CircRNF121 facilitates ovarian cancer progression through modulating microRNA-153/IGF2BP2/B3GNT6 axis

doi:10.21203/rs.3.rs-3250482/v1

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CircRNF121 facilitates ovarian cancer progression through modulating microRNA-153/IGF2BP2/B3GNT6 axis

https://doi.org/10.21203/rs.3.rs-3250482/v1

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Background

Increasing studies have evidenced that circular RNAs (circRNAs), a new class of non-coding RNAs, exert a vital role in controlling the progression of multiple human cancers.

Methods

The functional roles of circRNF121 in the initiation and progression of ovarian cancer (OC) warrant further investigation. In our study, RT-qPCR was utilized to determine the expression of circRNF121 in OC cancerous tissues and cells. Also, CCK-8 analysis was performed to detect the viability of CaSki and MS751 cells. The effects of circRNF121 on OC cells were explored using in vitro migration assay and in vivo subcutaneous xenograft tumor model. RT-qPCR and western blotting were used to determine the expression of a panel of genes relative to the EMT process. Luciferase reporter assay were conducted to confirm the specific interaction between circRNF121 and miRNA-153 or between miRNA-153 and IGF2BP2. RNA-Immunoprecipitation (RIP) assay confirmed the binding efficacy of IGF2BP2 and B3GNT6.

Results

In our study, we firstly demonstrated circRNF121 was upregulated in OC cancerous tissues and cells, and the expression of circRNF121 was negatively correlated with patient’s survival. Functionally, circRNF121 can function as an oncogene to promote OC progression both in vitro and in vivo. Mechanistically, circRNF121 worked as a sponge for miRNA-153 to regulate IGF2BP2 expression. Besides, RIP and qPCR analysis suggested that IGF2BP2 can promote B3GNT6 expression to drive OC progression.

Conclusions

CircRNF121 promotes OC progression through modulating the miR-153/ IGF2BP2/B3GNT6 axis, thereby providing a promising therapeutic target for OC treatment.

Mini-abstract:

CircRNF121 promotes OC progression through modulating the miR-153/ IGF2BP2/B3GNT6 axis.

circRNF121

ovarian cancer

microRNA-153

IGF2BP2

B3GNT6

m6A modification

Ovarian cancer (OC), a heterogeneous disease that originates in the tissues of an ovary, is one of the most common gynecological cancers that is generally diagnosed at a late stage as result of lacking the effective diagnostic method^{1, 2}. As previously described, approximately 70% of OC patients are diagnosed at an advanced stage ³. Owing to a high rate of recurrence, patients with OC usually exhibits poor prognosis, including unfavorable 5-year survival rate. As a result, great efforts are extremely needed to illustrate the underlying mechanism of OC progression as well as its specific and sensitive therapeutic targets.

Circular RNAs (circRNAs), a subgroup of noncoding RNAs that were first found in pathogens, exert a vital role in controlling gene expression, and possess impressively diverse functions in a variety of biological processes^4–6. CircRNAs in animals are generally derived by backsplicing that joins a downstream 5' splice site with a 3' splice site of an upstream exon ⁷. Given their covalently closed-loop structures, circRNAs are more stable than linear RNAs in cells that show resistant to exonuclease R^{5, 8}. A growing body of studies have documented circRNAs are aberrantly expressed in a wide range of human cancers such as colorectal cancer (CRC), hepatocellular carcinoma (HCC) and gastric cancer^9–11. Notably, circRNAs, acted as competing endogenous RNAs (ceRNAs), can bind to miRNAs, thereby abolishing the negative regulation of miRNAs on their targets, which eventually impact tumorgenesis ^{6, 12, 13}. Recently, circRNAs have been demonstrated to obviously impact OC progression by sponging multiple miRNAs^14–16. However, the functions of dysregulated circRNAs during OC progression are far from clear and require further exploration.

