LncRNA RHPN1‐AS1 inhibition induces autophagy and apoptosis in prostate cancer cells via the miR‐7‐5p/EGFR/PI3K/AKT/mTOR signaling pathway

LncRNA RHPN1‐AS1 (RHPN1‐AS1) has been confirmed to promote tumor progression in multiple cancers and is upregulated in prostate cancer (PCa), but whether it has an effect on PCa progression remains unclear. In this study, we found that PCa patients with high RHPN1‐AS1 expression had a shorter survival time, and RHPN1‐AS1 was significantly upregulated in PCa tissues and cells. Based on informatics analysis we predicted that miR‐7‐5p binds to 3′UTR of RHPN1‐AS1 and epidermal growth factor receptor (EGFR) and verified it by luciferase reporter gene assay. Subsequently, we transfected PCa cells with RHPN1‐AS1 overexpression vector (RHPN1‐AS1), knockdown plasmids (sh‐RHPN1‐AS1) and/or miR‐7‐5p mimics or inhibitor and/or overexpression vector (EGFR) or small interfering RNA of EGFR (si‐EGFR) or its control, and found that overexpression of RHPN1‐AS1 inhibited miR‐7‐5p expression and promoted EGFR expression, silencing RHPN1‐AS1 inhibited proliferation and invasion, and induced G2/M arrest, apoptosis and autophagy in PCa cells. 3MA (an inhibitor of autophagy)‐mediated autophagy inhibition attenuated RHPN1‐AS1 inhibition‐induced apoptosis. Overexpression miR‐7‐5p or silencing EGFR promoted LC3‐I to LC3‐II conversion, enhanced autophagy activity, induced cleaved‐caspase‐3 expression and apoptosis in PCa cells. Furthermore, overexpression of RHPN1‐AS1 promoted phosphorylation of phosphatidylinositol 3‐kinase (PI3K), AKT and mTOR, inhibited LC3‐I to LC3‐II conversion and reduced apoptosis in PCa cells, while GSK2126458 (an inhibitor of PI3K) reversed the effect of RHPN1‐AS1 on PCa cells. In summary, RHPN1‐AS1 acted as a ceRNA of miR‐7‐5p to upregulate EGFR expression, silencing RHPN1‐AS1 suppressed PCa tumor progression by inducing autophagy and apoptosis in PCa cells through the miR‐7‐5p/EGFR/PI3K/AKT/mTOR pathway.


| INTRODUCTION
Prostate cancer (PCa) is considered the most common urological malignancy in males and is the leading cause of cancer-related death in the world. Prostate cancer ranks 7th for incidence and 10th for mortality among male malignancies in China. 1,2 However, as China begins to enter an aging society, the impact of PCa on the lifestyle and life quality of Chinese men has become increasingly prominent.
Moreover, the incidence of PCa has been on the rise in recent years due to changes in diet and lifestyle. 3,4 Approximately 10% of newly diagnosed patients present with locally advanced PCa, and 5% showed distant metastasis because of the insidious symptoms. 5 Although improvements in PCa diagnostic strategies and multiple therapeutic approaches have led to a significant reduction in PCa related deaths over the past three decades, the prognosis of patients is still not optimistic. Therefore, early diagnosis and intervention of PCa will help to reduce its harm to patients and improve the therapeutic effect. However, even with the in-depth study of PCa, the pathogenesis and causes of PCa have not been fully understood. 6 This situation has led a large number of scholars to continuously search for molecular indicators for early diagnosis of PCa, molecular targets for PCa treatment and molecular markers for the prognosis of PCa.
In recent years, long non-coding RNAs (lncRNAs), mainly transcribed from the genomic intergenic regions have become a research focus in cancers transcriptome. Functionally, they have been demonstrated to be participants in various biological processes through various mechanisms from transcription level to post-transcription level. 7 The oncogenic or tumor suppressive roles of lncRNAs have been well characterized in tumorigenesis. 8 To date, several lncRNAs have been defined as PCa specific. 9 Nevertheless, investigation of the function and mechanism of novel lncRNAs is necessary to find functional biomarkers in PCa progression. RHPN1-AS1 is a lncRNA that was differentially expressed in PCa patients and the potential function of RHPN1-AS1 in multiple tumor progression has been reported. 10 For example, RHPN1-AS1 increased the expression of leucine zipper/EF hand transmembranecontaining-1 (LETM1) in ovarian cancer via inhibiting miR-596 expression to activate the FAK/PI3K/AKT signaling pathway to promote tumorigenesis and metastasis. 11 Other studies also demonstrated the oncogenic role of RHPN1-AS1 in cholangiocarcinoma, 12 cervical cancer, 13 colorectal cancer, 14 gastric cancer, 15 and hepatocellular carcinoma. 16 Nevertheless, the molecular mechanism of RHPN1-AS1 regulation in PCa tumor progression remains unclear.
Autophagy, a cellular self-eating process, is a catabolic process that delivers cellular proteins and organelles to lysosomes for subsequent degradation. 17 Autophagy plays a key role in the metabolic regulation of cells. 18 Although the process of autophagy improves cell survival under harsh conditions, in many cases autophagy can induce autophagic programmed cell death, also known as type II cell death. 19,20 Autophagy and apoptosis pathways are interconnected, and loss of homeostatic balance between these two pathways may lead to cell death. 18,19,21 Previous reports have shown that the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, as a key regulator of autophagy, is involved in the initiation and promotion of a range of pathological diseases, including various tumors. 22 In parallel, reports have shown that the PI3K/AKT-mTOR signaling pathway is upregulated by loss of phosphatase in 30%-50% of patients with PCa has been reported. 23 Targeting PI3K/ AKT-mTOR mediated autophagy is an important therapeutic strategy for PCa and is important for inhibiting tumor cell growth and metastasis in PCa. 23,24 Therefore, it is necessary to gain insight into the molecular mechanisms of autophagy and apoptosis in PCa, search for novel biomarkers for detection, and improve current therapeutic strategies.
In this study, we explored the effect of RHPN1-AS1 on the proliferation, apoptosis and autophagy of PCa cells and its mechanism, expecting to provide a new theoretical basis for the treatment of PCa.

