Regulation of AUF1 alternative splicing by hnRNPA1 and SRSF2 modulate the sensitivity of ovarian cancer cells to cisplatin

doi:10.21203/rs.3.rs-3584984/v1

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Regulation of AUF1 alternative splicing by hnRNPA1 and SRSF2 modulate the sensitivity of ovarian cancer cells to cisplatin

https://doi.org/10.21203/rs.3.rs-3584984/v1

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Ovarian cancer is one of the most prevalent gynecologic malignancies, with a poor prognosis due to its late-stage diagnosis, frequently presenting as highly advanced disease with concomitant drug resistance. Alternative splicing plays an important role in the evolution, development and disease of organisms. Tumor-specific alternative splicing events related to prognosis might be a great significance for tumor diagnosis and prediction and may even become effective targets for tumor therapy. AU-rich element RNA-binding protein 1 (AUF1) is a nucleic acid binding protein that is also known as heterogeneous ribonucleoprotein D (hnRNPD). Alternative splicing of the AUF1 precursor mRNA produces four different mRNA splicing variants, which encode four different molecular weights of protein isoform, p37, p40, p42 and p45 respectively. In this study, we demonstrated that different isoforms of AUF1 played a bidirectional role in ovarian cancer. In ovarian cancer, p37 isoform played a “cancer promoter” role, p42 and p45, especially p45 played a “cancer suppressor” role. The competitive binding of phosphorylated hnRNPA1 and O-GlcNAc modified SRSF2 on exon 2 and exon 7 of AUF1 regulated the alternative splicing of AUF1 and mediated the decrease of cisplatin responsiveness in ovarian cancer. This study provides a new target for exploring the drug resistance mechanism of ovarian cancer.

Biological sciences/Cancer/Oncogenes

Health sciences/Diseases/Cancer/Gynaecological cancer/Ovarian cancer

AU-rich element RNA-binding protein 1 (AUF1)

alternative splicing

ovarian cancer

cisplatin

Ovarian cancer is the second most common cause of gynecologic cancer death in women around the world. Due to the concealment of the incidence and the lack of perfect early diagnosis methods, about 70% of the patients were diagnosed at an advanced stage^[1–3]. The current treatment largely relies on debulking surgery and platinum-based chemotherapy. However, in clinical practice, some patients are insensitive to platinum, and some patients relapse after treatment and eventually become resistant to platinum, which has a serious impact on the quality of life and survival rate of patients^[4–6]. At present, there is no specific drug for the treatment of recurrent platinum-resistant ovarian cancer. Therefore, it is imperative to improve the chemotherapy sensitivity and overcome platinum resistance in the management of ovarian cancer.

Alternative splicing (AS) is a common mechanism of gene expression regulation in the human, representing a crucial co-transcriptional modification process that generates mature mRNA and holds significant importance for precise gene expression control and protein diversity^[7–8]. Alternative splicing plays an important role in cell growth and development, some tumor-specific alternative splicing events, such as alternative splicing of CD44, FGFR2, RAC1 or MST1R has been ascribed to the acquisition of tumor invasiveness ^[11–12]. Therefore, alternative splicing might be a great significance for tumor diagnosis and prediction and may even become effective targets for tumor therapy.

AUF1 (AU-rich element RNA-binding protein 1), also known as heterogeneous nuclear ribonucleoprotein D (hnRNPD), is an important member of the AU-rich element (ARE) binding protein family. I It can specifically bind to various ARE located in the 3’-untranslated region (3’-UTR) of mRNA, thereby modulating mRNA stability and translation efficiency, and playing a pivotal role in the intricate regulation of gene expression^[13–14]. A large number of previous studies have shown that AUF1 is abnormally expressed in various tumors and plays a bidirectional role in different tumors. It has been reported that AUF1 promotes progression of colon and breast cancer^[15–17], while inhibits progression of liver and kidney cancer^[18–19]. Four different mRNA splicing variants are generated from AUF1 pre-mRNA by alternative splicing, which encode four protein isoforms with different molecular weight, respectively known as p37, p40, p42 and p45. The subcellular localization of AUF1 protein isoforms were different, p37 and p40 were in the nucleus and cytoplasm, while p42 and p45 were mainly located in the nucleus ^[20]. Although the mechanism of these differential distribution was still not clear, studies have shown that AUF1 isoforms play different roles in the regulation of gene expression, and may be affected by other factors^[13]. Thereby, the contradiction and diversity of AUF1 function may at least partially be ascribed to the different localization of different subtypes in cells.