MicroRNAs (miRNAs), a class of small non-coding single-stranded RNAs that act as crucial regulators in posttranscriptional gene silencing, can exert vital roles in modulating many biological processes such as cell metabolism, apoptosis, cell growth and invasion ^{17, 18}. Mechanically, miRNAs can induce translational repression via interaction with the 3’ untranslated region (3’ UTR) of target mRNAs, thereby decreasing targeted gene expression^{19, 20}. Moreover, studies have discovered high-frequency genomic variations in miRNA loci in ovarian cancer ²¹, thus suggesting that targeting miRNAs can be of greater benefits in the diagnosis and treatment of OC.

In this study, we found that the expression level of circRNF121 was upregulated in OC cancerous tissues and cell lines. Functionally, circRNF121 can promote OC progression both in vitro and in vivo. Mechanistically, we uncovered that circRNF121 can repress microRNA-153 expression to regulate IGF2BP2. A deeper exploration suggested that IGF2BP2 can promote B3GNT6 expression, thus ultimately driving OC progression. In summary, our findings revealed that circRNF121 can promote OC progression through modulating the miR-153/IGF2BP2/B3GNT6 axis, thereby indicating that targeting circRNF121 could be a promising therapeutic approach in OC treatment.

2.1 Specimen collection

OC cancer tissues and their matched adjacent normal ovary tissues were obtained from Changzheng Hospital between 2017 and 2020. Totally, 32 pairs of tissues were analyzed in this study. Patients with OC didn’t experience systemic treatment of chemotherapy or radiotherapy before surgery. All of patients had got the written informed consent. The study followed the ethics committee of Changzheng Hospital guidance (19JSZ07). Clinical stage of OC was evaluated in accordance with the criteria of FIGO.

2.2 Cell culture

Five OC cell lines, including Hela, CaSki, C-33A, ME-180, MS751 and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured with RPMI-1640 medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum in a humidified atmosphere at 37°C supplement with 5% CO₂.

2.3 Quantitative real time PCR assay

Total RNAs from tissues or cultured cells were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. A reverse transcription kit (Takara, Dalian, China) was used to reverse transcribe total RNA into cDNA according to the manufacturer’s protocol. Meanwhile, a stem-loop RT-qPCR method was used to generate miRNAs. qRT-PCR was conducted in an ABI StepOnePlusTM real-time PCR system (Applied Biosystems, Foster City, USA). U6 and GAPDH were applied as internal controls. The gene-specific primers are all purchased from Sangon Biotech (Shanghai, China).

2.4 Plasmid and transfection

The siRNA oligos targeting circRNF121, miRNA-153 and IGF2BP2 and the paired scrambled siRNAs were generated by GenePharma (Shanghai, China). Plasmids for ectopic expression of microRNA-153, IGF2BP2 as well as the matched control plasmids were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen) was utilized to perform cellular transfection according to the manufacturer’s recommendations. After 48 hours of transfection, cells were harvested for the following experiments.

2.5 Cell Counting Kit-8 (CCK-8) assay

A CCK-8 kit (Doindo, Japan) was used to detect the viability of CaSki and MS751 cells. Briefly, cells were seeded into a 96-well plate with a density of 1*10⁴. After 0, 24, 48, 72 and 96 h of culture, cells were incubated with the CCK-8 solution for 2 hours. The final OD value was measured at 450nm using an automatic microplate reader (Synergy4, BioTek).

2.6 Cell Migration Assay

The transfected OC cells were plated into the upper chamber (8-µm) with a density of 5×10⁴ (Corning, USA). The lower chamber was supplemented with DEME medium containing 10% FBS. Subsequently, after 48 hours of migration, penetrating cells were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Beyotime Biotechnology) for 10 min. For each sample, five or six fields were randomly selected to calculate the number of migrating cells under an inverted microscope with 100× magnification.

