Ring finger protein 19A is overexpressed in non‐small cell lung cancer and mediates p53 ubiquitin‐degradation to promote cancer growth

Abstract The expression pattern, biological functions and the related mechanisms of the ring finger protein 19A (RNF19A) in non‐small cell lung cancer (NSCLC) remain poorly understood. This study aimed to explore the role of RNF19A, as well as the underlying potential mechanism, in the development of NSCLC. Here, we found that RNF19A was overexpressed in NSCLC tissues, and RNF19A expression in NSCLC tissue samples was associated with NSCLC carcinogenesis and poor outcome. RNF19A promoted the proliferation of NSCLC cells and inhibited apoptosis. RNF19A reduced p53, p21 and BAX expression and induced Cyclin D1, CDK4, CDK6 and BCL2 expression. The inhibitory effect of RNF19A knockdown on proliferation was partially rescued by p53 silencing. RNF19A interacted with p53, shortened p53 half‐life and mediated p53 ubiquitin‐degradation. Collectively, we suggest that RNF19A plays a critical oncogenic role in lung carcinogenesis by disrupting the function of p53. RNF19A may serve as a new biomarker and/or target for NSCLC management.

and cycle control. 7 Misregulation of RBR proteins often leads to diverse diseases, including various cancers. For instance, RBR E3 ligases, including Parkin, 8 RNF144A, 9,10 RBCK1 11,12 and RNF216 13 are involved in lung, breast and colorectal cancers through ubiquitinmediated degradation, indicating the vital function of RBR proteins in carcinogenesis.
Ring finger protein 19A (RNF19A), also known as Dorfin, is a poorly understood member of RBR E3 ligases, which carries three highly conserved domains including two RING finger motifs and an IBR motif at its N terminus. 14 The biological functions and action mechanisms of RNF19A remain largely unknown. Available literature confirms the independent function of RNF19A by controlling protein quality, and its potential involvement in the development of neurodegenerative diseases. [15][16][17][18] It was reported that the mRNA levels of RNF19A increases in the serum of patients suffering from prostate cancer. 19 However, to date, there are no studies on the intracellular RNF19A expression pattern, as well as its direct role and mechanism, in cancers. Here, we aimed to explore the biological functions, clinical application and underlying molecular mechanisms of RNF19A in NSCLC.

| Bioinformatic analyses
Differences in the transcriptional expression of RNF19A between NSCLC tissues and their corresponding non-tumour samples were evaluated based on the data obtained from the Oncomine database (http://www.oncom ine.org) 20 The conditions were set as follows: Data type, mRNA; P-value < 0.01; fold change >1.5; gene rank, all.
The prognostic value of RNF19A in NSCLC was analysed via Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cance r-pku.cn/) 21 The median gene expression level, 95% confidence intervals (CIs), HRs and P values were retrieved from the GEPIA database. A P-value < 0.05 was considered to denote statistically significant results.

| Immunohistochemistry (IHC)
The resected specimens were fixed using a 10% formaldehyde solution, embedded in paraffin and cut into 4 μm thick serial sections.
After incubating at 70℃ for 2 hours, the sections were dewaxed and rehydrated in xylene and graded alcohol. A citrate solution (0.01 mol/L) (Maixin-Bio) was used for antigen retrieval under high temperature and pressure for 3 minutes. The activity of endogenous peroxidase was blocked via incubation with 0.3% H 2 O 2 for 20 minutes, and then, each section was blocked in 5% goat serum for 30 minutes.
All the sections were incubated with a drop of anti-RNF19A rabbit polyclonal antibody (1:50 in 2% BSA; #PA5-54861; Invitrogen, Carlsbad, CA, USA) overnight at 4°C. After soaking in 1 × PBS thrice for 5 minutes, the sections were incubated with the substrate provided in the Elivision TM super HRP (Mouse/Rabbit) IHC Kit (KIT9921; MaiXin) according to the manufacturer's instructions. Next, 3,3'-diaminobenzidine (DAB) staining and haematoxylin counterstaining were performed. Finally, the sections were dehydrated using gradient ethanol, cleared by dimethylbenzene and mounted with a neutral gum seal tablet. Immunohistochemical staining results were evaluated by two experienced pathologists in a double-blinded manner. The scoring system for positively stained cancer cells was established as described previously. 22 Briefly, based on the staining intensity, cells characterized by no staining, weak staining, moderate staining and strong staining were scored as 0, 1, 2 and 3, respectively. According to the range of positive cell distribution, tissues characterized by 0%, 1-30%, 31-70% and 71-100% positive cells were scored as 0, 1, 2 and 3, respectively. The two scoring results were multiplied to give a total score ranging from 0 to 9. Tissues with total scores of 0-3 were considered to show low expression of RNF19A, and those with scores of 3-9 were considered to show high expression of RNF19A.

