RASA1 inhibits the progression of renal cell carcinoma by decreasing the expression of miR-223-3p and promoting the expression of FBXW7

Renal cell carcinoma (RCC) is a common cancer with approximately 65,000 new cases each year [1]. RCC makes up 2–3% of all adult cancers and ranks eighth among the most common causes of death associated with cancers [2]. Thus, RCC greatly affects quality of life and patient life span while burdening health and economic systems owing to increased risks of metabolic syndrome and cardiovascular and kidney diseases in late stages [3]. Additionally, the incidence and death rate of RCC is increasing worldwide [4]. Thus, studying the molecular basis of RCC is essential for the development of new treatments for RCC patients.

Ras small GTPases exert regulatory effects in various signaling cascades as well as in different cellular processes [5,6]. Roughly 30% of human cancers have mutations in Ras genes (K-Ras, H-Ras, and N-Ras) making them constantly active in their GTP-bound form [7], giving rise to constitutively active Ras signal transduction even without extracellular stimuli. RAS p21 protein activator 1 (RASA1), also known as p120-RasGAP, is a RasGAP protein. In addition to its RasGAP domain, RASA1 has two Src homology 2 (SH2) domains, an SH3 domain, a Pleckstrin homology (PH) domain, and a Calcium-dependent phospholipid-binding (C2) domain. It functions as a signaling scaffold protein regulating pivotal signal cascades [8,9]. RASA1 has also been implicated in many biological processes including actin filament polymerization, blood vessel development, and cell apoptosis and movement [10]. Mice deficient in RASA1 have aberrantly growing blood vessels and exhibit large-scale neuronal apoptosis and embryonic death at E10.5 [11]. In mouse endothelial cells, loss of RASA1 increases endothelial proliferation and tube formation [10]. Human RASA1 germline mutations are related to an autosomal dominant disorder, capillary malformation-arteriovenous malformation (CM-AVM), featuring malformed atypical capillaries [12]. Despite its physiological functions, the role of RASA1 in tumor formation, and specifically in RCC, has not yet been elucidated.

The purpose of the present study was to inquire into the functions of RASA1 in the occurrence and progression of RCC and to explore its potential mechanisms, in order to provide novel protocols for the diagnosis and therapy of RCC.

Renal cancer tissues and corresponding non-cancerous tissues were collected from renal cancer patients who underwent surgical resection at the First Affiliated Hospital of Xinjiang Medical University from 2016 to 2018. All clinical specimens were preserved at -80°C until use. No routine treatments were performed before surgery. All research subjects provided written informed consent in advance, and this project was approved by the Institutional Review Board from the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University in accordance with the Code of Ethics in the Declaration of Helsinki.

ACHN, SN12C, 786-0, SKRC-39, A-498 and HTB-46 (RCC cells lines), HK-2 (a normal proximal tubule epithelial cell line), and HEK293T cell lines were purchased from BeNa Culture Collection. The culture medium used contained 10% fetal bovine serum (FBS). Cells were incubated at 37°C in an environment with 5% CO₂.

Using manufacturer’s instructions, TRIzol reagent (Invitrogen) was used for total RNA extraction from RCC tissues and cells. Through determination via a NanoDrop 2000 (Thermo Fisher Scientific), 200 ng of RNA from each sample was used for reverse transcription with the ReverTra Ace qPCR RT Kit. THUNDERBIRD SYBR® qPCR Mix (Toyobo) was utilized for the calibration of mRNAs in the three groups through qRT-PCR. Reaction conditions were as follows: 94°C for 2 min, 94°C for 10 s, 56°C for 30 s, 72°C for 1 min, and 72°C for 10 min. The reaction was conducted three times. Finally, with GAPDH and U6 as endogenous controls, mRNA expression levels were normalized to GAPDH expression and quantified via the 2^−ΔΔCt method. In addition, miRNA expression was normalized to U6 expression and also quantified via the 2^−ΔΔCt method. The qRT-PCR primers used in the present study are listed in Table 1.

