A Black Phosphorus Nanosheet-based RNA Delivery System for Prostate Cancer Therapy by Increasing the Expression Level of Tumor Suppressor Gene PTEN via CeRNA Mechanism

doi:10.21203/rs.3.rs-3844285/v1

Download PDF

Research Article

A Black Phosphorus Nanosheet-based RNA Delivery System for Prostate Cancer Therapy by Increasing the Expression Level of Tumor Suppressor Gene PTEN via CeRNA Mechanism

https://doi.org/10.21203/rs.3.rs-3844285/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Prostate cancer (PCa) has a high incidence in men worldwide, and almost all PCa patients progress to the androgen-independent stage which lacks effective treatment measures. PTENP1, a long non-coding RNA, has been shown to suppress tumor growth through the rescuing of PTEN expression via a competitive endogenous RNA (ceRNA) mechanism. However, PTENP1 was limited to be applied in the treatment of PCa for the reason of rapid enzymatic degradation, poor intracellular uptake, and excessively long base sequence to be synthesized. Considering the unique advantages of artificial nanomaterials in drug loading and transport, black phosphorus (BP) nanosheet was employed as a gene-drug carrier in this study.

Results

The sequence of PTENP1 was adopted as a template which was randomly divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs, and loaded onto polyethyleneimine (PEI)-modified BP nanosheets to construct BP-PEI@RNA delivery platforms. The RNAs could be effectively delivered into PC3 cells by BP-PEI nanosheets and elevating PTEN expression by competitive binding microRNAs (miRNAs) which target PTEN mRNA, ultimately exerting anti-tumor effects.

Conclusions

Therefore, this study demonstrated that BP-PEI@RNAs is a promising gene therapeutic platform for PCa treatment.

black phosphorus nanosheets

RNA delivery

ceRNA mechanism

PTEN

prostate cancer

Prostate cancer (PCa) is the second most common malignancy and is the fifth cause of cancer death in men worldwide [1]. In recent years, endocrine therapy, and radiotherapy along with surgical operation have contributed to a reduction in mortality, however, one-third of PCa patients will recur, advance to castration-resistant PCa stage, and develop distant organ metastases, which lack effective therapeutics and result in shorter overall survival [2]. Thus, there is a thirst for a novel and more effective strategy for PCa treatment currently.

Gene therapy strategies have demonstrated superior application potential to treat cancer diseases by altering the expression levels of target genes [3]. However, it suffers from limitations due to its poor intracellular uptake and its rapid enzymatic degradation under an abiotic internal environment [4]. In recent years, artificial nanomaterials have emerged as a promising vehicle for gene therapeutic agent delivery to overcome the inherent limitations of gene therapy [5]. And, it is demonstrated that nanomaterials as gene therapeutic agent carriers could deliver the gene drug cargo into cells by “Trojan Horse” effect, and possess the ability to reduce the degradation rate of gene therapeutic agents [6].

PTEN is a typical tumor suppressor gene with PCa. Inactivation of PTEN due to genomic deletion or mutation is widely identified within primary prostate tumor samples and particularly within castration-resistant tumors [7]. Inhibition of PTEN expression results in activation of the PI3K-AKT pathway which plays a powerful role in promoting tumor progression, and cancer cells are ultrasensitive to even a subtle decrease in PTEN levels and activity, making PTEN an important therapeutic target for PCa therapy [8]. Recently, several studies have confirmed that long non-coding RNA PTENP1, the pseudogene of PTEN, which contains a highly homologous region upstream of the 3′UTR of PTEN could act as an “endogenous sponge” to share miRNA binding sites to block the degradation effect of miRNA on the PTEN mRNA by post-transcriptional control [9–11]. Thus, elevating the content of PTENP1 within the cells could improve the amount of PTEN proteins through ceRNA mechanism.

To this end, we employed a two-dimensional biodegradable nanomaterial BP nanosheets to design a gene-drug nanocarrier, which showed excellent biocompatibility [12]. The gene drugs (RNA segments) were designed to increase the expression level of PTEN by ceRNA mechanism and were synthesized according to the sequence of lncRNA PTENP1 as a template. To improve the loading efficiency of the negatively charged RNA segments, the surface of BP nanosheets was modified with positively charged PEI, so that the RNA segments could be loaded onto the BP-PEI nanosheets by electrostatic adsorption. We uncovered that BP-PEI could effectively load and transport RNA segments to the interior of the cell, meanwhile, protect RNA segments from enzymatic degradation. Finally, BP-PEI@RNAs inhibited prostate tumor progression by improving the expression level of tumor suppressor gene PTEN. Taken together, our results revealed the potential of a BP-based RNA delivery system that could be a promising method for gene therapy in the treatment of PCa.

