Ultrasound Microbubbles-mediated Microrna-505 Regulates Cervical Cancer Cell Growth via AKT2

Background: The gene-loaded microbubbles (MBs) combined with ultrasound resulting in increased delivery eciency, may be a novel method of gene delivery. We explored the effects of ultrasound and microbubbles (USMB)-mediated microRNA (miR)-505 on cervical cancer (CC) development. Methods: miR-505 mediated by USMB was prepared. The effect of miR-505 on its transfection eciency was studied by RT-qPCR. The effect of miR-505 on HeLa cell proliferation was evaluated by MTT and colony formation assays. Flow cytometry was used to study cell cycle changes, Hoechst was utilized to detect apoptosis. Through the wound healing and Transwell assay, the migration and invasion ability of HeLa cells were measured. The target gene of miR-505 was predicted, and its expression in CC was detected. The target relationship and the effect of the target gene on HeLa cells were further veried. Results: USMB-miR-505 showed higher transfection eciency than miR-505 alone. miR-505 inhibited HeLa cell malignant episodes, which were reinforced by USMB treatment. miR-505 targeted AKT2. AKT2 was highly expressed in CC, and overexpression of AKT2 signicantly reversed the inhibitory effect of miR-505 mediated by USMB on HeLa cell malignant biological behaviors. Conclusion: USMB-miR-505 inhibited HeLa cell malignant biological behaviors by targeting AKT2.


Introduction
Cervical cancer (CC) is an aggressive gynecological malignancy with a high risk of recurrence and death, mainly in women [1,2]. It is the 4th most prevalent female cancer and causes approximately 265,700 annual deaths worldwide [3][4][5]. The incidence varies across the world, with the highest incidence rate in Eastern Africa and lowest incidence in Western Asia [6]. Over 70% of CC cases diagnosed in developing countries are locally invasive or metastatic, and the diagnosis of early stage CC is di cult, thus leading to high mortality [7]. Numerous efforts have been made to prevent CC that include the expansion of human papillomavirus vaccine coverage and primary screening [8], which eventually reduce the disease in many developed countries [9,10]. Fortunately, radical hysterectomy has gained increasing popularity in the last twenty years and enjoyed broad acceptance across Europe and the Americas [11]. Although there is great progress in diagnostic and therapeutic strategies, the survival rate for CC patients remains poor [12].
Thus, this study aims to identify novel biomarkers and develop non-invasive therapies for the diagnosis and prognosis of CC. microRNAs (miRs) participate in most biological processes like apoptosis and epithelial-to-mesenchymal transition (EMT) by regulating gene expression [13]. miRs and genes can serve as biomarkers for prognosis and treatment of CC [14]. miR-505 suppressed EMT and metastasis phenotypes in HK-1 cells and prevented macroscopic lung metastases [13]. But the role of miR-505 in CC is less studied.
Microbubbles (MBs) were originally developed for ultrasound imaging, and now they are considered as ultrasound-assisted gene delivery tools to disturb cell membranes and accelerate gene entering into cells [15]. Behaviors of MBs under ultrasonic irradiation will lead to short-term membrane permeability of surrounding cells, thus promoting targeted local administration without cell damage [16]. The combination of ultrasound and microbubbles (USMB) is an emerging approach for non-invasive enhancement of uptake of drugs and genes [17,18]. Microbubbles promote ultrasound-mediated gene transfer e ciency in cell culture and tumor transplantation of hindlimb, and may selectively transfer therapeutic genes to disease sites [19]. USMB-mediated miR (USMB-miR) delivery has been considered as an effective tool for treatment of cancer and cardiovascular diseases [20,21]. Interestingly, USMB-miR-133a prevented tumor growth and improved survival of breast cancer mice, and USMB-mediated miR-767 silencing offered a novel therapeutic strategy for non-small cell lung cancer treatment [22,23]. However, the mechanism of USMB-miR-505 in CC remains unclear. We performed serials of molecular and histological experiments to evaluate the relevance of USMB-miR-505 in CC development.

Sample collection
From March 2018 to January 2019, cancer and adjacent normal tissues of 20 CC patients undergoing surgery in the First Hospital of Huai'an A liated to Nanjing Medical University were collected. The patients aged between 45 and 69 years old. They were free of any other malignant tumors and did not receive preoperative radiotherapy and chemotherapy. The tissues were immediately preserved at − 80 °C.