siRNAs targeting RASA1 or FBXW7 (F-box and WD repeat domain containing 7), scrambled siRNAs, miR-223-3p inhibitor, miR-223-3p NC inhibitor, miR-223-3p mimic, and miR-223-3p NC mimic were obtained from GenePharma. The pcDNA3.1 plasmid (Thermo Fisher Scientific) was utilized for RASA1 or FBXW7 overexpression. Prior to transfection, 786-0 and ACHN cells were trypsinized (0.25%) and inoculated in six-well plates with 1 × 10⁵ cells per well. In the case of 80–90% cell fusion, original medium was replaced with fresh medium lacking serum and antibiotics. Transfection was conducted using Lipofectamine 2000 (Life Technologies Corporation (Gaithersburg, MD, U.S.A.)), followed by cultivation of the transfected cells at 37°C in an environment with 5% CO₂ for 48 h.

Through bioinformatical analysis (miRDB, http://www.mirdb.org/), the targeted association between miR-223-3p and FBXW7 was predicted. Enzymes were used to digest the FBXW7 3′UTR using XbaI. Subsequently, with the use of Lipofectamine 2000 reagent (Invitrogen), HEK293T cells were treated with a pGL3-control luciferase reporter gene vector provided by Promega (Madison, WI, U.S.A.) containing FBXW7-wt/FBXW7-mut and miR-223-3p mimics/miR-223-3p control. Finally, after 48 h of treatment, a Dual-Glo Luciferase Assay System (Promega) was utilized to examine the luciferase activity in cell lysates following manufacturer's protocols. Each assay was implemented in triplicate at the minimum.

In a six-well plate, 10 μl of CCK-8 solution (Dojindo) was added to each fell following cell transfection for 0, 24, 48, and 72 h. Next, the RCC cells underwent 2 h of incubation in a humid chamber with 5% CO₂ at 37°C. Then, a plate luminometer from Bio-Rad was employed to determine the optical density of the cells at 450 nm. The assay was implemented in triplicate at the minimum.

To analyze the cell cycle, cells were collected from different groups at 24–48 h after transfection, and a cell suspension was obtained by digestion of the cells. The supernatant was removed following centrifugation, the cells were washed in PBS twice to discard any residue, and the cells were immobilized in ethanol (75%) for 4 h at 4°C. Later, the immobilized cells underwent centrifugation and PBS washes, followed by mixing with 40 μg propidium iodide (PI) and 1 ml of a 100 μg RNase staining solution from BD Biosciences, and incubation at room temperature for 15 min. Cell cycle analysis was conducted using a FACSCalibur flow cytometer subsequent to staining, and FACSDiva was adopted for statistical data analysis. In apoptosis assays, transfected cells were obtained from each group and underwent trypsinization (0.25%) 24–48 h later. Following inoculation into 96-well plates (2 × 10⁴ cells per well), 5 μl PI, 200 μl HEPES and 5 μl Annexin V/FITC were added and the mixtures were incubated for 25 min at room temperature. A FACSCalibur FCM from BD Biosciences was used for observing apoptosis. The above three assays were independently implemented to reduce errors, and data were analyzed via FACSDiva software.

Matrigel (BD Bioscience) and serum-free DMEM were thoroughly mixed and placed in a Transwell chamber (Corning Incorporated). Cells in serum-free medium at a concentration of 2.5 × 10⁴ cells/ml were placed into the upper chamber (500 μl for each chamber) of 24-well invasion chambers, while culture medium containing 20% FBS was added to the lower chamber. A 4% paraformaldehyde solution was utilized to immobilize the cells, and 0.1% Crystal Violet was used to stain RCC cells after 24–48 h of incubation. Pictures were taken in each chamber, and the cells in 5–10 independent fields were counted. The assay was repeated in triplicate at the minimum.

A BCA Protein Assay Kit from Pierce was utilized to measure the protein concentration of each sample. Following isolation via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% gel, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane provided by Millipore (Billerica) for 120 min. Next, TBST with 5% skim milk was used to seal the membrane, followed by 1 h of incubation with primary antibodies (anti-FBXW7 and anti-GAPDH) from Abcam and secondary antibody (anti-rabbit IgG H&L) from Abcam at 37°C. An ECL system from Life Technology was used to visualize the antibody signals. Differences in the relative protein levels among samples were detected by GAPDH density as an endogenous control.