Synthesis RNAs

Total mRNA of RWPE-1 cells (human normal prostate epithelial cell) was extracted by Trizol method and reverse transcribed into cDNA, and all the steps and PCR setup were performed according to the guide of Fast-Quant RT Kit (TianGen, China) to obtain cDNA. Then, the cDNA was used as a template for the PCR to synthesize target RNAs. The sequence of PTENP1 gene (GeneID:11191) was sequentially divided into four segments as templates to synthesize four RNAs (RNA1, RNA2, RNA3, RNA4) by PCR method, the primers were summarized in the supplementary materials (Table S1). Then, the RNAs were gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega Corporation, USA), and stored at -80 ℃.

Construction, Transformation, Amplification and Extraction of the Plasmid

Four RNAs were inserted into pGEM-T Easy Vector System I (Promega, USA) to synthesize plasmids, respectively. Then, 2 µg plasmid was mixed with 100 µL of competent Escherichia coli and an ice bath was performed for 30 minutes. It was placed in a 42 ℃ water bath with thermal shock for 90 s, and immediately placed in an ice bath for 2 min. And 900 µL Luria-Bertani (LB) liquid medium was added, which was prepared by adding 10 g tryptone (OXOID, UK), 10 g NaCl (Solarbio, China), 5 g yeast extract (OXOID, UK), deionized water to 1000 mL, and then autoclaved and added 10 mg ampicillin. And then the mixture was stirred at 225 rpm at 37 ℃ for 1 h, after which the bacterial solution was planted on an agar plate containing 100 µg/mL ampicillin, which was then inverted and kept at 37 ℃ overnight. After colonization, bacterial colonies of approximately 1 mm were selected with a sterile ring in 25 mL LB liquid medium containing 100 µg/mL ampicillin and shaken at 180 rpm at 37 ℃ for 16 h. Part of the bacterial solution obtained was used for sequencing to verify the sequence. The rest was centrifuged at 4 ℃ at 6000 rpm for 10 min. After discarding the supernatant, the QIAGEN Plasmid Midi Kit (QIAGEN, Germany) was used to extract the plasmids in the bacterial solution.

Linearization, Purification and Recovery of the Plasmid

A 50 µL plasmid linearization system was used for plasmid linearization. Specifically, 5 µL NcoI-HF (New England Biolabs, USA), 5 µL CutSmart Buffer (New England Biolabs, USA) and 10 µg plasmid were added into the enzyme-free EP tube, and enzyme-free water was added to the system to 50 µL. After full mixing, the linearized plasmid was obtained after incubation at 37 ℃ for 6 h. The linearized plasmid was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega Corporation, USA), and stored at -20 ℃.

Synthesis of RNA by in vitro Transcription

Linearized DNA was synthesized into RNA through in vitro transcription. Specifically, 10 µL of 5× transcription buffer (Thermo Fisher Scientific), 10 mM of ATP/GTP/CTP/UTP (Thermo Fisher Scientific) 1 µL each, 1 µg of linearized DNA, 1.25 µL RNAase inhibitor (Thermo Fisher Scientific), 1.5 µL T7 polymerase (Thermo Fisher Scientific), DEPC water to 50 µL were added into the enzyme-free EP tube; then, the system was incubated at 37 ℃ for 2 h, and RNA was transcribed and then stored at -20 ℃ for later use.

Construction of BP-PEI@RNAs

BP nanosheets were centrifuged at 4 ℃ (16000 rpm, 60 min) and washed three times with enzyme-free water to obtain a well-dispersed BP aqueous dispersion solution. Polyetherimide (PEI) (2.5k, 10mg/mL, BP to PEI volume ratio of 1:1.5) was then added to the solution and sonicated in ice water bath for 3–4 h to obtain BP-PEI. BP-PEI was resuspended and incubated with RNA (BP to RNA mass ratio of 1:0.4) at 4°C for 10 min with continuous rotational mixing to obtain BP-PEI@RNAs platform.

Characterization of BP@RNA

The diameter and surface characteristics of BP nanosheets, BP-PEI and BP-PEI@RNAs were characterized by transmission electron microscope (TEM, JEOL JEM-2100 F, Japan) and atomic force microscope (AFM, Dimension Icon, Germany). Raman spectroscopy analysis of them was performed using a Raman spectrometer (Horiba scientific LabRAM HR evolution). The hydrated particle size and zeta potential were analyzed by nanoparticle size analyzer (Zetasizer Nano ZS90, Malvern, UK).