In this study, we demonstrate that the expression levels of the AUF1 isoforms were different in the cisplatin-resistant or cisplatin-sensitive ovarian cancer tissues and cells. The current study demonstrated that isoform p37 played a “tumor-promoter” role, while p45 and p42 isoforms played a “tumor-suppressor” function in ovarian cancer. Our data supported that the competitive binding of hnRNPA1 and SRSF2 to the regions flanking exon 2 and exon 7 to regulate the alternative splicing of AUF1 pre-mRNA. Furthermore, the current study demonstrated that phosphorylation of hnRNPA1 and O-GlcNAc modification of SRSF2 regulated their recruitment to AUF1 pre-mRNA. Investigating the role of the AUF1 isoforms in cisplatin responsiveness may provide a new therapeutic strategy for the treatment of cisplatin resistant ovarian cancer.

Different expression of AUF1 isoforms in cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues and cell lines

Western blot showed that AUF1 was highly expressed in most ovarian cancer tissues, and there was a significant difference in expression between the sensitive and resistant patient tissues. Among the 24 cisplatin sensitive ovarian cancer tissues, the expression of p37 isoforms was generally low, and p45 isoforms was highly expressed in the 15 tissues. While among the 12 cisplatin resistance ovarian cancer tissues, p37 isoforms was highly expressed in 10 tissues, and only 2 tissues had high expression of p42 isoforms (Fig. 1A). AUF1 produces four protein isoforms by alternative splicing, whole exon subtype p45, exon 2 deletion isoform p42, exon 7 deletion isoform p40, and exon 2 and 7 deletion isoform p37 (Fig. 1B). RT-qPCR showed that there was no significant difference in the total RNA expression of AUF1 between sensitive and resistant tissues, but the expression of mRNA containing exon 2 or exon 7 in sensitive tissues was significantly higher than that in drug resistant tissues (Fig. 1C). In addition, agarose gel electrophoresis showed that the expression of RNA variant 1 containing exons 2 and 7 was significantly higher in sensitive tissues than that in drug-resistant tissues, while the expression of RNA variant 4 with exon deletion was significantly lower than that in drug-resistant tissues (Fig. 1D). TCGA alternative splicing database TSVdb confirmed expression of different AUF1 RNA variants in primary and recurrent solid tumors (Fig. 1E). In addition, p45, p42 and p40 isoforms were highly expressed in SKOV3 and A2780 cells, while the expression of p37 isoform was significantly up-regulated in SKOV3/DDP and A2780/DDP cells (Fig. 1F). RT-qPCR results showed that the total RNA expression of AUF1 in sensitive cells was similar to that in drug resistant cells, and the expression of mRNA containing exon 2 or exon 7 in sensitive cells was significantly higher than that in drug resistant cells (Fig. 1G). Full-length variant 1 was highly expressed in sensitive cells, while variant 4 with deletion of exons 2 and 7 were highly expressed in drug-resistant cells (Fig. 1H).