BALB/c nude mice were obtained from Xinjiang Medical University. Animal experiments took place in SPF Animal Laboratory at Xinjiang Medical University. All animal experiments were approved by the Changji Branch Hospital of the First Affiliated Hospital of Xinjiang Medical University. Female athymic BALB/c nude mice were injected with control, NC or RASA1-transfected ACHN cells in the subcutaneous tissue of the right flank. Six nude mice were used per group. Tumor growth was monitored by 2D measurements using electronic calipers starting from the seventh day after tumor transplantation, once every 7 days for a total of 28 days. All surgeries were performed under sodium pentobarbital anesthesia via intraperitoneal injection (40 mg/kg) and all efforts were made to minimize suffering. At 4 weeks post-injection, the mice were anesthetized with 40 mg/kg sodium pentobarbital and then killed by 10% formalin perfusion fixation of central nervous system; death was confirmed by complete cessation of heartbeat and breathing. The tumor tissues were isolated and weighed.

Data from each assay implemented thrice were presented as mean ± standard deviation (SD). The comparison between two different groups with parametric variables was conducted via Student’s t-test, while the comparison among multiple groups was performed via one-way ANOVA. GraphPad Prism 6.0 was adopted for statistical processing. P<0.05 indicated statistically significant differences.

RASA1 mRNA was expressed at lower levels in RCC tissue samples than in adjacent (paracancerous) tissue (Figure 1A). In RCC cell lines, we also found that RASA1 expression was decreased compared to the HK2 cell line (Figure 1B). Additionally, the RASA1 protein expression, as detected by Western blot, was lower in RCC tumor tissue samples compared with adjacent tissue (Figure 1C).

The transfection efficiency of the RASA1 over-expression plasmid was verified in 786-0 cells (Figure 2A). Then, a Western blot was utilized to detect the overexpression of RASA1 and the expression of Caspase 3. Previous studies have reported that Caspase-3 is involved in cell apoptosis. Here, no differences were found in RASA1 and Caspase-3 protein expression between the control cells and NC cells, after RASA1 transfection. However, the expression of RASA1 and Caspase-3 proteins in cells with RASA1 overexpression was strikingly increased relative to the NC cells, indicating that RASA1 may regulate Caspase-3 expression and promote cell apoptosis (Figure 2B). To confirm the effect of RASA1 on cell apoptosis, a flow cytometry assay was used to detect apoptosis following RASA1 overexpression. The results showed that the apoptosis rate increased after RASA1 overexpression (Figure 2C). These experiments were also performed in ACHN cells and the same results were observed (Figure 2D–F).

Next, we aimed to verify the influence of RASA1 on cell proliferation, migration, and cell cycle progression. A CCK-8 assay was conducted which demonstrated that RASA1 overexpression significantly decreased the viability of 786-0 cells (Figure 3A). Furthermore, a cell colony formation assay disclosed that the rate of colony formation was lower in 786-0 cells with RASA1 overexpression (Figure 3B). In addition, CCK-8 results showed that overexpression of RASA1 in 786-0 cells lowered their proliferation ability with a time gradient compared to the NC cells (Figure 3C). A Transwell assay uncovered that RASA1 overexpression drastically reduced cell migration (Figure 3D). Likewise, RASA1 overexpression greatly decreased cell proliferation, colony formation, and migration in ACHN cells (Figure 3E–H). Finally, flow cytometry results revealed that RASA1 overexpression increased the RCC cell proportion in G0/G1 phase in both 786-0 and ACHN cells (Figure 4A,B).