Fluorescent labeling of RNA

In the process of RNA preparation by in vitro transcription, 6-FAM- or Cy3-labeled RNA was obtained by in vitro transcription with 6-FAM-labeled or Cy3-labeled ribonucleotide as fluorescent substrate, followed by repeating steps of RNA synthesis.

Uptake and subcellular distribution of BP@RNA

When PC3 cells reached 50%-60% confluence, they were exposed to 6-FAM- and Cy3-labeled BP-PEI@RNA (1µg/mL BP; 0.4 µg/mL RNA), respectively. The cellular uptake of BP-PEI@RNA-6FAM in PC3 cells was observed by CLSM at different time points (1, 3, and 6 hours post exposure). The subcellular localization of PEI@RNA-Cy3 in PC3 cells was detected by co-staining with lysosomes and observed by CLSM at different time points (6, and 24 h post exposure).

Live/ Dead staining

When the confluence reached 50%-60%, PC3 cells were exposed to BP-PEI@RNA (1µg/mL BP; 0.4 µg/mL RNA), and the cells were then stained with the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Proteintech, China) at 24 h post exposure. The fluorescence signal of the cells was observed by CLSM.

Cell viability test

PC3 cells were cultured in 96 well plates, when the confluence reached 50%-60%, these cells were exposed to BP-PEI@RNA (1µg/mL BP; 0.4 µg/mL RNA) for 24 hours, and then the cell viability was tested by Cell Counting Kit-8 assay (MedChemExpress, USA).

Autophagy, apoptosis and cell cycle test

PC3 cells were cultured in RPMI 1640 medium (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin). When 50%-60% confluence was reached, these cells were exposed to BP-PEI@RNA (1µg/mL BP; 0.4 µg/mL RNA) for 24 hours. PC3 cells were harvested and stained with Lyso Tracker Green DND-26 (Cell Signaling Technology, USA), Annexin V-FITC apoptosis assay kit (Beyotime Biotechnology, China) and Cell Cycle Staining Kit (Abbkine, China).

RT-qPCR

PC3 cells incubated with BP-PEI@RNA for 24 hours were harvested. The miRNA of PC3 cells was extracted and inversed into cDNA according to the guide of FastQuant RT Kit (With gDNase) (TianGen, China). RT-qPCR was performed using Universal SYBR Green qPCR Supermix (US EVERBRIGHT, China). U6 was used as an internal control. The expression of miRNAs was quantified by 2^−ΔΔCT method. The primers were summarized in Table S2.

Western-blot

PC3 cells were harvested and washed 3 times with RPMI 1640 medium. Then, these cells were lysed by RIPA lysis buffer (Pierce IP Lysis Buffer, USA) and the protease inhibitor cocktail was added to inhibit the degradation of total proteins. Protein concentration was detected by BCA protein assay kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Equal proteins were used for sodium dodecyl sulfate-polyacrylamide gel and then incubated with different primary antibodies (anti-β-actin, anti-GAPDH, anti-PTEN, anti-P-PI3K, anti-PI3K, anti-P-AKT, anti-AKT, anti-LC3-Ⅰ, anti-LC3-Ⅱ, anti-p62, anti-cleaved caspase 3, anti-cleaved caspase 8, anti-cleaved caspase 9, anti-Bax and anti-Bcl-2) (Proteintech, China) at 4 ℃ overnight and then incubated with secondary antibodies at room temperature for 1 hour. Finally, the expression of different proteins was visualized by BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).

Animal experiment

With the approval of the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd., male Balb/C nude mice at the age of 4 weeks were purchased from HFK Co. (Beijing, China) and then received subcutaneous injection with 1×10⁷ PC3 cells on day 1. On day 14, all mice were randomly divided into four groups (Cont, BP-PEI, Free-RNA2 and BP-PEI@RNA2 group) and received intra-tumor injection with PBS, BP-PEI, Free-RNA2 and BP-PEI@RNA2 every 5 days for 4 times. The tumor volume was measured every 3 days. Finally, the mice were sacrificed and the tumors were harvested for weight measurement and histological examination.

Tunel staining

Tumor tissues were sectioned and stained with CoraLit-594 TUNEL Assay Apoptosis Detection Kit (Proteintech, China) for cellular apoptosis detection. All the steps were performed under the guidance of the kit and the cells undergoing apoptosis were observed by CLSM.

Immunohistochemistry staining

Paraplast-fixed tumor tissues were sectioned, deparaffinized, dehydrated, and antigen retrieved in citrate buffer (pH = 6.0). The sections were then incubated with different primary antibodies (anti-PTEN, anti-Ki67, anti-p62, anti-caspase 8, and anti-Bcl-2) (Proteintech, China) at 4 ℃ overnight. The sections were then incubated with biotin-conjugated secondary antibodies for 30 minutes at room temperature, and protein expression was visualized by 3-3-diaminobenzidine (DAB) staining and observed under an optic microscope.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 10 software and presented as mean ± SD. Comparisons were performed using an independent t-test or one-way ANOVA test and differences between groups were considered statistically significant at P < 0.05.