2.7 Western blotting

In general, CaSki and MS751 cells were lysed using RIPA buffer (Beyotime Biotechnology) containing 1🞩 protease inhibitor cocktail (PIC; Sigma, USA) for 30 mins. A BCA Assay Kit (Beyotime Biotechnology) was utilized to determine the protein concentration following the manufacturer’s recommendations. Next, the protein samples were prepared by mixing with protein loading buffer (Beyotime Biotechnology). Afterward, the protein was electrophoresed using a SDS-PAGE gel and subsequently transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After 1h of incubation with 5% fat-free milk, these membranes were incubated with specific primary antibodies overnight at 4°C. After 12 hours, after 3 times of washing, above membranes were co-incubated with the secondary antibodies for 1 hour at room temperature (RT). The final signals were visualized using an ECL Western blot kit (Thermo Fisher Scientific, USA). GAPDH was applied as controls. The primary and secondary antibodies used were listed as following: GAPDH (2118, Cell Signaling, 1:5000), E-Cadherin (3159, Cell Signaling, 1:1000), N-Cadherin (4061, Cell Signaling, 1:1000), Vimentin (ab188499, abcam, 1:10000), IGF2BP2 (ab128175, abcam, 1:1000), anti-rabbit IgG H&L (HRP) (1:5000, 7074, Cell Signaling).

2.8 RNA-Immunoprecipitation (RIP)

Immunoprecipitation assays of m6A and IG2BP2 were conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s recommendations. In general, CaSki and MS751 cells were lysed and collected. Afterward, magnetic beads that were previously coated with 5µg of anti-m6A and anti-IGF2BP2 primary antibodies were incubated with the supernatants overnight at 4°C. Next, the RNA-protein complexes were washed for 6 times. Finally, the immunoprecipitated RNAs were extracted and the relative expression of B3GNT6 was determined using real-time PCR.

2.9 Target predication

The miRDB database (http://mirdb.org/) was used to predict the targets of circRNF121 and microRNA-153.

2.10 Dual luciferase reporter assay

The fragments of wild-type or mutant circRNF121 (or IGF2BP2) containing the binding site were generated through chemical synthesis. Afterward, the obtained fragments were inserted into pGL3-Control Vector (Promega, Madison). Lipofectamine 2000 was used to co-transfect the obtained luciferase reporter vectors with miR-153 negative control or miR-153 mimics into CaSki and MS751 cells. After 48 hours of transfection, the activities of firefly luciferase and Renilla luciferase were determined using a Dual-Luciferase Reporter Assay system (Promega). And the firefly luciferase activity was normalized to the Renilla luciferase activity.

2.11 Animal experiment

All animal experiments were conducted following the guidelines of our Animal Care and Use Committee. a total of 12 BALB/c-nu/nu mice (male, 4–5 weeks old) were randomly assigned into 2 subgroups. Then, the mice were subcutaneously injected with stably knockdown CaSki cells or the matched controls. After 28 days, the tumors were excised.

2.12 Statistical analysis

All experimental data was analyzed using GraphPad Prism 7.0 software and presented as

mean ± standard deviation (SD). Standard t-test was used to compare the differences between two different groups. Kaplan-Meier was introduced to evaluate the prognosis of patients. *P < 0.05, **P < 0.01, *** P < 0.001.

3.1 circRNF121 is upregulated in OC cancer tissues and cells

With the aim to illustrate the functional role of circRNF121 during OC progression, the expression level of circRNF121 in OC tissues and paired normal tissues was firstly evaluated using real-time PCR analysis. As depicted in Fig. 1A, the expression of circRNF121 was significantly upregulated in OC tissues relative to the matched normal tissues. Additionally, patients with high circRNF121 showed an obviously lower survival, thus suggesting that circRNF121 could be a prognostic marker for OC (Fig. 1B). Consistently, the expression of circRNF121 was higher in all OC cells relative to normal ovarian epithelial cells (Fig. 1C). Moreover, CaSki and MS751 cells were selected for the following experiments.

