Ribosomal protein L22‐like1 promotes prostate cancer progression by activating PI3K/Akt/mTOR signalling pathway

Abstract Prostate cancer (PCa) is one of the most common malignancies in men. Ribosomal protein L22‐like1 (RPL22L1), a component of the ribosomal 60 S subunit, is associated with cancer progression, but the role and potential mechanism of RPL22L1 in PCa remain unclear. The aim of this study was to investigate the role of RPL22L1 in PCa progression and the mechanisms involved. Bioinformatics and immunohistochemistry analysis showed that the expression of RPL22L1 was significantly higher in PCa tissues than in normal prostate tissues. The cell function analysis revealed that RPL22L1 significantly promoted the proliferation, migration and invasion of PCa cells. The data of xenograft tumour assay suggested that the low expression of RPL22L1 inhibited the growth and invasion of PCa cells in vivo. Mechanistically, the results of Western blot proved that RPL22L1 activated PI3K/Akt/mTOR pathway in PCa cells. Additionally, LY294002, an inhibitor of PI3K/Akt pathway, was used to block this pathway. The results showed that LY294002 remarkably abrogated the oncogenic effect of RPL22L1 on PCa cell proliferation and invasion. Taken together, our study demonstrated that RPL22L1 is a key gene in PCa progression and promotes PCa cell proliferation and invasion via PI3K/Akt/mTOR pathway, thus potentially providing a new target for PCa therapy.

Recently, ribosomal proteins were reported to be involved in cancer progression. 8,9 Ribosomal protein L22-like1 (RPL22L1), a component of the 60S subunit, is regulated by RPL22. 10 Several studies showed that RPL22L1 is involved in the regulation of cellular functions, including cell proliferation, migration, invasion and apoptosis, and is associated with ovarian cancer, colorectal cancer and hepatocellular carcinoma. [10][11][12] In addition, Liang et al. found that RPL22L1 may be a promising biomarker for PCa. 13 However, the role and potential mechanism of RPL22L1 in PCa remain unclear.
Various molecular mechanisms contribute to the progression of PCa, among which PI3K/Akt/mTOR pathway is one of the primary causes. 14,15 PI3K/Akt/mTOR pathway has been reported to be related to the formation and progression of PCa, biochemical recurrence after radical prostatectomy, and drug-resistance. 16,17 Additionally, several studies found that this signalling pathway's inhibitor has the potential to be a therapeutic agent for hormonesensitive PCa and CRPC. [18][19][20] Accordingly, it is necessary to further investigate the mechanism of PI3K/Akt/mTOR pathway in PCa progression, providing clinical benefit to patients with PCa.
The purpose of this study is to investigate the role of RPL22L1 in PCa progression and its potential mechanisms, thus providing a potential new target for PCa therapy.

| Cell culture and lentivirus transfection
The PCa cell lines (LNCaP, 22Rv1, DU145 and PC3) were purchased from Shanghai Cell Bank and cultured in DMEM supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C. The stable cell lines shRPL22L1-PC3 and overexpressed RPL22L1-LNCaP were obtained by transfection of RPL22L1 shRNA lentiviral particles and RPL22L1 lentiviral activation particles, respectively.

| CCK-8 and cell colony formation
For the CCK-8 assay, relative cell viability was monitored after seeding PC3 and LNCaP cells (2000 cells per well) in 96-well plates, respectively, as described in our previous study. 21 The values of light absorbance were tested by the microplate reader. For the assay of cell colony formation, as in previous studies, 22

| Wound-healing and invasion
For the wound-healing assay, PC3 cells were cultured in serum-free medium, and then, a micropipette tip was used to create a cell-free space as previously described. 23 Photographs were taken with a microscope after 48 h. For the invasion assay, as described in the previous study, 24 approximately 2.5 × 10 4 PC3 and LNCaP cells were seeded separately into transwell chambers, and after 24 h of culture, the cells that passed through the membrane were stained with 0.5% crystal violet solution and counted.