BP-PEI@RNA Construction and Physicochemical Characterization

The BP nanosheets adopted in this study were prepared as previously study reported [13]. To enhance the loading efficiency of RNAs, PEI with a positive charge was pre-modified on the surface of BP nanosheets before RNAs loading. Considering that the sequence of PTENP1 is too long to be high fidelity synthesized in vitro, thus, the whole length of PTENP1 sequence was sequentially divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs (RNA1, RNA2, RNA3, RNA4; Fig S1). Four RNA fragments were loaded on the BP-PEI, respectively, and BP-PEI@RNA2 was randomly chosen as a representative to illustrate the physicochemical properties and subcellular localization of BP-PEI@RNA. The physicochemical characterization data of BP, BP-PEI and BP-PEI@RNA were shown in Fig. 1a-f. BP nanosheets were ultrathin nanosheet layers under observation by transmission electron microscopy (TEM) and it seemed that PEI modification and RNA loading did not change the morphology of BP nanosheets (Fig. 1a). The atomic force microscopy (AFM) showed the average lateral size of BP, BP-PEI and BP-PEI@RNA mainly distributed in the range of 100–400 nm, with a thickness of about 20–30 nm (Fig. 1b-c). The typical Raman spectral data manifested the same representative band of BP, BP-PEI and BP-PEI@RNA (~ 360.8 cm^− 1, ~ 438.7 cm^− 1 and ~ 465.8 cm^− 1) as shown in Fig. 1d. BP nanosheets and RNA were negatively charged with an average zeta potential of about − 33.33 ± 1.72 mV and − 23.20 ± 1.92 mV, and presented a positive surface charge of 37.83 ± 1.07 mV and 23.43 ± 0.55 mV post PEI modification and RNA loading, respectively (Fig. 1e). The hydration particle size of BP nanosheets was around 200 nm, and it increased to around 300 nm and 500 nm post modified with PEI and further loaded with RNA, respectively (Fig. 1f).

BP Nanosheets Could Efficiently Transport RNA Segments into PC3 Cells and Further into the Lysosomes

To verify whether BP nanosheets could transport RNA segments to the cell interior, confocal laser scanning microscopy (CLSM) analysis was performed after PC3 cells were treated with BP-PEI@RNA for 1, 3 and 6 hours. The fluorescent molecular probe 6-carboxyfluorescein (6-FAM) was adopted to label nucleotides (in green) when RNA was synthesized. As shown in Fig. 2a, CLSM images revealed a mass of RNA entering into the cytoplasm (indicated by white arrows) at 3 h post BP-PEI@RNA treatment, and much more RNA accumulated in the cytoplasm at 6 h post BP-PEI@RNA treatment. Thus, we confirmed that BP-PEI nanosheets could effectively transport RNA into the interior of the cell. To further visualize the location of BP-PEI@RNA after it entered into the cell, co-staining of lysosomes and RNA was performed. RNA was labeled with sulfo-cyanine3 (Cy3) in red and lysosomes were stained in green using Lyso-Tracker. As shown in Fig. 2b, a mass of red fluorescence was observed which indicated as BP-PEI@RNA at 6 h and 24 h post BP-PEI@RNA treatment. The red fluorescence and green fluorescence merged at 6 h post BP-PEI@RNA treatment, which indicated that BP-PEI@RNA was located in the lysosome. However, the red fluorescence was scattered throughout the cytoplasm and no longer located in the lysosome at 24 h post BP-PEI@RNA treatment. Hence, we speculated that BP-PEI@RNA first entered the lysosome, and then was released into the cytoplasm [14, 15]. Additionally, there is no red fluorescence signal was observed with RNA-CY3 alone treatment cells, demonstrating that RNA segment could not get into the interior of the cells in this study. Afterwards, we endeavored to address the resultant influence of BP-PEI@RNA on the proliferation, autophagy and apoptosis of PC3 cells in vitro and in vivo.