To explore the underlying mechanism of RASA1 in cells, the effect of RASA1 on the expression of miRNAs closely related to kidney cancer was examined. Here, we assessed miR-132, miR-630, miR-223-3p, miR-508-3p, miR-486, and miR-296-3p [13–19]. RASA1 overexpression reduced the expression of miR-223-3p (Figure 5A), but had no notable impacts on the expression of other miRNAs tested (Supplementary Figure S1). Next, 786-0 cells were transfected with a miR-223-3p mimic or inhibitor to alter miR-223-3p expression (Figure 5B). A CCK-8 assay showed that miR-223-3p partially reversed the inhibitory effect of RASA1 on cell proliferation (Figure 5C). The effect of miR-223-3p on cell proliferation was also seen in ACHN cells (Figure 5D–F). Further, based on a Transwell assay, miR-223-3p could partially attenuate the inhibition of cell migration caused by RASA1 overexpression (Figure 5G). The above results indicate that RASA1 might inhibit cell proliferation and migration by regulating miR-223-3p.

Through bioinformatic analysis, FBXW7 was identified as a potential target gene of miR-223-3p (Figure 6A). There’re some other potential target genes of miR-223-3p, which have high binding scores, but we found they are not dysregulated in RCC tissues. Therefore, FBXW7 was chosen for the further study. A dual luciferase reporter gene assay confirmed a binding association between FBXW7 and miR-223-3p (Figure 6B,C). Further, we detected the expression level of FBXW7 mRNA and protein level in RCC tissues. FBXW7 mRNA and protein were down-regulated in RCC tumor tissues compared with adjacent tissues (Figure 6D,E). Interestingly, the mRNA and protein levels of FBXW7 were pronouncedly decreased after up-regulation of miR-223-3p expression, but increased when miR-223-3p was down-regulated (Figure 6F–I), confirming that miR-223-3p could modulate the expression of FBXW7. In RCC cell lines, the expression of FBXW7 was generally reduced compared with HK2 cells (Figure 6J). By Pearson correlation analysis, the expression of FBXW7 was found to be negatively correlated with miR-223-3p expression, but remarkably positively correlated to the expression of RASA1 in renal cancer tissues, indicating that RASA1 might up-regulate FBXW7 expression by down-regulating miR-223-3p expression (Figure 6K,L).

As shown in Figure 7A, overexpression of RASA1 inhibited tumor growth, while down-regulation of RASA1 promoted tumor growth in vivo, which was confirmed by the detection of tumor volume and weight (Figure 7B,C). Additionally, overexpression of RASA1 greatly up-regulated the RASA1 protein expression, which was down-regulated with an siRNA targeting RASA1 in tumor tissues (Figure 7D). Furthermore, Western blot and IHC results revealed that the up-regulation of RASA1 greatly reduced the expression of Ki67, which was up-regulated when RASA1 was silenced (Figure 7E,F).

Currently, 14 RasGAPs have been identified in mammals [20], and several of them are epigenetically modified in various cancers [21], including NF1 [22], DAB2IP [23], RASAL1 [24], and RASAL2 [25]. Within the RasGAP family, the RASA subfamily is composed of five RasGAPs [21]: RASA1/p120GAP, three GAP1 members (RASA2/GAP1m, RASA3/GAP1IP4BP and RASA4/CAPRI), and RASA5/SynGAP. The RASA group inversely modulates Ras signaling and could therefore curb tumor formation in cancer pathogenesis. The RASA1 inactive mutation, called a driver mutation, together with the NF1 mutation defines a genetically new subclass of non-small cell lung cancer [26,27]. Allelic deletion of RASA1 is common in triple-negative breast cancer (BC) with TP53 mutations, and its decrement gives rise to the malignant phenotype of breast cells [28]. However, the role of RASA1 in RCC remains unclear. The present study disclosed that RASA1 was down-regulated in RCC tissues and cells. Furthermore, we found that the up-regulation of RASA1 could inhibit cell proliferation and migration while promoting cell apoptosis. Caspases are cysteine-containing aspartate-specific hydrolases and are the primary executor of cell apoptosis [29]. Caspase-3 is a key protease in mammalian cell apoptosis and is one of the most important proteins in caspase family [30,31]. Here, we demonstrated that RASA1 could promote cell apoptosis by regulating Caspase-3 expression.