| Histopathology observation and immunohistochemistry
The experiments involving human tissue specimens in this study were in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Histopathology observation was performed as previously described. 25 Briefly, tissue samples were fixed in 10% neutral buffered formalin and then embedded in paraffin. Afterwards, the samples were cut to a thickness of approximately 4 μm and subjected to haematoxylin-eosin (HE) staining. Immunohistochemistry (IHC) was performed as described in our previous study. 26 In short, tissue samples were treated with

| In vivo assay
In vivo assay was approved by the Animal Ethics Committee of Weifang Medical University. Five-week-old male nude mice (BALB/c) were divided into two groups and subcutaneously injected with control PC3 cells (5 × 10 6 cells/mouse) and RPL22L1 low-expressing PC3 cells (5 × 10 6 cells/mouse), respectively. The growth of the tumour was measured every 1 week, and tumour volume was calculated using the following formula: volume (mm 3 ) = 4π/3 × (width/2) 2 × (length/2). After 5 weeks, mice were executed under deep anaesthesia. Tumours were collected, weighed and subjected to histopathology observation and IHC testing.

| Statistical analysis
Data were analysed by anova or independent sample t-test using SPSS 19.0 software. Experiments were repeated three times. Data were expressed as mean ± SD, and p < 0.05 represents statistical significance.

| Identification of RPL22L1 by bioinformatics tool and IHC
The gene expression profile (GSE55945) was downloaded from GEO and analysed by GEO2R tool, which showed that RPL22L1 expression was significantly elevated in PCa tissues ( Figure 1A).
Subsequently, RPL22L1 expression was analysed by Oncomine, UALCAN and GEPIA database. The results showed that the expression of RPL22L1 was significantly higher in PCa tissue than in the normal prostate tissue ( Figure 1B,C), and RPL22L1 expression was strongly associated with PCa prognosis ( Figure 1D). In addition, RPL22L1 expression was further validated by IHC. The results suggested that RPL22L1 was localized in the nucleus and cytoplasm and was significantly more expressed in the PCa tissue compared with the normal prostate tissue ( Figure 1E). The results above suggested that RPL22L1 may be a pivotal gene in the progression of PCa. the RPL22L1 overexpression cell line. As shown in Figure 2B, the expression of RPL22L1 in RPL22L1-shRNA group was significantly lower than that in the control group, while the expression of RPL22L1 in RPL22L1-overexpression group was significantly higher than that in the control group. These results proved that RPL22L1 knockdown and overexpression cell lines were successfully established.

| RPL22L1 promotes PCa cells proliferation, migration and invasion
To assess the role of RPL22L1 in PCa cells, CCK-8, colony formation, wound-healing and transwell assay were carried out. As shown in Figure 3A,B, knockdown of RPL22L1 significantly inhibited PC3 cell proliferation, whereas overexpression of RPL22L1 significantly promoted LNCaP cell proliferation (p < 0.05). Similarly, wound-healing results suggested that knockdown of RPL22L1 significantly inhibited the migratory ability of PC3 cells (p < 0.05, Figure 3C). However, LNCaP cells adhered to each other and wound-healing assay was difficult to perform, so the data for LNCaP were not shown. Transwell assay results proved that the invasion ability was remarkably suppressed by RPL22L1 knockdown, while significantly increased by RPL22L1 overexpression (p < 0.05, Figure 3D). In addition, apoptosis assay showed that knockdown of RPL22L1 elevated apoptosis in PC3 cells compared with the control (p < 0.05, Figure S1). Taken together, these results demonstrated that RPL22L1 promotes PCa cells proliferation, migration and invasion.

| RPL22L1 activates PI3K/Akt/mTOR pathway in PCa cells
Given that PI3K/Akt/mTOR signalling pathway is closely related to PCa progression, we examined whether RPL22L1 activated PI3K/Akt/ mTOR pathway in PCa cell lines. As shown in Figure 4, the expression of PI3K, Akt and mTOR was unchanged, but their phosphorylation level was decreased significantly in RPL22L1-knockdown PC3 cells compared with the control group (p < 0.05). Conversely, in RPL22L1overexpressed LNCaP cells, the level of p-PI3K, p-Akt (Ser473) and p-mTOR was elevated remarkably (p < 0.05). These data demonstrated that RPL22L1 activates PI3K/Akt/mTOR pathway in PCa cells.