Implication of AUF1 in the cisplatin responsiveness in cisplatin-sensitive ovarian cancer cells

In order to explore the role of AUF1 in cisplatin resistance, AUF1 was knocked down using CRISP/Cas9 system in SKOV3 and A2780 cells (Fig. 2A). Knockdown of AUF1 increased the survival of both SKOV3 (cisplatin > 5ng/ml) and A2780 (cisplatin > 2ng/mL) cells exposed to cisplatin treatment (Fig. 2B). AUF1 knockdown significantly decreased apoptosis of SKOV3 and A2780 cells upon exposure to 20ng/ml and 10ng/ml cisplatin, respectively (Fig. 2C). AUF1 knockdown had no obvious effects on colony formation under normal culture condition, while increased colony formation of SKOV3 (Fig. 2D) and A2780 (Fig. 2E) cells upon continuous cisplatin exposure. AUF1 knockdown had no obvious effects on the size and weight of xenograft tumors in SKOV3 cells (Fig. 2F). Ki-67 immunostaining confirmed no marked alteration of SKOV3 cell proliferation in xenograft tumors (Fig. 2G). On the contrast, treatment of the mice burdening xenograft tumors with cisplatin demonstrated that knockdown of AUF1 significantly reduced the tumor volume after 6 days with cisplatin treatment (Fig. 2H).

Implication of AUF1 in maintenance of cancer stem cell-like features in cisplatin-resistant ovarian cancer cells

In order to explore the role of AUF1 in cisplatin-resistant ovarian cancer cells, AUF1 was knockdown using CRISP/Cas9 system in cisplatin-resistant ovarian cancer cells SKOV3/DDP and A2780/DDP (Fig. 3A). AUF1 knockdown decreased ovarian cancer stem cell marker CD133 expression (Fig. 3A). Knockdown of AUF1 had no obvious effects on the survival of SKOV3/DDP and A2780/DDP cells exposed to cisplatin treatment (Fig. 3B). Apoptosis was also not affected by AUF1 knockdown regardless of cisplatin exposure(5µg/ml) (Fig. 3C). The number of clones formed by AUF1 knockout cell lines was significantly decrease than control cells at the ability of the clone formation, but there was no significant difference in clonal formation ability in AUF1 knockdown cell lines exposure to 0.5µg/ml cisplatin (Fig. 3D-E). AUF1 knockdown led to a significant reduction in the size and weight of xenograft tumors (Fig. 3F). In addition, Ki-67 immunostaining confirmed reduction of SKOV3/DDP cell proliferation by AUF1 knockdown in nude mice (Fig. 3G). Furthermore, knockdown of AUF1 significantly decreased spheroid formation capacity of SKOV3/DDP and A2780/DDP cells (Fig. 3H).

Assignment of distinct functions to AUF1 isoforms in ovarian cancer cells

To explore the role of different AUF1 isoforms in ovarian cancer cells, individual AUF1 isoforms were reintroduced into ovarian cancer cisplatin-sensitive cells with AUF1 knockdown (Fig. 4A). p45 isoform significantly decreased viability of SKOV3 and A2780 cells with AUF1 knockdown (Fig. 4B). p42 isoform also decreased viability of SKOV3 cells with AUF1 knockdown, while p40 or p37 isoform demonstrated no effects (Fig. 4B). Consistent with alteration in cell viability, p45 increased apoptosis in both SKOV3 and A2780 cells with AUF1 knockdown, p42 increased apoptosis of SKOV3 with AUF1 knockdown, no alteration was observed upon restoration of p40 or p37 isoform (Fig. 4C). p37 isoform significantly improved colony formation of cells under normal culture condition, while p42 and p45 suppressed colony formation under cisplatin exposure (Fig. 4D-G). In vivo xenograft demonstrated that p37 isoform increased the size and weight of tumors (Fig. 4H), which was further confirmed by Ki-67 immunostaining (Fig. 4I).

The expression of distinct AUF1 isoforms was also restored in cisplatin-resistant ovarian cancer cells with AUF1 knockdown (Fig. 5A). p45 isoform also decreased viability (Fig. 5B), while increase apoptosis (Fig. 5C) of SKOV3/DDP and A2780/DDP with AUF1 knockdown. Similar like in cisplatin-sensitive cells (Fig. 4D-G), p37 isoform increased colony formation of cisplatin-resistant ovarian cancer cells with AUF1 knockdown under normal culture condition, while p45 isoform decreased colony formation of cells with cisplatin exposure (Fig. 5D-G). Similarly, p37 isoform increased the size and weight (Fig. 5H), as well as proliferation (Fig. 5I) of xenograft tumors derived from SKOV3/DDP.