| Cell culture, transfection and transduction
Human non-small cell lung carcinoma cell lines A549, H292, H460, H661, H1299 and SK-MES-1 were purchased from the Cell Bank of the China Academy of Sciences (Shanghai, China). Human bronchial epithelial cells (HBE) were obtained from the ATCC. According to the ATCC protocol, HBE cells were grown in DMEM, while A549, H292, H460, H661 and H1299 cells were grown in RPMI 1640 medium, and SK-MES-1 cells were cultured in MEM. Ten per cent foetal bovine serum was added to all media, and all cells were cultured at 37°C in a 5% CO 2 -containing atmosphere. In vitro transfection of two small interfering RNAs (siRNAs) and RNF19A expression plasmids were performed using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer's protocol. Cells were transfected with siRNA targeting RNF19A and scrambled control siRNA (Ribobio, Guangzhou, China) for 72 hours. The siRNA sequence against RNF19A was as follows: siRNF19A-1, 5'-GATCCATTCTGAATTCCTA-3'; siRNF19A-2, 5'-GCAAGTAGATATTGAGTCA-3'. An RNF19A DNA fragment was cloned into the PCMV3 vector, containing a FLAG sequence, which was obtained from Sino Biological.

| MTT and colony formation assays
The viability of lung cancer cells was analysed using the MTT assay.
Briefly, the treated A549, H460 and H1299 cells were seeded in 96 well microplates at a density of 3000, 1500 and 2000 cells per well, respectively. The cells were incubated with a 10% MTT solution (100 µl/well) for 3.5 hours, and the absorbance was measured at 490 nm. Measurements were taken once per day for five consecutive days. For the colony formation assay, approximately 500 cells/well were plated into a six-well plate and incubated for 8-12 days. The colonies were fixed with cold methanol for 15 minutes, stained with 0.5% crystal violet solution for 30 minutes, and finally, colonies consisting of more than 50 cells were counted using ImageJ.  and anti-GAPDH (#60004-1-Ig) antibodies were obtained from Proteintech. All primary antibodies were diluted 1:1000, except for GAPDH which was used at 1:10 000.

| Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (TransGen Biotech, Beijing, China), and the cDNA was reverse transcribed using the FastQuant RT Kit (TIANGEN Biotech, Beijing, China) following the manufacturer's instructions. qRT-PCR was performed using a 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). The reaction conditions for qRT-PCR were as follows: Pre-degeneration at 95°C for 15 minutes; followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing at 60°C for 32 seconds. GAPDH was used as an internal control, and relative gene expression was calculated using the 2 −ΔΔCt method.
Sequences of designed primers were as follows: For RNF19A,

| Protein half-life detection
After being transfected with 75 nmol/L siRNF19A or siControl for 24 hours, A549 and H460 cells were treated with 3 μg/ml cycloheximide (CHX) and collected after 0, 0.5, 1, 1.5, 2 and 2.5 hours of treatment. Protein was extracted for SDS-PAGE and Western blotting using anti-RNF19A or anti-p53 antibody. GAPDH served as an internal reference and the half-life of p53 was estimated.