3.2 circRNF121 facilitates OC progression

To investigate the potential function of circRNF121 in OC initiation and progression, circRNF121 was firstly silenced via transfection of small interfering RNA. The knockdown efficiency was confirmed using real-time PCR analysis (Fig. 2A). As presented in Fig. 2B-2C, circRNF121 silencing obviously suppressed the viability of two OC cells, thereby highlighting the significance of circRNF121 in supporting OC cell proliferation. It is well accepted that metastasis is a major contributing cause for cancer-related deaths. Therefore, we further investigated the impacts of circRNF121 on the metastatic potential of CaSki and MS751 cells. Notably, silencing of circRNF121 led to a significant decrease of in vitro migration in both CaSki and MS751 cells, thus suggesting circRNF121 could be a vital contributor for OC cell metastasis. Subsequently, a panel of genes were examined to illustrate the potential targets involved in the anti-OC properties of circRNF121. The epithelial-mesenchymal transition (EMT), well-recognized as a physiological and multistep process in which epithelial cells lose their features and acquire mesenchymal characteristics, facilitate the initiation of cancer metastasis. More interestingly, we found that disruption of circRNF121 induced E-cadherin expression but repressed N-cadherin and Vimentin expression, thus hinting a potential role of circRNF121 in regulating the EMT process (Fig. 2E-2H). Next, we explored whether circRNF121 impacted OC development in an in vivo context. Interestingly, knockdown of circRNF121 resulted in a considerable decrease of tumor volumes (Fig. 2I). In summary, our study revealed that circRNF121 facilitated OC progression both in vitro and in vivo.

3.3 circRNF121 promotes OC progression via inhibition of microRNA-153

With the aim of exploring whether circRNF121 acts as a sponge for miRNAs in OC progression, miRDB database (http://www.mirdb.org/) was utilized to predict the potential binding microRNAs of circRNF121. Interestingly, microRNA-153 was found. Accordingly, we reported silencing of circRNF121 increased the expression of microRNA-153 in both CaSki and MS751 cells (Fig. 3A). Besides, the dual-reporter luciferase assay was performed to verify the targeted interaction between microRNA-153 and circRNF121. As depicted in Fig. 3B-3D, our study revealed that overexpression of miRNA-153 can obviously decrease the luciferase activity of a vector harboring the wild-type circRNF121 but not in the reporter carrying mutant circRNF121 (Fig. 3B-3D). Moreover, miRNA-153 expression was remarkably decreased after transfection of siRNA against miRNA-153, and this inhibitory effect was reversed after circRNF121 silencing (Fig. 3E). Consistently, disruption of miRNA-153 increased the viability of two OC cells, and this promotion was reversed by co-transfection of siRNA against circRNF121 (Fig. 3F-3G). Also, our study found that knockdown of miRNA-153 repressed E-cadherin expression but induced N-cadherin and Vimentin expression in both CaSki and MS751 cells, and these impacts could be reversed after deletion of circRNF121(Fig. 3H-3K). Taken together, we suggested that circRNF121 sponged miR-153 to regulate the EMT process of OC cells, thus ultimately facilitating OC progression.