Regulation of AUF1 splicing by phosphorylation of hnRNPA1 and O-GlcNAc glycosylation of SRSF2 in ovarian cancer cells

p37 isoform of AUF1 was distinguished from other isoforms by skipping both exons 2 and 7. RNA pulldown assays followed by protein profiling were performed to screen for proteins recruited to RNA fragments flanking Exon2 and Exon7 regions, and alteration of SRSF2, hnRNPA1 and RBMX enrichment were screened out. RNA pulldown confirmed the increase in hnRNPA1 enrichment, while decrease in SRSF2 enrichment in both SKOV3/DDP and A2780/DDP cells (Fig. 6A). Cisplatin-resistant and cisplatin-sensitive ovarian cells expressed hnRNPA1 and SRSF2 with similar extents (Fig. 6A). Recent studies have shown that O-GlcNAc glycosylation, which is closely related to phosphorylation, was associated with cisplatin resistance in ovarian cancer phosphorylation^[21–22]. We then performed the phosphorylation and O-GlcNAc modification changes in cisplatin-resistant ovarian cancer cells by quantitative protein spectroscopy, and identified reduction of SRSF2 O-GlcNAc modification at Ser101 site (Fig. 6B), while increment of hnRNPA1 phosphorylation at Ser95 site (Fig. 6C) in cisplatin-resistant ovarian cancer cells. Immunoprecipitation assay with Lectin gel or pan-Ser-phosphorylated antibodies confirmed that phosphorylation of hnRNPA1 was increased, while glycosylation of SRSF2 was decreased in cisplatin-resistant ovarian cancer cells (Fig. 6D). It has been reported that hnRNPA1 undergoes phosphorylation at Ser95 site by DNA-PK^[23–24], we then focused on SRSF2 O-GlcNAc modification. Wild-type (WT) or S101A non-O-GlcNAc mutant SRSF2 prokaryotic expression vectors were constructed, and recombinant proteins were purified (Fig. 6E). In vitro O-GlcNAc glycosylation reaction demonstrated that WT SRSF2 underwent O-GlcNAc modification, while S101A mutant SRSF2 couldn’t be modified (Fig. 6F). To explore whether modification of hnRNPA1 or SRSF might affect their recruitment to Exon 2 or Exon 7 regions of AUF1 pre-mRNA, RNA pulldown experiments were performed using potential hnRNPA1 (Fig. 6G) or SRSF2 (Fig. 6H) binding site mutant RNA fragments. RNA pulldown verified that potential hnRNPA1 binding mutation significantly blocked the interaction of hnRNPA1 with RNA fragments flanking Exon 2 or Exon 7 region of AUF1 (Fig. 6G). In addition, S95D phosphorylation mimic mutation significantly increased, while S95A non-phosphorylation mutation significantly prohibited binding of hnRNPA1 to RNA fragments of AUF1 (Fig. 6G), indicating that phosphorylation at Ser95 site is critical for recruitment of hnRNPA1. RNA pulldown also confirmed that potential SRSF2 binding mutation blocked interaction of SRSF2 with Exon 2 or Exon 7 containing RNA probe (Fig. 6H). S101A non-O-GlcNAc mutant and wild-type SRSF2 demonstrated similar interactive capacity (Fig. 6H), indicating that O-GlcNAc modification is not required for SRSF2 recruitment. To study the potential correlation between hnRNPA1 and SRSF2, HEK293 cells were co-transfected with distinct hnRNPA1 and SRSF2. RNA pulldown found that WT and S95D hnRNPA1 significantly repressed recruitment of SRSF2 (Fig. 6I).