| Co-immunoprecipitation and ubiquitination assays
Co-immunoprecipitation and ubiquitination assays were carried out as described previously. 23 Briefly, the cell lysates were blocked with 40 μl Protein A + G agarose beads (Beyotime Biosciences) at 4°C for 2 hours and immunoprecipitated with 4-10 μg mouse pri-

| Statistical analysis
The SPSS 19.0 statistical software (SPSS Inc) and the GraphPad Prism 7.0 software (GraphPad Software, Inc) were used to analyse the experimental data. The correlation between RNF19A expression and clinicopathological characteristics was examined using the F I G U R E 1 RNF19A is highly expressed in NSCLC tissues and correlates with poor prognosis. A, Expression of RNF19A in eight matching NSCLC and non-tumour tissues was detected via Western blotting. T, tumour tissue; N, non-tumour tissue. *P < .05. B, IHC staining representative images of RNF19A in NSCLC and non-tumour specimens. a, bronchiole tissue; b, alveolar tissue; c, squamous cell carcinoma tissue; d, adenocarcinoma tissue. C, RNF19A mRNA expression comparison between NSCLC and non-tumour tissues from the Oncomine database. *P < .05, **P < .01 and ***P < .001. D, RNF19A expression correlates with poor prognosis in patients with NSCLC, obtained from GEPIA data sets. *P < .05, ** P < .01 and ***P < .001. E, Immunoblot of RNF19A confirms high expression of RNF19A in NSCLC cell lines. IHC, immunohistochemistry; NSCLC, non-small cell lung cancer chi-squared test. Data were presented as the mean ± standard error (SE), and quantitative analysis was performed using Student's t test if not stated otherwise. The level of significance was set at *P < .05, ** P < .01 and ***P < .001.

| RNF19A is highly expressed in NSCLC and is associated with poor patient outcome
To explore the RNF19A expression pattern in NSCLC, we examined the expression of RNF19A in matching cancerous and normal tissues from eight patients with NSCLC via Western blotting.
RNF19A expression levels were notably increased in six out of eight NSCLC tissues compared with those in the normal adjacent tissues ( Figure 1A P < .05)

| RNF19A represses p53 expression and regulates p53 downstream signalling
Our results showed that RNF19A supported the growth of NSCLC cells. We further explored the possible mechanisms of RNF19A-  Figure 3A). These data suggested that RNF19A might inhibit the p53 signalling pathway in NSCLC cells.

| RNF19A exerts tumour-promoting effects through p53
The above results suggested that RNF19A promoted cellular proliferation and inhibited apoptosis in A549 and H460 cells, which en- To further confirm whether p53 is essential for the oncogenic role of RNF19A, we carried out a rescue experiment by repressing p53 expression in RNF19A siRNA-transfected cells. As shown in Figure 4D-E, p53 knockdown dramatically reduced the proliferation inhibition effect of RNF19A knockdown in A549 and H460 cells. In addition, p53 knockdown reversed the effect of RNF19A knockdown on the expression of p53 downstream proteins ( Figure 4F). These data suggested that RNF19A promoted NSCLC growth at least partially through p53.

| RNF19A interacts with p53 to promote p53 ubiquitination
The above results suggested that RNF19A knockdown and overexpression increased and decreased p53 expression at the protein level, respectively, while p53 expression did not change, indicating that RNF19A might regulate p53 at the post-transcriptional level.
As RNF19A is an E3 ubiquitin ligase that can achieve ubiquitination independently, we speculated that RNF19A might interact with p53 and participate in its ubiquitination and degradation. To confirm this hypothesis, RNF19A-silenced A549 and H460 cells were treated with the proteasome inhibitor MG132, and p53 expression was detected via Western blotting. In line with our hypothesis, MG132 rescued the elevated expression of p53 due to RNF19A knockdown ( Figure 5A). This suggested that RNF19A promoted p53 degradation via the proteasome pathway. We next examined the half-life of endogenous p53 in A549 and H460 cells which were treated with CHX after RNF19A siRNA transfection.
We observed that endogenous p53 in RNF19A-knockdown cells had a longer half-life than in the scramble siRNA-treated group ( Figure 5B).
We then performed endogenous co-immunoprecipitation in A549 cells to determine whether RNF19A interacts with p53. The results showed that precipitated p53 immunocomplexes included RNF19A ( Figure 5C).
Next, plasmids carrying exogenous FLAG-RNF19A and p53 were co-transfected into A549 cells; then, FLAG and p53 were immunoprecipitated, respectively. We found that exogenous RNF19A and p53 co-immunoprecipitated in both assays ( Figure 5D). To further assess  whether RNF19A regulates p53 ubiquitination, RNF19A was overexpressed or knocked down in A549 cells that were pre-treated with MG132 to block proteasome-dependent degradation. We observed that RNF19A overexpression distinctly induced p53 ubiquitination, while RNF19A knockdown reduced p53 ubiquitination ( Figure 5E).