3.4 CircRNF121/miR-153/IGF2BP2 axis mediate the EMT process in OC progression

Using bioinformatic studies, we found that IGF2BP2 could be a potential target for miR-153. As a result, the dual-reporter luciferase assay was conducted to confirm whether IGF2BP2 was a direct target of miR-153. As presented in Fig. 4A-4C, ectopic overexpression of miRNA-153 can obviously decrease the luciferase activity of a vector carrying wild-type IGF2BP2. By contrast, the luciferase reporter harboring mutant IGF2BP2 was unchanged after overexpression of miR-153, thus indicating miR-153 can directly suppress IGF2BP2 expression. Consistently, enforced expression of miR-153 repressed the expression level of IGF2BP2 in both 2 OC cells (Fig. 4D). As depicted in Fig. 4E, RT-qPCR analysis demonstrated a successful overexpression of IGF2BP2 in both CaSki and MS751 cells. Consistently with previous results, silencing of circRNF121 led to a considerable reduction of IGF2BP2, and this inhibitory effect was reversed after overexpression of IGF2BP2 (Fig. 4F). Finally, enforced expression of IGF2BP2 can effectively inhibit the expression of E-cadherin and promote the expression of N-cadherin and Vimentin in circRNF121-silenced CaSki and MS751 cells, thereby suggesting that circRNF121/miR-153/IGF2BP2 axis promoted OC progression via regulation of the EMT process (Fig. 4G-4H).

3.5 IGF2BP2 induce OC progression via promotion of B3GNT6 expression

m6A modification, as the most prevalent posttranscriptional modification in eukaryotic RNAs, has been reported to exert a considerably important role in regulating cancer progression due to its ability to control RNA fate. As previous described, IGF2BP2, as a m6A reader that can recognize m6A-modified transcripts, is highly connected with the initiation, metastasis and treatment response of many malignancies. Moreover, a very recently study have revealed that IGF2BP2 promotes pancreatic carcinoma progression via promotion the mRNA stability of B3GNT6 in a m6A-dependent manner. As a result, RIP assays were performed to determine whether IGF2BP2 can modulate B3GNT6 to promote OC progression. As shown in Fig. 5A, IGF2BP2 directly bound to the B3GNT6 transcripts. Also, the knockdown efficiency of IGF2BP2 was confirmed using real-time PCR and immunoblotting (Fig. 5B-5C). Consistently, disruption of IGF2BP2 also resulted in a decreased expression of B3GNT6, thereby suggesting IGF2BP2 can induce OC progression via promotion of B3GNT6 expression. Finally, IGF2BP2 silencing can result in a significantly reduced binding of IGF2BP2 to B3GNT6 mRNA, further supporting IGF2BP2 targeted B3GNT6 to facilitate OC progression.

OC is one of the most common gynecological malignancies with a higher mortality threatening female health worldwide². Although the applications of advanced technologies, including cytoreductive surgery and platinum-based chemotherapy, are widely developed for OC treatment. However, the therapeutic treatments for the advanced OC patients are still unfavorable due to tumor recurrence and chemotherapy resistance. Therefore, great efforts are extremely needed to develop novel biological hallmarks and therapeutic targets for OC treatment. Emerging studies have shown that noncoding RNAs such as circRNAs and miRNAs play a crucial role in regulating OC progression ^{14, 15, 22–24}. In our study, we firstly confirmed that circRNF121 was upregulated in OC tissues and cells. Moreover, OC patients with a higher circRNF121 expression exhibited an unfavorable survival, thereby suggesting that circRNF121 could be a potential prognostic marker for OC. Also, we found circRNF121 can promote OC cell proliferation and migration, thus indicating circRNF121 could be a potential therapeutic target for OC treatment. Mechanistically, recent literature pointed out circRNAs can work as competing ceRNAs through competitively binding to miRNAs, thus abolishing the inhibitory effect on targeted mRNAs²⁵.

Interestingly, our miRNA-targeting prediction studies reported that circRNF121 can interact with miRNA-153, thereby suggesting that circRNF121 might function as a miRNA sponge to regulate miRNA-153 expression. Indeed, we found that knockdown of circRNF121 repressed miRNA-153 expression. Moreover, the luciferase reporter assay suggested that miRNA-153 was a direct target of circRNF121. Also, a growing body of studies have documented that miR-153 was aberrantly expressed in a wide range of human cancers, including non-small-cell lung cancer, breast cancer, CRC, etc^{26, 27}. Moreover, Zuo et al., have reported that miR-153 can serve as a tumor repressor in breast cancer progression via inhibition of cell growth and migration as well as the EMT process²⁸. Also, miRNA-153 was also found to inhibit OC cell proliferation and to suppresses the EMT process by downregulating SET7 and ZEB2²⁹. Functionally, our study suggested that downregulation of circRNF121 suppressed OA progression, and this inhibitory effect was effectively abolished by decreasing miRNA-153. In summary, we provided an evidence that circRNF121 promoted OC progression via sponging miRNA-153.