BP-PEI@RNAs Treatment Inhibited the Proliferation and Viability of PC3 Cells by Elevating the Expression Level of PTEN

Firstly, the cytotoxicity of BP-PEI was determined by cell count analysis at the concentrations of 0.2, 0.5, 1.0, 2.0 and 5.0 µg/mL (Fig. 3a), and it showed a positive correlation with the treatment dose. Finally, we here selected 1.0 µg/mL as the treatment dose of BP-PEI in the following experiments. Next, four different pieces of RNAs (RNA1, RNA2, RNA3 and RNA4) were loaded onto BP-PEI at the mass ratio of 2:1, respectively. Cell counting analysis, Cell Counting Kit-8 analysis, and cell dead/living staining were adopted to evaluate the inhibition effects of BP-PEI@RNAs on the cellular viability and death of PC3 cells. The data showed that four pieces of BP-PEI@RNAs were all able to inhibit the proliferation of PC3 cells and induce cell death, and there was no significant difference among them (Fig. 3b-e). Meanwhile, the cellular viability of PC3 cells exposed to free RNA was also evaluated and the results suggested that free RNA treatment had no effect on the cellular viability of PC3 cells (Figure S2), probably the reason that free RNAs could not enter into cells by itself and easy to be degraded. Furthermore, western blot analysis was adopted to determine the expression level of the target protein PTEN and its downstream effector molecules (PI3K and AKT) which play a crucial role in cellular proliferation. We found that four BP-PEI@RNAs treatments were all able to elevate the expression level of PTEN, and decreased the expression level of P-PI3K and P-AKT which were the activated state of PI3K and AKT (Fig. 3f). These data suggested that BP-PEI@RNAs established in this study have an obvious inhibitory effect on the proliferation of PC3 cells by increasing the expression level of tumor suppressor gene PTEN, and four different BP-PEI@RNAs showed almost equal efficiency.

BP-PEI@RNAs Treatment Induced Cell Cycle Arrest of PC3 Cells

Since PTEN is involved in the regulation of cell cycle progression which is the origin of rapid proliferation of cancer cells [16], increasing the expression of PTEN to induce cell cycle arrest is considered as an effective anti-tumor method. Here, cell cycle analysis was performed on PC3 cells treated with BP-PEI@RNAs. As shown in Fig. 4a-b, exposure of PC3 cells to four BP-PEI@RNAs respectively resulted in an increasing number of cells distributed in the G0/G1 phase and fewer cells distributed in the S and G2/M phases. Thus, cell cycle analysis demonstrated that BP-PEI@RNAs treatment effectively induced cell cycle arrest on PC3 cells. In consideration of cell cycle is regulated and controlled by a family of proteins known as cyclins and cyclin-dependent kinases (CDKs), the expression levels of cyclins and CDKs were evaluated by western blot analysis and the result was presented in Fig. 4c. Cyclin A2 is synthesized at the onset of the S phase and the cyclin A2-CDK2 complex in the nucleus is implicated in the initiation and progression of DNA synthesis [17]. Consistent with this, the expression of cyclin A2 and CDK2 was dramatically decreased in PC3 cells post treated with BP-PEI@RNAs for 24h. Corresponding with the finding that activated cyclin B1-CDK1 promotes several early events of mitosis, such as chromosome condensation, nuclear envelope breakdown, and spindle pole assembly [18, 19], the activation of cyclin B1 and CDK1 was suppressed in PC3 cells post the treatment of BP-PEI@RNAs. Meanwhile, reduced cyclin D1 expression was detected in PC3 cells after the treatment with BP-PEI@RNAs, which was consistent with the finding that mutations, amplification and overexpression of cyclin D1 are frequently observed in a variety of tumors and may contribute to tumorigenesis [20].

BP-PEI@RNAs Treatment Promoted the Autophagy and Apoptosis of PC3 Cells

Autophagy and apoptosis, two programmed cell death pathways, play important roles in maintaining cellular and organismal homeostasis [21]. Here, we investigated the effect of BP-PEI@RNAs treatment on autophagy and apoptosis of PC3 cells in vitro. After being treated with BP-PEI@RNAs respectively, the intracellular autophagic vesicles, which are proportional to the intensity of cellular autophagy, were stained with a fluorescent probe and observed by CLSM. As shown in Fig. 5a, the number of intracellular autophagic vesicles (in blue) was significantly increased after BP-PEI@RNAs treatment compared with the BP-PEI group and the cont. group. This illustrated that BP-PEI@RNAs induced cellular autophagy within PC3 cells. Furthermore, the expression levels of autophagy-related proteins in PC3 cells were detected by western blot, as shown in Fig. 5b, which showed that BP-PEI@RNAs treatment led to a significant increase with the LC3-II/LC3-I ratio (which is positively correlated with the number of autophagosomes, reflecting the autophagic activity of the cells) compared with the cells in other groups, while p62 protein, degraded by lysosomal enzymes in autophagic vesicles when autophagy is activated, was significantly decreased. Moreover, the apoptosis of PC3 cells post BP-PEI@RNAs treatment was detected by flow cytometry assay with Annexin V-FITC staining as shown in Fig. 5c-d. BP-PEI@RNAs treatment increased the apoptosis rate of PC3 cells compared with the cells in the BP-PEI group and the cont. group. However, free RNA treatment could not change the apoptosis rate of PC3 cells (Figure S3). Furthermore, the expression levels of apoptosis-related proteins were detected by western blot assay, and the expression levels of caspase-8, caspase-3, caspase-9 and Bax proteins (pro-apoptotic proteins) were significantly increased, while the expression levels of Bcl-2 protein (anti-apoptosis protein) were significantly decreased after BP-PEI@RNAs treatment within PC3 cells (Fig. 5e). These data demonstrated that BP-PEI@RNAs treatment could promote the autophagy and apoptosis of PC3 cells.