| D ISCUSS I ON
In this paper, we have identified RNF19A as a novel onco-driver in NSCLC based on two observations: (1) RNF19A was highly expressed in NSCLC tissues, and its overexpression was positively correlated with poor patient outcome in NSCLC; (2) RNF19A increased NSCLC cell proliferation and survival. These results suggested that RNF19A promoted NSCLC development. Previous work has confirmed that several RBR E3 ligases are involved in the regulation of tumour progression by modulating the degradation of tumour promoters or suppressors. 24 However, the expression pattern, functional implication and prognostic value of RNF19A in NSCLC have been poorly defined.
The canonical homo-tetrameric p53a protein, also known as p53 or the 'Guardian of the genome', 25 is a powerful and well-known tumour suppressor, encoded by TP53. p53 is a vital transcription factor that plays a crucial role in several cell cycle regulation pathways and induces apoptosis when necessary. 26 The loss of p53 usually leads to tumorigenesis [27][28][29] and promotes the occurrence and development of tumours. 30,31 In this study, we showed that RNF19A not only decreased p53 expression at the protein level but also regulated the downstream signalling of p53, suggesting that RNF19A might promote NSCLC development by decreasing p53 function. The investigation of the potential mechanism indicated that RNF19A (1) promoted prolif- kinds of BCL2 pro-apoptotic family members. 35  Our study also has some limitations. First, we were unable to analyse the prognosis of our collection of specimens, because the original specimens were collected between 2014 and 2016, and not enough time had passed to collect information. Therefore, prognostic information could only be downloaded from biological databases for further analysis. Second, we have evidence that p53 is at least partially involved in lung tumorigenesis by RNF19A; however, we should not rule out other targets of RNF19A in this complex biological process.
In conclusion, our study has revealed the clinical significance and biological function of RNF19A in NSCLC. RNF19A decreases p53 expression and its downstream signalling, binds to p53 and promotes its ubiquitination, thereby promoting NSCLC growth and progression. RNF19A may thus act as a new biomarker and target for NSCLC prognosis and therapy.

ACK N OWLED G EM ENTS
We would like to thank Editage (www.edita ge.cn) for Englishlanguage editing.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Yu Cheng
https://orcid.org/0000-0001-5528-2058 Xueshan Qiu https://orcid.org/0000-0001-9481-8249 F I G U R E 5 RNF19A interacts with p53 and promotes p53 ubiquitination. A, RNF19A-silenced A549 and H460 cells were treated with MG132 and DMSO (control). RNF19A and p53 expression levels in the treated cells were detected via Western blotting. B, RNF19A-silenced A549 and H460 cells were treated with CHX at the indicated time points. RNF19A and p53 expression levels in the treated cells were detected via Western blotting and the half-life of p53 was calculated. C, Endogenous p53 and RNF19A form a protein complex in A549 cells. D, RNF19A co-immunoprecipitates with p53 in A549 cells transfected with p53 and FLAG-tagged RNF19A. E, A549 cells transfected with RNF19A expression plasmid or the RNF19A siRNA along with HA-ubiquitin (Ub). Levels of p53 ubiquitination were detected via immunoprecipitation using the anti-p53 antibody, followed by anti-HA immunoblotting