Alteration of SRSF2 O-GlcNAc and hnRNPA1 phosphorylation in cisplatin-resistant ovarian cancer tissues

Immunohistochemical staining showed that AUF1 was mainly located in the nucleus of cisplatin-sensitive ovarian cancer tissues, while it was distributed in both the nucleus and cytoplasm of cisplatin-resistant clinical tissues. Consistent with previous results, O-GlcNAc modification was significantly decreased in cisplatin-resistant ovarian cancer tissues (Fig. 7A-B). Consistent with reduction of O-GlcNAc transferase OGT, as well as total O-GlcNAc modification, O-GlcNAc glycosylation of SRSF2 was decreased in cisplatin-resistant tissues (Fig. 7B). On the contrary, upregulation of hnRNPA1 phosphorylation was observed in cisplatin-resistant tissues (Fig. 7B).

The abnormal expression of AUF1 has been observed in various types of cancers, and it exerts a tumor-promoting effect specifically in colon cancer and breast cancer^{[13–15−16]}, while inhibits tumor in liver cancer and kidney cancer ^[18–19]. Alternative splicing of AUF1 precursor mRNA generate 4 different mRNA splicing variants, resulting in the production of four protein isoforms (p37, p40, p42 and p45) with varying molecular weights. AUF1 isoforms exhibit distinct functional roles in the regulation of gene expression, and their expression may be influenced by various factors^[13]. Our study demonstrated that p37 was the predominant isoform in cisplatin-resistant ovarian cancer cell, whereas p45 was predominantly expressed in cisplatin-sensitive ovarian cancer cell. Furthermore, our current investigation revealed distinct functional roles of individual AUF1 isoforms in ovarian cancer.

The p45 isoform has been implicated in the responsiveness of ovarian cancer to cisplatin, as knockdown of AUF1 decreased the sensitivity of cisplatin-sensitive ovarian cancer cells, while restoration of the p45 isoform increased the responsiveness of both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells with AUF1 knockdown. On the other hand, p37 isoform was implicated in the cancer stem cell-like features of ovarian cancer, as knockdown of AUF1 decreased some cancer stem cell like features, including colony formation, spheroid formation, in vivo tumorigenesis, as well as CD133 expression, in cisplatin-resistant ovarian cancer cells. Importantly, restoration of the p37 isoform enhanced cancer stem cell-like characteristics in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Consequently, the differential expression of distinct AUF1 isoforms within diverse cellular contexts may underlie its dualistic impact as either a “promoter” or a “suppressor” in cancer. Targeted inhibition of the p37 isoform could potentially offer a viable therapeutic approach for ovarian cancer patients exhibiting elevated AUF1 expression.

Alternative splicing serves as a pivotal mechanism in the regulation of eukaryotic gene expression and contributes significantly to the generation of proteome diversity. It was mediated by multiple RNA cis-acting elements and trans-acting factors and played an important role in tumor development and progression^[25–26]. The present study demonstrates that trans-acting factors hnRNPA1 and SRSF2 competitively recruit to AUF1 pre-mRNA, potentially contributing to the alternative splicing of AUF1 in ovarian cancer cells. Dysfunctional SR and hnRNP proteins in various tumors result in aberrant splicing, leading to the formation of different isoforms that may either promote or inhibit apoptosis or proliferation processes^[27]. The current study did not observe any significant changes in the expression of hnRNPA1 and SRSF2 in ovarian cancer. However, the current study demonstrated that phosphorylation of hnRNPA1 and O-GlcNAc modification of SRSF2 played a regulatory role in their recruitment to the AUF1 pre-mRNA.