BP-PEI@RNAs Treatment Decreased the Content of miRNAs within PC3 Cells Which Targeted Inhibiting the Expression of PTEN

It was reported that long non-coding RNA PTENP1 could act as an “endogenous sponge” to share miRNA binding sites to block the degradation effect of miRNAs on the PTEN mRNA through ceRNA mechanism (the scheme was shown in Fig. 6a) [22]. In this study, we aimed to elevate the PTEN expression level by RNAs with the sequence derived from PTENP1. Therefore, to further illustrate the mechanism of how the BP-PEI@RNAs elevated the expression level of PTEN in PC3 cells, the content of several miRNAs (miR-17, miR-19a, miR-19b, miR-21, miR-26a, miR-26b, miR-214, miR-216 and miR-217) which were validated could bind PTEN mRNA and participate its degradation process in previous studies were quantified by RT-Qpcr [22–25]. As shown in Fig. 6b-i, treated with all of four different BP-PEI@RNAs respectively, the content of miR-17, miR-19a, miR-26a and miR-26b within PC3 cells were significantly decreased. The expression level of miR-21 was also decreased in PC3 cells treated with BP-PEI@RNAs (except BP-PEI@RNA1). In addition, the expression levels of miR-214, miR-216 and miR-217 were downregulated within PC3 cells in BP-PEI@RNA3, BP-PEI@RNA2, BP-PEI@RNA2, 3 treatment groups, respectively. (Fig. 6g-i). These data suggested that BP-PEI@RNAs possibly reduced the degradation of PTEN mRNA in PC3 cells by sponging with miRNAs, thereby increasing the intracellular PTEN expression level and ultimately exerting tumor suppressive effects.

BP-PEI@RNAs Inhibited Tumor Growth in the PC3 Tumor Models

Further, the tumor suppression effect of the BP-PEI@RNAs in vivo was determined within PC3 tumor-bearing nude mouse models, and BP-PEI@RNA2 was adopted in this section as a representative. Fourteen days post PC3 cells subcutaneous injection when the tumors grew to the size of about 50 mm³, BP-PEI@RNA2 was intra-tumor injected every 5 days for 4 times and the changes of the tumor volume were monitored. As shown in Fig. 7a ~ c, BP-PEI@RNA2 intra-tumor injection could effectively inhibit tumor growth when compared with the mouse models in other groups. TUNEL fluorescence staining was adopted to detect the apoptosis rate within the tumor tissues, and many more apoptotic cells (red fluorescence) were observed in the tumor tissue section which was treated with BP-PEI@RNA2 than that in the other three groups (Fig. 7d). Next, immunohistochemical staining was adopted to evaluate the expression level of PTEN protein and proteins which play a vital role in the biological process of tumor proliferation (Ki67), apoptosis (Caspase8 and Bcl2) and autophagy (P62). As shown in Fig. 7e, BP-PEI@RNA2 intra-tumor injection notably enhanced the content of PTEN protein in the tumor tissues, thus demonstrating the efficacy of BP-PEI@RNA2 in increasing the expression level of PTEN protein in vivo. Meanwhile, BP-PEI@RNA2 treatment inhibited the expression level of Ki76 and Bcl2 which could promote cell proliferation and inhibit cell apoptosis respectively, and enhanced the expression level of Caspase 8 and P62 which were involved in promoting cell apoptosis and autophagy respectively. Furthermore, western blot analysis was adopted to evaluate the changes in the levels of PTEN and its downstream cascade effector molecules which are involved in cell proliferation (PI3K and AKT), autophagy (LC3 and p62) and apoptosis (Caspase 3/ 8/ 9 and Bcl-2), and two tumor tissues were randomly selected in each group (Fig. 7f, g and h). Similar observations were found as the data in vitro, the expression level of PTEN protein was elevated and the activated form of PI3K and AKT (P-PI3K and P-AKT) were decreased post BP-PEI@RNA2 treatment. In addition, BP-PEI@RNA2 treatment facilitated the expression of cleaved-caspase 3/ 8/ 9 and suppressed the expression of Bcl-2. Moreover, autophagy-related protein LC3 was elevated and p62 was decreased post BP-PEI@RNA2 treatment. Thus, these data confirmed the therapeutic effect of BP-PEI@RNA on PCa by enhancing the expression level of tumor suppress gene PTEN in vivo.

