	5 (27.8)	13 (72.2)
Pregnancy
Yes	53	20 (37.7)	33 (62.3)	0.436
No	47	17 (45.9)	20 (54.1)
HPV infection
Yes	93	35 (40.0)	58 (60.0)	0.942
No	7	2 (47.7)	5 (52.3)
Clinical Stage (FIGO)
I	13	9 (69.2)	4 (30.8)	0.028*
II	44	16 (36.4)	28 (63.3)
III	43	12 (27.9)	31 (72.1)
Tumor size
T1	84	36 (42.9)	48 (57.1)	0.002*
T2-4	16	1 (6.3)	15 (93.8)
FIGO, International Federation of Gynecology and Obstetrics; * P < 0.05, Statistically significant

3.2 IPO7 plays an oncogenic role in CC

To determine the function of IPO7, this study employed two cervical cancer (CC) cell lines, SiHa and HeLa, exhibiting high levels of IPO7 expression (Supplementary Fig. 2A-2C). Subsequently, IPO7 knockdown experiments were performed in these cell lines (Fig. 2A). Following IPO7 knockdown, the CC cells exhibited reduced cell proliferation (Fig. 2B, P < 0.001), decreased colony formation (Fig. 2C and 2D, P < 0.01), and impaired migration and invasion (Fig. 2H-2I, P < 0.01, Supplementary Fig. 2D and 2E), along with increased apoptosis (Fig. 2E-2G, P < 0.01).

Furthermore, we established HeLa cell lines with stable IPO7 knockdown and evaluated their effect on tumorigenesis using subcutaneous xenograft models in nude mice. The group with IPO7 shRNA exhibited markedly slower tumor growth and lower tumor burden compared to the control shRNA group (Fig. 3A-3D). IHC analysis revealed decreased expression of the proliferation marker Ki67 in the IPO7-shRNA xenografts (Fig. 3E and 3F). This result indicated that IPO7 promoted the proliferation of HeLa cells. In summary, cellular and in vivo experiments collectively support the oncogenic function of IPO7 in CC cells.

3.3 MSI2 is a potential target protein transported by IPO7 in CC

As a nuclear transport protein, IPO7 is responsible for the import of cargo proteins into the cell nucleus. Thus, we investigated the molecular mechanism of IPO7 in CC by focusing on nuclear-cytoplasmic transport. We manipulated IPO7 expression and isolated nuclear-cytoplasmic fractions from both IPO7 knockdown and control groups. Subsequently, mass spectrometry analysis identified differentially expressed proteins. The analysis identified 74 proteins that were downregulated in the cell nucleus following IPO7 knockdown (fold change > 2 and P < 0.05). These proteins are considered potential IPO7 cargo proteins (Fig. 4A). The enrichment analysis indicated that these proteins are involved in processes such as protein localization, Rho GTP cycling, and translation regulation (Fig. 4B). Additionally, we performed enrichment analysis using Metascape (http://metascape.org/) on the top 200 genes most correlated with IPO7 expression in the TCGA- CESC database. This analysis showed that IPO7 is predominantly linked to mRNA processes involving transport and regulation (Fig. 4C).

Among the candidates, the RNA-binding protein Musashi-2 (MSI2) attracted our attention. MSI2 is a member of the Musashi family, which includes MSI1 and MSI2, and possesses two RNA-recognition motifs (RRM1 and RRM2) at the N-terminus¹³. MSI2 can bind to the 3'-end of target RNAs and modulate the stability and translation of mRNA of proteins implicated in tumorigenesis, including SMAD3, PTEN and others¹⁴. MSI2 is considered a potential IPO7 cargo protein in this study.