| RPL22L1 promotes PCa cell growth and invasion in vivo
To further verify the effect of RPL22L1 on PCa cell growth and invasion in vivo, xenograft tumour assay was carried out. As  shown in Figure 5A,B, PC3 cells with low RPL22L1 expression showed a weaker proliferative capacity. The tumour volume and weight were significantly decreased in the RPL22L1 low expression group compared to the control group (0.36 ± 0.19 vs. 0.63 ± 0.27 g, p < 0.05). IHC analysis revealed that the phosphorylation levels of Akt and mTOR were significantly lower in the RPL22L1 low expression group than the control group ( Figure 5C).
Additionally, histopathological analysis showed that tumour cells in the RPL22L1 low expression group had a weaker ability to invade into adjacent tissues compared with the control group, indicating that RPL22L1 enhanced PCa cell invasion ( Figure 5D).
To sum up, these data proved that RPL22L1 promotes PCa cell growth and invasion in vivo.

| RPL22L1 contributes to PCa progression through PI3K/Akt/mTOR pathway
To determine whether RPL22L1 exerts oncogenic effects in PCa through PI3K/Akt/mTOR pathway, LY294002, an inhibitor of PI3K/ Akt pathway, was used to block this pathway. As shown in Figure 6A,

| DISCUSS ION
RPL22L1 has been suggested to play an essential role in the progression of various tumours. [10][11][12][13] However, its function and underlying molecular mechanism in PCa are still poorly understood. In this study, we found that RPL22L1 was significantly more expressed in PCa tissues than in normal prostate tissues. Further evidence from cell function experiments indicated that RPL22L1 significantly promoted PCa cell proliferation, migration and invasion. In addition, in vivo experiments showed that the low expression of RPL22L1 inhibited the growth and invasion of PCa cells in nude mice.
In recent years, there is increasing evidence that the abnormal expression of ribosomal proteins is closely related to tumorigenesis. 8,28 Clarifying the role and mechanism of ribosomal proteins in tumour progression will provide new targets for tumour therapy.
RPL22L1, a component of the ribosomal 60 S subunit, was determined for the first time in mouse brain tissue. 29 Several studies in related fields have demonstrated that RPL22L1 is highly expressed in various tumours, including PCa, 13 colorectal cancer 12 and ovarian cancer. 11 Likewise, our results showed that RPL22L1 expression was significantly upregulated in PCa and was strongly associated with patient prognosis, suggesting that RPL22L1 may be a pivotal gene in the PCa progression.
Currently, there are limited studies on the function of RPL22L1 in cancer progression. It has been reported that RPL22L1 promotes cell proliferation, migration and invasion in colorectal cancer 12 and hepatocellular carcinoma. 10 Liang et al. also found that the low expression of RPL22L1 inhibited PC3 cell proliferation and invasion in vitro. 13 It should be noted that only PC3 cell was used to construct the low-expressing cell line in their study, so the results need further confirmation. In the present study, PC3 and LNCaP cells were screened from four PCa cell lines to construct RPL22L1 low and overexpressed cell lines, respectively. PI3K/Akt/mTOR pathway is a critical carcinogenic pathway involved in tumorigenesis and progression in a wide variety of tumours, including PCa, 14   found that RPL22L1 activated ERK pathway to induce atypical epithelial-to-mesenchymal transition (EMT) in hepatocellular carcinoma. 10 We speculate that ERK pathway may also be involved in PCa progression promoted by RPL22L1. Taken together, these results demonstrated that RPL22L1 contributes to PCa cell proliferation and invasion primarily by PI3K/Akt/mTOR pathway. However, it is not known how RPL22L1 regulates PI3K/Akt/mTOR pathway, which will be further investigated.
In conclusion, our study demonstrated that RPL22L1 is a key promoter of PCa and promotes PCa cell proliferation and invasion through PI3K/Akt/mTOR pathway, thus potentially providing a new target for PCa therapy.

ACK N O WLE D G E M ENTS
The study was supported by Natural Science Foundation of Shandong Province (ZR2020MH263, ZR2014CL034) and National Natural Science Foundation of China (11802209).

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 on request from the corresponding author.