O-GlcNAc glycosylation is a dynamic post-translational modification at Serine/Threonine sites, which is tightly regulated by the O-GlcNAc glycosyltransferase (OGT) and its hydrolase (OGA) ^[28]. The levels of OGT and O-GlcNAc glycosylated proteins were found to be decreased in cisplatin-resistant ovarian cancer cells, indicating their significant involvement in the development of cisplatin resistance^[22]. The present study validated the decrease in OGT and O-GlcNAc proteins in cisplatin-resistant ovarian cancer. Our findings demonstrate a significant reduction in O-GlcNAc glycosylation of SRSF2 at Ser101 in cisplatin-resistant cells, suggesting that O-GlcNAc modification may regulate cisplatin resistance through alternative splicing of AUF1 to generate p45 or p37 isoforms mediated by SRSF2. It showed that the Ser95 site of hnRNPA1 was a potential phosphorylation site of DNA-PK^[29]. Cisplatin can directly or indirectly interact with purine bases on DNA, leading to the formation of cisplatin-DNA complexes that disrupt the structure and function of DNA. The involvement of DNA-PK is crucial in recombination repair. Revealing the involvement of DNA-PK in DNA repair may elucidate one of the mechanisms underlying cisplatin resistance^[30]. The current study demonstrated that phosphorylation of hnRNPA1 at S95 site was significantly increased in cisplatin-resistant ovarian cancer. In addition, phosphorylation at Ser95 regulated recruitment of hnRNPA1 to AUF1 pre-mRNA to compete with SRSR2. Therefore, the phosphorylation of hnRNPA1 mediated by DNA-PK and O-GlcNAc glycosylation of SRSF2 might potentially regulate the alternative splicing of AUF1 and contribute to cisplatin resistance in ovarian cancer.

AUF1 could bind to a variety of mRNAs and non-coding RNAs and regulate their stability. AUF1 is commonly associated with the instability of target mRNAs, but it could also play a role in stabilizing mRNAs in some cases^[31–32]. The localization of AUF1 isoforms in cells is different ^[33–34]. The nuclear localization sequence of the p37 isoform was blocked by binding with the 14-3-3σ protein, resulting in its retention in the cytoplasm.^[35]. Immunohistochemical staining showed different localization of AUF1 in ovarian cancer cells. AUF1 was mainly distributed in the nucleus in cisplatin-sensitive tissues, while distributed in both cytoplasm and nucleus in cisplatin-resistant tissues. The differential localization of AUF1 may impact its target range, thereby contributing to its distinct functions in ovarian cancer.

Collectively, this study demonstrated the competitive interaction of hnRNPA1 and SRSF2 with AUF1 pre-mRNA to regulate its alternative splicing, which was modulated by phosphorylation and O-GlcNAc modification, respectively. Furthermore, our findings revealed that the p37 isoform exerted a “cancer promoter” role while the p45 isoform acted as a “cancer suppressor” in ovarian cancer. Further clarification of specific target RNAs regulated by AUF1 isoforms may offer potential therapeutic targets for cisplatin-resistant ovarian cancer.

Clinical samples

The tissue specimens used in this study were collected from 36 patients with ovarian cancer who underwent surgical resection in Shengjing Hospital of China Medical University from June 2014 to July 2017. None of the patients received radiotherapy or chemotherapy before operation. Platinum sensitivity was defined according to the recurrence within 6 months after the first cisplatin chemotherapy. The patients with recurrence within 6 months were classified as platinum resistant group (n = 12) and those without recurrence within 6 months as platinum sensitive group (n = 24). The tissues were frozen with liquid nitrogen and stored in a deep freezer at -80°C for follow-up experimental analysis. The study was approved by the Institutional Review Committee of China Medical University.

Cell culture

Human ovarian cancer cell lines SKOV3 and A2780, as well as their cisplatin resistant cohorts SKOV3/DDP and A2780/DDP were maintained in RPMI1640 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma) and 100 IU/ml of penicillin, 100 µg/ml of streptomycin (Sigma). The cells were maintained in a humidified atmosphere at 37°C with 5% CO2.

Western Blot and immunoprecipitation

Total cellular proteins were extracted using lysis buffer containing 20 mM Tris-HCl, 150mM NaCl, 2mM EDTA, 1% Triton-X100 and protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Extracted proteins were quantified using the BCA protein assay kit. 20µg of total proteins was separated using 10% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). For immunoprecipitation, cell lysates were pre-cleared with protein A/G magnetic beads, the protein A/G magnetic beads were treated by various antibodies, then incubated overnight at 4°C. The immunoprecipitants were washed three times with lysis buffer and analyzed by Western blot analysis using the indicated antibodies.