We utilized the NLS-mapper website (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi ) to predict the NLS of MSI2, identifying a highly conserved sequence (Fig. 4D) with a score of 5, indicating its potential for nucleocytoplasmic shuttling. Furthermore, MSI2 shows increased expression in CC (Fig. 4E) and is positively correlated with IPO7 expression (Fig. 4F, P < 0.001, R = 0.38). Further analysis showed decreased MSI2 protein levels after IPO7 knockdown (Fig. 4G), while qPCR analysis showed no significant change in MSI2 mRNA levels following knockdown (Fig. 4H). After isolating nuclear and cytoplasmic fractions, we performed individual analyses of MSI2 expression. The findings showed a reduction in MSI2 expression in both the nucleus and cytoplasm subsequent to IPO7 knockdown (Fig. 4I). The reduction in MSI2 expression could be attributed to hindered nuclear entry and subsequent degradation of the protein. This hypothesis is supported by the identification of seven ubiquitination sites on the MSI2 protein, as predicted using the SMART website (http://smart.embl-heidelberg.de/ ) (Fig. 5D). To further investigate, UB-HA was introduced into both normal and IPO7 knockdown CC cells, followed by immunoprecipitation (IP) experiments to evaluate the ubiquitination level of MSI2. The results indicated elevated MSI2 ubiquitination upon IPO7 knockdown, implying that this could be a potential cause for the decreased cytoplasmic levels of MSI2 (Fig. 4J).

3.4 MSI2 directly interacts with IPO7, which exerts oncogenic effects by transporting MSI2

In this study, we examined the interaction between IPO7 and MSI2. Initially, Co-IP experiments and IF assays were conducted to confirm a direct interaction (Fig. 5A-5C). Subsequently, we used the SMART website for predicting the structure of the MSI2 protein and generated a corresponding model diagram (Fig. 5D). This model indicated that the nuclear localization sequence (NLS) is predominantly located in the RRM1 region of MSI2. Then, we deleted the NLS fragment from the MSI2 DNA sequence to observe its effects on binding to IPO7 in cell cultures. A significant reduction in the binding affinity between MSI2 (del-NLS) and IPO7 was observed, particularly in HeLa cells where almost no binding was detected (Fig. 5E and 5F). The results demonstrate the importance of the NLS region in mediating the interaction between IPO7 and MSI2, suggesting that IPO7 likely facilitates the nuclear transport of MSI2.

To investigate whether the oncogenic effects of IPO7 are mediated through the transportation of MSI2, we conducted experiments with the overexpression of IPO7 and simultaneous knockdown of MSI2 in two cell lines (SiHa and HeLa). Firstly, we assessed the efficacy of MSI2 knockdown and selected the most potent shRNA sequence for further experiments (Supplementary Fig. 3). MSI2 knockdown did not influence IPO7 mRNA expression levels, and IPO7 overexpression had no impact on MSI2 mRNA levels in both cell lines (Fig. 6A-6D). Nevertheless, the overexpression of IPO7 and knockdown of MSI2 simultaneously resulted in a notable decrease in both cell proliferation capacity and colony formation ability (Fig. 6E-6I, P < 0.01). These results suggest that IPO7 exerts its oncogenic effects through the transportation of MSI2 into the nucleus.

3.5 MSI2 acts as an oncogene by directly interacting with c-MYC and is co-transported into the nucleus by IPO7.

To further investigate how MSI2 promotes cancer progression, we RNA-sequenced IPO7-knockdown HeLa cells and performed a GSEA analysis (Fig. 7A), which showed that the knockdown primarily led to the enrichment of MYC TARGET V1 and V2 (Fig. 7B and 7C). The MYC gene, a well-known oncogene, plays a pivotal role in the formation and progression of many tumors. Although our mass spectrometry analysis failed to detect changes in c-MYC expression following IPO7 knockdown, we identified a direct interaction between the MYC family and IPO7 using the BioGRID database (Supplementary Fig. 4A). Analysis of the TCGA cervical cancer database revealed a positive correlation between MYC and IPO7 expression levels (Supplementary Fig. 4B), and IPO7 expression levels was also positively correlated with MYC target pathways (Supplementary Fig. 4C). After confirming that IPO7 can bind to MSI2 and translocate it into the nucleus, we investigated whether MSI2 also interacts directly with c-MYC. Co-IP experiments validated a direct interaction between MSI2 and c-MYC in Hela and SiHa cells (Fig. 7D and 7E), leading us to suggest that IPO7 may synergistically transport the MSI2 and c-MYC complex into the nucleus, thereby promoting oncogenic effects. Furthermore, we found that MSI2 knockdown reduced the expression of c-MYC at both the mRNA and protein levels (Fig. 7F-7I). Given that MSI2 is an RNA-binding protein, we examined whether it influences the stability of MYC mRNA. Following treatment of MSI2-knockdown cells with actinomycin D for 0, 3, and 6 hours, qPCR analysis showed that MSI2 knockdown significantly decreased the mRNA stability of c-MYC compared to the control group (Figs. 7J and 7K). Therefore, we suggest that IPO7 may systematically transport the MSI2-c-MYC complex into the nucleus, where MSI2 plays a crucial role in maintaining the stability of MYC, potentially facilitating the nuclear import of c-MYC to exert oncogenic activities.