MicroRNAs, non-coding RNAs with 19–22 nucleotides, inversely modulate translation and are implicated in a number of cellular processes. Augmentation of certain miRNAs has been intimately correlated with diverse diseases, including diabetes, obesity, cancers, and cardiovascular diseases [32,33]. Aberrant modulation of miRNAs is an important component in cancers due to both the oncogenic and tumor-suppressive functions of miRNAs [34]. miR-223 is found in the q12 segment of the human X chromosome, whose expression is modulated by transcription factors such as C/EBPa, PU.1, and NFI-A [35]. miR-223 is implicated in modulating functions of the cardiovascular system, human immune system, muscle, and bone [36]. Additionally, miR-223 also functions as a tumor suppressor gene in different tumors. A study by Fabris et al. ascertained that overexpression of miR-223 could inhibit BC relapse through the mediation on the EGF signaling pathway, implying that miR-223 overexpression also has crucial impacts on BC progression [37]. Additionally, it has been ascertained that miR-223-3p promotes cell proliferation and metastasis by down-regulating SLC4A4 in RCC [15]. Here, the expressions of RCC-correlated miRNAs were examined upon RASA1 overexpression and only miR-223-3p had altered expression. In order to confirm whether RASA1 regulates miR-223-3p in RCC cells, a rescue experiment was conducted. We found that RASA1 inhibits cell migration and proliferation but boosts cell apoptosis by inhibiting miR-223-3p expression.

Reports show that miRNAs participate in cell differentiation, apoptosis, and proliferation by targeting downstream mRNAs [38]. It was found in this work that F-box/WD repeat-containing protein 7 (FBXW7) is a target gene of miR-223-3p, and is expressed at lower levels in RCC tissues and cell lines. FBXW7 is pivotal for modulating cell growth and differentiation as well as cell cycle progression in human cells, and its decrement is able to drive cancer cells to proliferate [39,40]. Deletion of FBXW7 results in poor prognosis in multiple cancers [41,42]. Several recent studies have shown that FBXW7 is the target of miRNAs and functions in the regulation of cancers. For instance, miR-223 regulates acute lymphoblastic leukemia by suppressing FBXW7 expression [43]. Additionally, miR-27a promotes lung cancer cell growth by repressing FBXW7. These results imply that FBXW7 acts as a tumor suppressor in human cancers [44]. In the present study, we confirmed an association between miR-223-3p and FBXW7 by dual-luciferase reporter and found that miR-223-3p regulates the expression of FBXW7 in RCC cell lines. Moreover, we also found FBXW7 was overexpressed in RCC tumor tissues and that miR-223-3p and FBXW7 mRNA expression is negatively correlated, while RASA1 and FBXW7 mRNA levels are positively correlated. Hence, miR-223-3p may directly regulate FBXW7. Finally, we demonstrated that RASA1 functions in RCC by regulating miR-223-3p. Taken together, we hypothesize that RASA1 has a role in the development of RCC by decreasing miR-223-3p expression, thus increasing FBXW7 expression.

In conclusion, RASA1 is lowly expressed in RCC tissues and cell lines and has a role in RCC occurrence and progression by inhibiting the expression of miR-223-3p and enhancing the expression of FBXW7. However, the present study has several limitations. First, the sample size was small. Further studies using a large sample size will validate the results shown here. Second, the present study found that RASA1 can regulate miR-223-3p, but its specific mechanism has not yet been elucidated. Future work will aim to elucidate the mechanism behind RASA1 regulation.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the National Natural Science Foundation of China [grant number 81760452].

All authors have participated in study design and manuscript preparation. Rui-Li Zhang performed all experiments, Ainiwaer Aimudula, and Jiang-Hong Dai performed the statistical analysis and drafted the manuscript. Yong-Xing Bao designed the work and revised the manuscript. All authors have approved the final article.

This study protocol was approved by The First Affiliated Hospital of Xinjiang Medical University. All patients provided written informed consent.

FBXW7

F-box/WD repeat-containing protein 7

lncRNA

long non-coding RNA

RASA1

RAS p21 protein activator 1

RCC

renal cell carcinoma