Cell culture
Hela cells at logarithm phase from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) were cultured at 37 °C with 5% CO 2 in Dulbecco's modi ed Eagle medium (DMEM, Gibco; Waltham, MA, USA) with 10% fetal bovine serum (FBS, Gemini Bio-products, West-Sacramento, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). The media were renewed every 2 days. Cells were subcultured when cells reached 80% con uency.
After 1 µg NC mimic or miR-505 mimic was mixed with 2 µL Lipofectamine 2000 (Invitrogen), cells were suspended again with 500 µL RPMI-1640. The mixed solution was put to the cells at 50 µL/well. Next, cells in the miR-505-MB group were treated with miR-505-MB at 50 µL/well and subjected to 10 Mhz ultrasound for 30 minutes. Based on the treatment in the miR-505-MB group, cells in the miR-505-MB + OE-NC or miR-505-MB + OE-AKT2 group were stably transfected with corresponding vectors using Lipofectamine 2000 after miR-505 expression was detected. After transfection and ultrasound exposure, cells were harvested for following experiments.

Colony formation assay
After 24 hours of transfection, 500 cells were seeded into the 6-well plates and cultured for two weeks.
The cell colonies were xed for 5 minutes with methanol and stained for 15 minutes with 0.1% crystal violet. Thereafter, cell colonies were counted and photographed.

Flow cytometry
Flow cytometry was utilized to detect cell cycles. After 48 hours of transfection, the cell cycle was evaluated by PI staining. Then cells were detached with trypsin, washed twice with PBS, and xed at 4 °C overnight in 70% cold ethanol. After twice washes with PBS, the cells were reacted with 50 µg/mL PI (Keygen) for 30 minutes. Cell cycle analysis was performed immediately using a FACS Calibur ow cytometer (BD, San Diego, USA). The proportion of cells in G1, S and G2 phases was detected.

Hoechst 33258 staining
The transfected cells were seeded at 1 × 10 5 cells/mL (3 mL per well) into 6-well plates. After the cells were incubated at 37 °C with 5% CO 2 for 24 hours, the media were removed. Cells were then xed with paraformaldehyde, washed with PBS, and stained with Hoechst 33258 (HY-15558, MedChemExpress, NJ, USA) in the dark. After 30 minutes, cells were observed under a uorescence microscope, and 5 elds were randomly chosen from each slide.
Wound healing assay Cells were plated into 6-well plates and cultured for 24 hours. Then, a 200 µL pipette tip was applied to scratch 3 parallel lines, and cells were washed twice in PBS. Then cells were cultivated at 37 °C, and photographed at 0 and 24 hours under a FSX100 microscope (Olympus) after wounding. Migration ratio was estimated by examining the change of scratch area.

Transwell assay
The invasion experiments were carried out in the Transwell chamber coated with Matrigel (BD Biosciences). Brie y, 500 µL DMEM with 10% FBS was supplemented to the basolateral chamber, while 2 × 10 5 cells were paved in the apical chamber. After 24 hours, cells passing through the Matrigel were xed with methanol and stained by 0.1% crystal violet, otherwise removed by cotton swabs. Finally. cells were counted and the stained cells represented the invasiveness.

Dual-luciferase reporter gene assay
The binding site between miR-505 and AKT2 was predicted from Starbase (http://starbase.sysu.edu.cn/), and ampli ed using PCR and cloned into pGL3 vector (Promega, Madison, WI, USA) to obtain AKT2 wildtype (WT). AKT2 mutant (MT) was obtained by mutating the binding site. These vectors were cotransfected with miR-505 mimic and its control into 293T cells (ATCC) by Lipofectamine 2000. After 48 hours of transfection, the luciferase activity was measured by the luciferase reporter system (Promega).

RNA immunoprecipitation (RIP)
RIP lysis buffer kit (Millipore) was used for RIP experiments. In short, HeLa cells were lysed in RIP lysis buffer, and RNA was precipitated with anti-AGO2 (Millipore) and anti-IgG (Millipore). TRIzol reagent was used to purify the immunoprecipitated RNA, and RT-qPCR was used to detect the gene expression.
Statistical analysis SPSS 22.0 (IBM Corp. Armonk, NY, USA) was used for data analysis. The obtained data of 3 independent experiments were described as mean ± standard deviation. Comparisons between group pairs were analyzed using paired t test or unpaired t test, while comparisons among multi-groups were analyzed through one-way or two-way analysis of variance (ANOVA). Pairwise comparison after ANOVA analysis was conducted using Tukey's multiple comparisons test. P value shall be based on a two-sided test. P < 0.05 was considered as a statistical difference.