3.6 Elevated MSI2 and IPO7 expression correlate with a worse prognosis in CC patients

To investigate the oncogenic effect of IPO7 via MSI2 transport, we conducted a comprehensive examination of MSI2 expression in CC. Analysis using the GEO database (GSE6791, GSE63514) revealed a significant upregulation of MSI2 expression in CC (Fig. 8A and 8B, *P < 0.05). IHC staining of clinical samples (Fig. 8C) showed a considerable elevation in MSI2 expression in CC compared to normal cervical tissues (Fig. 8D), especially in advanced-stage cases (Fig. 8E). The CCK-8 assay demonstrated a reduction in the proliferation capacity of both CC cell lines following MSI2 knockdown (Fig. 8F and 8G). Moreover, after MSI2 knockdown, the majority of cells exhibited arrest in the S phase (Fig. 8H and 8I, **P < 0.01). Survival analysis performed on CC patients from TCGA indicated that high MSI2 expression alone did not impact the prognosis (Fig. 8J). However, CC patients with high MSI2 expression alongside high IPO7 expression experienced the poorest prognosis (Fig. 8K and 8L, P = 0.00069). Our results strongly suggest that IPO7 may serve as a promising target for CC treatment.

In conclusion, our research demonstrates a correlation between elevated IPO7 levels and unfavorable outcomes in patients with CC. IPO7 contributes to the progression of CC by promoting cellular proliferation, migration, and invasion, and inhibiting apoptosis. This oncogenic effect is primarily due to the transportation of MSI2 and c-MYC into the nucleus by IPO7, which that recognizes and binds to the nuclear localization signal (NLS) region of MSI2. Thus, in the presence of Ran-GTPase, the MSI2-c-MYC complex is released from IPO7 and exhibits its oncogenic characteristics within the nucleus. The oncogenic effects of IPO7 occur through the presence of MSI2, as illustrated in Fig. 9. If the cytoplasmic transport of MSI2 is hindered, it is subject to degradation via ubiquitination

Appropriate subcellular localization is crucial for the functioning of biological macromolecules, such as proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of numerous macromolecules within the nucleus or cytoplasm⁵. Dysfunctional nuclear transport leads to changes in the physiological levels and spatiotemporal localization of tumor suppressors, proto-oncogenes, and other macromolecules, thereby affecting tumorigenesis and drug sensitivity in cancer cells¹⁵. Our study investigates the role of karyopherins, specifically IPO7, in the development of CC. By analyzing expression profiles of 27 karyopherins in the TCGA-CESC database and comparing them to control samples in the GEO database, we discovered IPO7 to be significantly upregulated in CC, exhibiting a significant prognostic correlation. IPO7 demonstrates elevated expression levels, as confirmed by histochemical analysis of our clinical samples. Consequently, we proceeded to conduct subsequent studies into IPO7's role in CC and its potential cargo proteins.

Recent studies¹⁶ have found that increased IPO7 expression in breast cancer is linked to poor prognosis, mainly by aiding USP22-AR nuclear transport. NUAK1, an AMPK-related kinase, plays a key role in tumorigenesis, relying on Importin β family transporters like KPNB1, IPO7, and IPO9 for nuclear transport¹⁷. Moreover, An NTS-derived myristoylated phosphomimetic peptide specifically blocked ERK1/2's interaction with IPO7, hindering ERK1/2's nuclear translocation¹⁸. These results, along with our study, demonstrates that IPO7 significantly contributes to tumorigenesis and tumor progression by transporting various cargo proteins. Knockdown of IPO7 significantly reduced cell proliferation, colony formation, migration, and invasion, and increased apoptosis in Hela and Siha cells. Furthermore, xenograft model experiments showed that IPO7 knockdown delayed tumor growth and decreased tumor burden, reinforcing its oncogenic role.