Besides, bioinformatic analysis as well as the luciferase reporter assay suggested that miRNA-153 was a negative regulator of IGF2BP2. As previous described, IGF2BP2, a well-known m6A reader, can recognize m6A-modified transcripts to modulate the stability of targeted RNAs, thereby exerting a crucial role in controlling the development of a variety of diseases, including diabetes, cancer progression and nonalcoholic fatty liver disease³⁰. Consistent with our prediction, the luciferase reporter assay suggested that IGF2BP2 was a direct target of miRNA-153. Functionally, knockdown of IGF2BP2 can abolish circRNF121-induced EMT process, thus highlighting the importance of circRNF121/miRNA-153/IGF2BP2 axis in OC progression.

B3GNT6, the protein that is essential for the synthesis of the core 3 structure of O-glycans, has been documented to be commonly dysregulated in many types of malignant tumors such as cervical cancer, prostate cancer and CRC³¹. Cao et al., showed that the oncogenic role of IGF2BP2 was mediated by modulating B3GNT6 mRNA stability³². Accordingly, B3GNT6 can affect EMT-MTE plasticity of CRC cells. Identically, our study confirmed IGF2BP2 promoted the expression of B3GNT6 via directly interaction with its transcripts, thereby contributing to the deterioration of OC.

Taken together, we confirmed circRNF121 was upregulated in OC cancerous tissues and cells. Moreover, circRNF121 can function as an oncogene to promote OC progression both in vitro and in vivo. Mechanistically, circRNF121 worked as a sponge for miRNA-153 to regulate IGF2BP2 expression. Additionally, silencing of circRNF121 led to EMT suppression, and its inhibitory effect was reversed by increasing IGF2BP2 expression. Also, RIP and qPCR analysis suggested that IGF2BP2 can promote B3GNT6 expression to drive OC progression. In summary, out results not only documented that circRNF121 promoted OC progression through modulating the miR-153/IGF2BP2/B3GNT6 axis, but also provided a promising therapeutic target for OC treatment.

Author Contribution

All authors made substantial contributions to the conception and design of this work. ZW: design, data analysis; HS and DH: carry out the experiments; XL: corrected manuscript; Manuscript writing: All authors; Final approval of manuscript: All authors.

Ethical Statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study followed the ethics committee of Changzheng Hospital guidance (19JSZ07).

Funding

This work was supported by PLA logistics research program (19jsz07)

Disclosure statement

The authors report there are no competing interests to declare.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [XL], upon reasonable request.

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CircRNF121 facilitates ovarian cancer progression through modulating microRNA-153/IGF2BP2/B3GNT6 axis

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Mini-abstract:

Figures

1. Introduction

2. Materials and Methods

2.1 Specimen collection

2.2 Cell culture

2.3 Quantitative real time PCR assay

2.4 Plasmid and transfection

2.5 Cell Counting Kit-8 (CCK-8) assay

2.6 Cell Migration Assay

2.7 Western blotting

2.8 RNA-Immunoprecipitation (RIP)

2.9 Target predication

2.10 Dual luciferase reporter assay

2.11 Animal experiment

2.12 Statistical analysis

3. Results

3.1 circRNF121 is upregulated in OC cancer tissues and cells

3.2 circRNF121 facilitates OC progression

3.3 circRNF121 promotes OC progression via inhibition of microRNA-153

3.4 CircRNF121/miR-153/IGF2BP2 axis mediate the EMT process in OC progression

3.5 IGF2BP2 induce OC progression via promotion of B3GNT6 expression

4. Discussion

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1