In summary, the sequence of long non-coding RNA PTENP1 was adopted as a template to synthesize four different RNA segments and was then loaded onto PEI-modified BP nanosheets to construct the BP-PEI@RNAs. This study revealed that BP-PEI nano-sheets could effectively transport RNAs into PC3 cells and acted as “endogenous sponges” to competitively bind miRNAs which could interact with PTEN mRNA and induce its degradation to elevate PTEN expression, and it could dramatically inhibit cell proliferation and enhance cell apoptosis, autophagy and cell cycle arrest to exert an obviously anti-tumor effect on PCa. It is a promising gene therapeutic platform for PCa treatment and provides a new perspective on tumor management.

Ethics approval and consent to participate

AII the animal experiments were complied with the guidelines Of the TianJin Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) CO., Ltd. (protocol number YSY-DWLL-2022258).

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported under grants from the National Natural Science Foundation of China (No. 22176142), Tianjin Medical University "Clinical Talent Training 123 Climbing Plan" and Young Elite Scientists Sponsorship Program by Tianjin (No. TJSQNTJ-2020-07).

Authors' contributions

J.Z. conceived the study and designed the experiment. S.S., L.L. and Q.F. performed all the experiments. M.M., N.Y., T.P. and S.G. participated in conducting the research. J.Z., S.S., L.L. and Q.F. drafted the manuscript and compiled all figures. J.Z. and X.Y. supervised the study, checked and revised the manuscript. All authors read and approved the final manuscript.