Subsequently, we aimed to elucidate the mechanisms underlying IPO7's tumor-promoting effects, focusing on the cargo proteins that IPO7 transports. Cells efficiently regulate protein synthesis and degradation¹⁹. Non-functional proteins are quickly degraded and removed. Therefore, we knocked down IPO7 expression and isolated nuclear and cytoplasmic proteins for mass spectrometry analysis. We selected proteins with reduced nuclear expression after IPO7 knockdown for further study. We systematically screened 74 potential cargo proteins and performed enrichment analysis. These proteins are predominantly involved in the regulation of mRNA and the nuclear-cytoplasmic transport process. Notably, elevated IPO7 expression primarily facilitates protein localization. Our identification of MSI2, a member of the Musashi protein family, as a potential IPO7 cargo protein was based on its function as an RNA-binding protein that regulates mRNA stability and translation in key oncogenic pathways¹³. The NLS-mapper website²⁰ facilitated the prediction of IPO7's probable NLS sequence and location. Notably, the conservation of the NLS sequence suggests its crucial role in IPO7 binding and transportation. Direct interactions between IPO7 and MSI2 were confirmed using Co-IP and IF assays. Intriguingly, deleting the NLS region in MSI2 significantly reduced its binding to IPO7, especially in HeLa cells. Moreover, proliferation and colony formation assays further supported IPO7's role in tumor promotion via MSI2.

The Musashi protein family, including MSI1 and MSI2, is a widespread and highly conserved group of RNA-binding proteins²¹. MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA. Notably, the first RRM exhibits a greater affinity towards RNA compared to the second RRM^{22, 23}. MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA²⁴. MSI2 is widely expressed in various tumors, including leukemia, gastric, colorectal, and triple-negative breast cancers^{25, 26, 27, 28}.

How does IPO7-mediated nuclear transport of MSI2 enhance its oncogenic effects? After RNA-sequencing HeLa cells with IPO7 knockdown, GSEA analysis revealed significant enrichment in the "MYC targets" pathway. Research shows that c-MYC is vital in CC, with MYC genes often being HPV integration targets²⁹. Microhomology aids HPV integration near MYC, boosting c-Myc expression³⁰. Microbial overgrowth, such as Prevotella, may also enhance persistent HPV-related cervical lesions by influencing c-Myc expression³¹. Can IPO7 facilitate MYC's nuclear transport? Using the BioGRID database, interactions between IPO7 and MYC were identified. Co-IP assays further confirmed that MSI2 directly binds to c-MYC, suggesting that IPO7 is capable of transporting both MSI2 and c-MYC into the nucleus. Additionally, reduced c-MYC expression at both the protein and RNA levels was observed following MSI2 knockdown, suggesting that MYC is targeted for degradation if it is not transported to the nucleus. Meanwhile, as an RNA-binding protein, MSI2 stabilizes MYC mRNA. Upon IPO7 knockdown, MSI2 fails to be transported into the nucleus, leading to its ubiquitination and reduced expression. Concurrently, MSI2 knockdown impedes MYC nuclear transport, thereby curtailing its oncogenic potential, and reduces MYC mRNA stability, resulting in decreased MYC expression.

Additionally, we found that overexpression of MSI2 promotes CC cell proliferation. MSI2 has been identified as a target for small molecule inhibitors such as largazole and Aza-9, which have effectively reduced MSI2 levels and suppressed the proliferation of non-small cell lung cancer (NSCLC), chronic myeloid leukemia, and colon cancer cells^{32, 33}. A thorough analysis of the TCGA database showed that patients with high MSI2 and IPO7 expression had the worst prognosis. Thus, targeting the IPO7-MSI2 axis could be a more effective treatment strategy for CC.