| Tissue samples
Twenty-one pairs of PCa tissue samples and adjacent normal tissues were collected from the Second Affiliated Hospital of Xi'an Jiaotong University between June 2019 and December 2020. This study was approved by the Ethics Committees of the Second Affiliated Hospital of Xi'an Jiaotong University, and was carried out in accordance with the principles of the Declaration of Helsinki. Each participant did not receive radiotherapy and chemotherapy before tissue collection and signed written informed consent before the study. All samples were stored at À80 C until required for further analysis.

| Cell culture
Human PCa cell lines PC-3, LNCaP, DU145 and normal prostate epithelial cell RWPE-1 were purchased from American Type Culture Collection (ATCC; Manassas, VA). All cells were cultured as recommended in DMEM (Gibco, Rockville, MD) containing 1% antibiotics (Gibco) and 10% FBS (Gibco) under 5% CO 2 at 37 C. Before using in this study, all cell lines were authenticated by STR profiling and tested for mycoplasma contamination in June 2019.

| RNA isolation of nuclear and cytoplasmic fractions
The RHPN1-AS1 content in the cell cytoplasm and cell nucleus of PC3 and DU145 cells was assayed following the protocol of PARIS™ Kit (AM1921, Invitrogen). After cells were treated with cell fractionation buffer and then centrifuged, RT-qPCR was applied to determine the expression levels of RHPN1-AS1 in cytoplasmic and nuclear fractions, with GAPDH (cytoplasmic control) and U6 (nuclear control) as respective controls.

| Bioinformatics analysis and dual-luciferase reporter gene assay
The online tool starBase v2.0 (http://starbase.sysu.edu.cn/) was used to predict the potential target genes and analyze the specific binding sites.
The relationship between miR-7-5p and RHPN1-AS1 or EGFR was verified by dual-reporter gene assay. In brief, the pmirGLO vectors (Promega, Madison, WI) containing the sequences of RHPN1-AS1 wild type with the miR-7-5p binding sites or RHPN1-AS1 mutant with the mutated miR-7-5p binding sites, or containing the sequences of wild type EGFR 3 0 untranslated region (UTR) with the miR-7-5p binding sites or mutant EGFR 3 0 UTR with the mutated miR-7-5p binding sites were obtained from Sangon Biotech and inserted into the pmir-GLO vectors (Promega, Madison, WI) for the construction of reporters. The 293T cells were co-transfected with above the pmir-GLO vectors or pmirGLO empty vectors and miR-7-5p mimic or NC mimic, respectively. After 48 h later, the luciferase activity was determined with the Dual-Luciferase Reporter Assay Kit (Promega).