USMB-miR-505 promoted miR-505 transfection e ciency in Hela cells
According to a previous report [25], miR-505 is poorly expressed in CC, which can inhibit the development of CC. The poor expression of miR-505 in CC was con rmed by RT-qPCR in our collected CC tissues (Fig. 1A). The MBs were opalescent suspension, and the homogeneous parts were spherical and distributed uniformly (Fig. 1B). The average particle size was about 2.16-4.68 µm (Fig. 1C).
USMB-miR-505 suppressed proliferation and promoted apoptosis of Hela cells MTT assay and colony formation assays examined cell proliferation. Results demonstrated that miR-505 mimic inhibited cell proliferation, while miR-505-MB strengthened the inhibitory effects of miR-505 mimic ( Fig. 2A/B). Flow cytometry detected cell cycle changes (Fig. 2C), and found that overexpression of miR-505 resulted in the increase of G1 phase and the decrease of S phase, especially in the miB-505-MB group. The expression of Bax and Bcl-2 was measured by ELISA kits (Fig. 2D). Compared with NC mimic group, Bax expression in the other two groups increased, and Bcl-2 expression decreased signi cantly, and miR-505-MB group showed more powerful effect in regulating Bcl-2 and Bax expression. Hoechst staining detect the apoptosis rate of HeLa cells, and found that overexpression of miR-505 led to the increase of the apoptosis rate of HeLa cells, especially in the miR-505-MB group (Fig. 2E).

USMB-miR-505 inhibited migration, invasion and EMT of Hela cells
In HeLa cells transfected with NC mimic, miR-505 mimic and miR-505-MB, the expression of EMT-related factors E-cadherin, N-cadherin and Vimentin was measured by RT-qPCR. Overexpression of miR-505, especially miR-505-MB led to the increase of E-cadherin and decreases of N-cadherin and Vimentin (Fig. 3A).
AKT2-WT and AKT2-MT were transfected with NC mimic or miR-505 mimic into 293T cells. miR-505 mimic signi cantly reduced the luciferase activity of AKT2-WT luciferase vector, but had no signi cant effect on AKT2-MT luciferase vector (Fig. 4D). RIP experiment found that anti-AGO2 signi cantly enriched miR-505 and AKT2 (Fig. 4E) compared with anti-IgG, which proved that miR-505 targeted AKT2 Overexpression of AKT2 compromised the inhibitory effects of USMB-miR-505 on Hela cells In the HeLa cells transfected with miR-505-MB + OE-NC, miR-505-MB + OE-AKT2, the effect of overexpression of AKT2 on the HeLa cells treated with miR-505-MB was studied.
MTT and colony formation assays showed that the vitality of HeLa cells treated with miR-505-MB increased after AKT2 overexpression (Fig. 5A), and the number of cell colonies increased (Fig. 5B). Flow cytometry showed that overexpression of AKT2 attenuated the effect of miR-505-MB on HeLa cell cycle arrest (Fig. 5C). RT-qPCR showed that overexpression of AKT2 resulted in the decrease of E-cadherin and Bax, and the increases of N-cadherin, vimentin and Bcl-2 in miR-505-MB treated cells (Fig. 5D). Wound healing and Transwell assays discovered that overexpression of AKT3 promoted migration and invasion of HeLa cells (Fig. 5E/F). ELISA found that overexpression of AKT2 resulted in the decrease of Bax and the increase of Bcl-2 (Fig. 5G). Hoechst staining con rmed the inhibition of AKT2 on HeLa cell apoptosis (Fig. 5H).