In this study, we observed that downregulating IPO7 led to decreased MSI2 protein expression in both the nucleus and cytoplasm. However, IPO7 knockdown did not affect MSI2 mRNA levels, implying a post-translational mechanism. Knockdown-induced IPO7 reduction impeded MSI2 nuclear transport, causing cytoplasmic degradation and loss of function. Notably, ubiquitination is a key mechanism for cellular protein degradation. Using the SMART database³⁴, we identified seven ubiquitination sites on MSI2. This evidence indicates that IPO7 is crucial for nuclear transport of MSI2, underscoring the complex precision of cellular protein regulation. Ubiquitination of MSI2 is commonly observed in tumors. For instance, MSI2's interaction with LncRNA LINC00942 inhibits ubiquitination, thereby enhancing the stability and expression of target mRNAs, including c-MYC²⁶. Intriguingly, our research revealed that MSI2 enhances c-MYC mRNA stability, and c-MYC can bind directly to MSI2. Both are transported to the nucleus by IPO7 to perform their functions.

In conclusion, our study identifies IPO7 as a pivotal factor in the progression of CC. Its oncogenic role is mediated by nuclear transport of MSI2 and c-MYC. Targeting the IPO7-MSI2 interaction may lead to new therapeutic strategies for CC.

CC cervical cancer

DSS disease-specific survival

GEO the Gene Expression Omnibus

IHC Immunohistochemistry

IP immunoprecipitation

IPO7 Importin 7

MSI2 Musashi 2

NESs Nuclear Export Signals

NLSs Nuclear Localization Signals

OS overall survival

PI propidium iodide

RRM RNA-recognition motifs

TCGA The Cancer Genome Atlas

TMA tissues microarray

Grant support: This work was supported by grants from the National Natural Science Foundation of China (82002730)

The authors declare no conflict of interest.

Ethics approval and consent to participate

All of patients provided written informed consent (ethic approval number: 2020-YS-075), and all the experiments, including animal work (ethic approval number: 2020 − 0204) were approved by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and complied with the Declaration of Helsinki.

Consent for publication

All authors have consented for publication

Funding

This research was supported by the National Natural Science Foundation of China (No: 82002730).

Authors' contributions

Q.Y.X., R.Z. and Y.B designed and supervised this study. W.Z.Z, J.C., and L.M. performed the data analysis, statistical analysis and finished the manuscript writing. Q.Y.X., and T.Q. performed the whole experiments. Z.H.A., Y.C.T and R.Z.J. provided data collection and technical support. W.Z.Z, T.Q., and J.W. contributed equally to this work. All authors participated in revising, reviewing, and approving the final submission.

Acknowledgements

We are grateful to Professor Zhigang Zhang and Lipeng Hu for their mentoring and assistance on this study. We are grateful to the Asia-Vector Biotechnology for assistance with our experiments.

Availability of data and material

The datasets utilized and/or examined in this research are accessible from the corresponding author upon request. The database of Cervical cancer data produced in this study can be accessed through the Gene Expression Omnibus (GEO) with accession numbers GSE9750, GSE7803, GSE6791, GSE63514, as well as the TCGA-CESC libraries.

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Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Clinical samples and database analysis

2.2 Culture of cell lines and transfection

2.3 Quantitative real-time PCR (qRT-PCR)

2.4 Western blotting analysis

2.5 Co-Immunoprecipitation (Co-IP)

2.6 Immunohistochemistry (IHC) analysis

2.7 Immunofluorescence (IF) assay

2.8 Cell proliferation assay

2.9 Colony formation assay

2.10 Cell migration and invasion assay

2.11 Cell apoptosis assay

2.12 Cell cycle assay

2.13 Mouse xenograft model

2.14 Ubiquitination assay

2.15 Liquid Chromatography–mass spectrometry

2.16 RNA-seq

2.17 mRNA stability assay

2.16 Statistical analysis

3. Result

3.1 IPO7 is highly expressed in CC and is associated with unfavorable prognosis

3.2 IPO7 plays an oncogenic role in CC

3.3 MSI2 is a potential target protein transported by IPO7 in CC

3.4 MSI2 directly interacts with IPO7, which exerts oncogenic effects by transporting MSI2

3.6 Elevated MSI2 and IPO7 expression correlate with a worse prognosis in CC patients

4. Discussion

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