AUF1 knockdown by CRISPR-Cas9 system

Lentivirus infection experiments were performed to stably knockdown AUF1 expression. Lentivirus was purchased from Nanjing Jikeyin Company. 1×10⁵ cells/well were inoculated in 6-well plates and cultured for 24 hours. The virus solution was added into the cell culture medium and cultured for 8h, then the culture medium was replaced with a normal medium containing 1µg/mL puromycin for 48h, and the protein was extracted and verified by Western blot.

Cell viability assay

Cells were seeded at 1.0 × 10⁴ per well into 96-well plates. After adhesion, cells were treated with the indicated concentrations of cisplatin. CCK8 (Dojindo Laboratories) assays was performed by exposing cells to different concentrations of cisplatin for 24 hours. 10 µl diluted CCK-8 solution was added to each well. Cells were incubated at 37°C for 4 hours, and the absorbance at 450 nm was determined with a microplate reader.

Colony formation assay

For the plate colony formation assay, 200 cells/well were seeded into six-well plates and cultured for 2 weeks. The cells were fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet. The colony number was determined under an optical microscopy.

RNA pull-down assay

RNA pull-down assays were performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific). Up to 50 pmol of biotinylated RNAs was mixed with 2 mg of protein lysates and 50 µl of streptavidin beads. After incubation and three washes, the streptavidin beads were boiled and used for the immunoblotting assay.

Immunohistochemical staining

Briefly, immunohistochemical intensity was assessed by multiplying the distribution areas (0–100%) at each staining intensity level by the intensities (0: negative; 1: weak staining; 2: moderate staining; 3: strong staining).

Nude mouse xenograft experiments

BALB/c-nu/nu mice (4–5 weeks old, female) were purchased from Liaoning Changsheng Biotechnology Co. Ltd. Mice were subcutaneously inoculated with the specified number of viable SKOV3 and SKOV3/DDP cells. The status of the mice and tumor formation were observed over time, and the mice were sacrificed after 28 days. The subcutaneous tumors were removed and photographed.

Detection of apoptotic cell death

To detect apoptotic cell death, cells were washed twice with PBS and then stained using the Annexin V/PI Apoptosis Detection Kit according to the manufacturer's instructions. After staining with Annexin V and PI for 30 minutes, samples were analyzed using a fluorescence-activated cell scanner flow cytometer.

Statistical analysis

ANOVA and post hoc Dunnett’s test were used to analyze the statistical significance differences in the most experiments, and the significance difference was defined as p < 0.05. All experiments were repeated 3 times, and data were expressed as mean ± SD (standard deviation) of representative experiments.

There is no conflict of interests in this work.

Funding

This work was partly supported by National Natural Science Foundation of China (NO.82003100) ,Liaoning Province Doctor Startup Fund Program Funded by Liaoning Province（NO.2021-BS-093）.

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Regulation of AUF1 alternative splicing by hnRNPA1 and SRSF2 modulate the sensitivity of ovarian cancer cells to cisplatin

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Abstract

Figures

Introduction

Results

Different expression of AUF1 isoforms in cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues and cell lines

Implication of AUF1 in the cisplatin responsiveness in cisplatin-sensitive ovarian cancer cells

Implication of AUF1 in maintenance of cancer stem cell-like features in cisplatin-resistant ovarian cancer cells

Assignment of distinct functions to AUF1 isoforms in ovarian cancer cells

Alteration of SRSF2 O-GlcNAc and hnRNPA1 phosphorylation in cisplatin-resistant ovarian cancer tissues

Discussion

Materials and Methods

Clinical samples

Cell culture

Western Blot and immunoprecipitation

AUF1 knockdown by CRISPR-Cas9 system

Cell viability assay

Colony formation assay

RNA pull-down assay

Immunohistochemical staining

Nude mouse xenograft experiments

Detection of apoptotic cell death

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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