Discussion
About 30%-35% of CC patients fail to completely recover from the treatment, including surgical resection and radiotherapy [26]. Additionally, the conventional clinical variables, such as parametrial involvement and lymph node metastasis, are not enough to predict curative effect or formulate supplementary therapy for CC patients after surgery [27]. The combination of drug-loaded USMB has been used in preclinical studies on drug and gene delivery to solid tumors, and ablation of blood vessels [28]. Based on these facts, we discussed the possible mechanism of USMB-miR-505 in the malignant episodes of CC cell. As expected, our results provided evidence that USMB-miR-505 further strengthened the inhibitory role of miR-505 in CC malignancy by targeting AKT2 (Fig. 6), which offered novel insights for CC treatment.
The abnormal expression of miRs is related to the pathological changes of human, including cancer, and some are regarded as potential prognostic markers in different tumors, such as CC [29]. In this study, we found poor miR-505 expression in CC. Consistently, miR-505 was poorly expressed in CC, and negatively correlated with tumor histology grade and lymph node metastasis [25,30]. Additionally, miR-940 inhibited CC cell proliferation, and USMB-miR-940 showed better effects [31]. However, the role of USMB treatment on miR-505 expression was not yet studied. Thus, MBs were synthesized by ultrasonic dispersion, and HeLa cells were transfected with USMB-mediated miR-505. We discovered that USMB-miR-505 had the most signi cant effect on miR-505 expression. miRs can intervene several aspects of CC, including proliferation, EMT and chemosensitivity [32]. A recent study unveiled that paclitaxel-miR-34a-USMBs are a promising anticancer strategy for treating CC [21]. In this study miR-505 blocked malignant episodes of Hela cells, which were reinforced by USMB treatment. EMT is a crucial mechanism of tumor cell invasion and cancer metastasis, by which epithelial cells obtain mesenchymal broblast-like properties [33]. Vimentin is a well-recognized metastasis marker [34], while E-cadherin could repress invasion and metastasis of epithelial cells [35]. EMT contributes to chemotherapy and radiotherapy resistance of CC cells, thus inhibiting EMT sensitizes CC cells sensitive to radiotherapy and drugs, and improves the survival rate of CC patients [32]. miR-505 mimic elevated Ncadherin expression but decreased E-cadherin in gastric cancer cells [36]. miR-505-5p was correlated with metastasis, and overexpression of miR-505-5p inhibited metastasis and EMT in CC cells [37]. In Ca-Ski and HeLa cells, miR-505 upregulation suppressed proliferation and tumorigenicity, indicating miR-505 may act as an inhibitor in CC [30]. Similarly, compared with liposome, MBs or ultrasound transfection, USMB-miR-940 further inhibited CC malignant episodes [31]. Our study may offer a novel approach for CC treatment from the miR-505 delivery by USMB treatment.
Furthermore, miR-505 targeted AKT2. There are three subtypes of AKT, namely AKT1, AKT2 and AKT3, among which AKT2 is responsible for tumor progression and metastasis through regulating EMT-related proteins [38]. Increased AKT2 expression was associated with the cervical lesion progression [39]. AKT2 was highly expressed in CC tissues, and AKT2 overexpression promoted the proliferation and colony formation ability in SiHa cells [40]. Overexpression of AKT2 compromised the inhibitory effects of USMB-miR-505 on Hela cells. It is known that activated Akt can promote the proliferation of cancer cells including CC [41]. AKT2 expression was required for EMT-like morphological changes [42]. Knockdown of AKT2 prevented cell proliferation and stimulated apoptosis in CC cells [43]. A recent study showed that AKT2 inhibition is potential for anti-cancer therapies for its function in EMT reversion, metastasis reduction and tumor recurrence prevention [44]. To sum up, AKT2 silencing is possible to prevent CC development.

Conclusion
In conclusion, our study provided substantial evidence that miR-505 overexpression repressed CC progression, and USMB mediated miR-505 reinforced the inhibitory effects of miR-505 in CC. These results indeed unveiled a promising approach for CC treatment. More works should be done in the future to identify the application value of our results. We will also make comprehensive investigation to gure out the downstream pathways involving in the bene cial roles of USMB-miR-505 in CC treatment.

Declarations
Ethics approval and consent to participate The study followed the Declaration of Helsinki and was approved by the ethics committee of the First Hospital of Huai'an A liated to Nanjing Medical University. All patients signed the informed consent.

Consent for publication
Not applicable.

Availability of data and materials
All the data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions LLX conceived the study and participated in its design and coordination;QZ, CHL andFH performed all experiments;XPLandLLX analyzed and interpreted the data; The draft was improved through discussion and editing by all the authors who read and approved the nal manuscript.  USMB-mediated miR-505 inhibited HeLa cell growth. A. HeLa cell viability detected by MTT assay. B. the ability of cell proliferation measured by colony formation assay. C, cell cycle detected by ow cytometry. D. Bax and Bcl-2 expression detected by ELISA; E, Hoechst staining detected the apoptosis rate. One-way ANOVA for data analysis in panels A/B/E; two-way ANOVA for data analysis in panels C/D; *p< 0.05vs. NC mimic; #p< 0.05vs. miR-505 mimic. C, the invasion of cells examined by Transwell assay. One-way ANOVA for data analysis in panels B/C; two-way ANOVA for data analysis in panel A; *p< 0.05vs. NC mimic; #p< 0.05vs. miR-505 mimic. miR-505 targeted AKT2. A, a binding site between miR-505 and AKT2. B, AKT2 expression in CC and adjacent normal tissues detected by RT-qPCR. C. WB detected AKT2 expression in HeLa cells; D,the activity of WT-AKT2 and MT-AKT2 luciferase detected by dual-luciferase assay; E, the binding relationship between miR-505 and AKT2 veri ed by RIP experiment. Paired t test for panel B, @@ p< 0.01 vs. the adjacent normal tissues; one-way ANOVA for data analysis in panel C; two-way ANOVA for data analysis in panes D/E; *p< 0.05vs. NC mimic; #p< 0.05vs. miR-505 mimic;%%p < 0.01vs.Anti-IgG.