Gandaglia G, Leni R, Bray F, Fleshner N, Freedland SJ, Kibel A, Stattin P, Van Poppel H, La Vecchia C: Epidemiology and Prevention of Prostate Cancer. Eur Urol Oncol 2021, 4(6):877-892.
Litwin MS, Tan H-JJJ: The diagnosis and treatment of prostate cancer: a review. 2017, 317(24):2532-2542.
Gregg JR, Thompson TC: Considering the potential for gene-based therapy in prostate cancer. Nat Rev Urol 2021, 18(3):170-184.
Whitehead KA, Langer R, Anderson DG: Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009, 8(2):129-138.
Zhou W, Cui H, Ying L, Yu XF: Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing. Angew Chem Int Ed Engl 2018, 57(32):10268-10272.
Wang H, Zhong L, Liu Y, Xu X, Xing C, Wang M, Bai SM, Lu CH, Yang HH: A black phosphorus nanosheet-based siRNA delivery system for synergistic photothermal and gene therapy. Chem Commun (Camb) 2018, 54(25):3142-3145.
Jamaspishvili T, Berman DM, Ross AE, Scher HI, De Marzo AM, Squire JA, Lotan TL: Clinical implications of PTEN loss in prostate cancer. Nat Rev Urol 2018, 15(4):222-234.
Lee YR, Chen M, Pandolfi PP: The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nat Rev Mol Cell Biol 2018, 19(9):547-562.
Liu M, Chen M-Y, Huang J-M, Liu Q, Wang L, Liu R, Yang N, Huang W-H, Zhang W: LncRNA weighted gene co-expression network analysis reveals novel biomarkers related to prostate cancer metastasis. BMC Medical Genomics 2022, 15(1):256.
Du Z, Sun T, Hacisuleyman E, Fei T, Wang X, Brown M, Rinn JL, Lee MG-S, Chen Y, Kantoff PW et al: Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer. Nature Communications 2016, 7(1):10982.
Gao X, Qin T, Mao J, Zhang J, Fan S, Lu Y, Sun Z, Zhang Q, Song B, Li L: PTENP1/miR-20a/PTEN axis contributes to breast cancer progression by regulating PTEN via PI3K/AKT pathway. Journal of Experimental & Clinical Cancer Research 2019, 38(1):256.
Zeng L, Zhang X, Liu Y, Yang X, Wang J, Liu Q, Luo Q, Jing C, Yu X-F, Qu GJC: Surface and interface control of black phosphorus. 2022, 8(3):632-662.
Bian S, Liu Q, Zhang X, Ma C, Zhang Y, Cheng Z, Kang Y, Lu W, Chu PK, Yu XF et al: Fabricating Black-Phosphorus/Iron-Tetraphosphide Heterostructure via a Solid-Phase Solution-Precipitation Method for High-Performance Nitrogen Reduction. Small (Weinheim an der Bergstrasse, Germany) 2022, 18(39):e2203284.
Zhao H, Zhang W, Liu Z, Huang D, Zhang W, Ye B, Hu G, Zhong H, Zhuang Z, Guo Z: Insights into the intracellular behaviors of black-phosphorus-based nanocomposites via surface-enhanced Raman spectroscopy. 2018, 7(10):1651-1662.
Wu Q, Yao L, Zhao X, zeng L, Li P, Yang X, Zhang L, Cai Z, Shi J, Qu G et al: Cellular Uptake of Few-Layered Black Phosphorus and the Toxicity to an Aquatic Unicellular Organism. Environmental Science & Technology 2020, 54(3):1583-1592.
Vermeulen K, Berneman ZN, Van Bockstaele DR: Cell cycle and apoptosis. Cell Prolif 2003, 36(3):165-175.
Yam CH, Fung TK, Poon RY: Cyclin A in cell cycle control and cancer. Cell Mol Life Sci 2002, 59(8):1317-1326.
Jackman M, Marcozzi C, Barbiero M, Pardo M, Yu L, Tyson AL, Choudhary JS, Pines J: Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint. J Cell Biol 2020, 219(6).
Gavet O, Pines J: Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J Cell Biol 2010, 189(2):247-259.
Wang J, Su W, Zhang T, Zhang S, Lei H, Ma F, Shi M, Shi W, Xie X, Di C: Aberrant Cyclin D1 splicing in cancer: from molecular mechanism to therapeutic modulation. Cell Death & Disease 2023, 14(4):244.
Fan YJ, Zong WX: The cellular decision between apoptosis and autophagy. Chin J Cancer 2013, 32(3):121-129.
Ghafouri-Fard S, Abak A, Shoorei H, Mohaqiq M, Majidpoor J, Sayad A, Taheri MJB, Pharmacotherapy: Regulatory role of microRNAs on PTEN signaling. 2021, 133:110986.
Baghaei F, Abdollahi A, Mohammadpour H, Jahanbin M, Naseri Taheri F, Aminishakib P, Emami Razavi A, Kharazifard MJJoOP, Medicine: PTEN and miR‐26b: Promising prognostic biomarkers in initiation and progression of Oral Squamous Cell Carcinoma. 2019, 48(1):31-35.
Du Y, Ding Y, Shi T, He W, Feng J, Mei Z, Chen X, Feng X, Zhang X, Jie ZJAbebS: Long noncoding RNA GAS5 attenuates cigarette smoke-induced airway remodeling by regulating miR-217-5p/PTEN axis. 2022, 54(7):1-9.
Lu R, Zhao G, Yang Y, Jiang Z, Cai J, Zhang Z, Hu HJJocb: Long noncoding RNA HOTAIRM1 inhibits cell progression by regulating miR‐17‐5p/PTEN axis in gastric cancer. 2019, 120(4):4952-4965.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
04 Feb, 2024
Reviews received at journal
01 Feb, 2024
Reviewers agreed at journal
12 Jan, 2024
Reviewers invited by journal
12 Jan, 2024
Editor assigned by journal
09 Jan, 2024
Submission checks completed at journal
08 Jan, 2024
First submitted to journal
07 Jan, 2024

You are reading this latest preprint version

A Black Phosphorus Nanosheet-based RNA Delivery System for Prostate Cancer Therapy by Increasing the Expression Level of Tumor Suppressor Gene PTEN via CeRNA Mechanism

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

Synthesis RNAs

Construction, Transformation, Amplification and Extraction of the Plasmid

Linearization, Purification and Recovery of the Plasmid

Synthesis of RNA by in vitro Transcription

Construction of BP-PEI@RNAs

Characterization of BP@RNA

Fluorescent labeling of RNA

Uptake and subcellular distribution of BP@RNA

Live/ Dead staining

Cell viability test

Autophagy, apoptosis and cell cycle test

RT-qPCR

Western-blot

Animal experiment

Tunel staining

Immunohistochemistry staining

Statistical analysis

Results and Discussion

BP-PEI@RNA Construction and Physicochemical Characterization

BP Nanosheets Could Efficiently Transport RNA Segments into PC3 Cells and Further into the Lysosomes

BP-PEI@RNAs Treatment Induced Cell Cycle Arrest of PC3 Cells

BP-PEI@RNAs Treatment Promoted the Autophagy and Apoptosis of PC3 Cells

BP-PEI@RNAs Inhibited Tumor Growth in the PC3 Tumor Models

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1