| Western blot analysis
Cells or tissues were lysed with lysis buffer (150 mM NaCl, 1.0% NP-40 or 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 8.0], and protease inhibitors). After incubation on ice for 30 min, the homogenate was centrifuged at 14,000 rpm for 30 min at 4 C, and the protein contents in the supernatant were quantified by BCA methods. Total protein was extracted from the supernatants, separated by 12% SDS-PAGE and transferred to PVDF membranes. The appropriate membranes were blocked by 5% BSA for 1 h at room temperature and incubated with primary antibodies,
Immunofluorescence staining was imaged using a fluorescence microscope (Olympus). Five representative fields were captured at Â200 magnification, and the numbers of cells expressing target proteins in the cytoplasm and the nucleus (overlapping with DAPI) cells were counted.

| Fluorescence in situ hybridization (FISH) analysis
RNA FISH probes were synthesized by Rochen Pharma (Shanghai, China). Briefly, PC3 cells were fixed in 4% formaldehyde for 15 min, washed with PBS, treated with pepsin, and dehydrated through graded ethanol. After the cells were air-dried, they were incubated with 40 nM FISH probe in hybridization buffer for 2 min at 80 C, followed by hybridization at 55 C for 2 h. Subsequently, the slides were washed with 0.1 Â SSC and dehydrated with graded ethanol. Then, the slides were mounted with Prolong Gold Antifade Reagent with DAPI, and a fluorescence microscope (DMI4000B, Leica) was used to observe the images.

| Transmission electron microscopy (TEM)
PCa cells were harvested after transfection with miR-7-5p mimic or NC mimic, fixed with ice-cold 2.5% glutaraldehyde in PBS for 2 h, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol (50%-100%) and acetone, and embedded in Epon. Representative areas were chosen for ultrathin sectioning and examined with a transmission electron microscope.

| Statistical analysis
The values are shown as the means ± SEM for triplicate experiments, and significant differences were calculated using the ANOVA test and Student's t-test. Levels of statistical significance were set at p < .05 or .01 as indicated.

| RESULTS
3.1 | RHPN1-AS1 is up-regulated in prostate cancer, and silencing RHPN1-AS1 inhibits prostate cancer cell proliferation, invasion, and induces apoptosis According to the statistics data collected from the GEPIA database, we found that the 245 PCa patients with high expression of RHPN1-AS1 had a shorter survival time than that of 245 PCa patients with low expression of RHPN1-AS1 ( Figure 1A). Moreover, 21 PCa patients were recruited in this study, and RHPN1-AS1 expression was detected in tissue samples collected from these patients. After RT-qPCR exanimation, we determined that RHPN1-AS1 was expressed higher in tumor tissue samples compared with adjacent normal tissues ( Figure 1B). Consistently, RHPN1-AS1 expressed at a higher level in PCa cell lines compared with normal epithelial cell PWPE-1 ( Figure 1C). Furthermore, we also observed that proliferation and invasiveness were significantly inhibited and apoptosis was markedly induced in PC3 cells that were transfected with sh-RHPN1-AS1 ( Figure 1D-F). These data suggested that RHPN1-AS1 potentially participated in tumorigenesis of PCa. The multiple layers of interaction between the processes of autophagy and apoptosis present, which is a seamless balance between life and death in response to given cellular stress. 19,25 Therefore, we verified whether apoptosis induced by silencing RHPN1-AS1 in PC3 and DU145 cells was related to autophagy. The results showed that after 12 h of silencing RHPN1-AS1, PC3 and DU145 cells exhibited a stark increase in discrete acidic vesicles, especially at 24 and 48 h, at which greater than 70% of the cells were MDC-positive, and an average of more than 15 AO puncta was dispersed throughout the cytoplasm in PC3 and DU145 cells with silencing RHPN1-AS1 ( Figure 2D,E). Next, autophagy activity was further validated by silencing RHPN1-AS1 induced LC3 puncta in PC3 and DU145 cells with immunofluorescence microscopy ( Figure 2F,G). All in all, based on the above results, we demonstrated that silencing RHPN1-AS1 simultaneously induced G2/M cell cycle arrest, autophagy and apoptosis. Furthermore, autophagy precedes apoptosis in RHPN1-AS1 silenced PC3 and DU145 cells, but the relationship between silencing RHPN1-AS1 induced autophagy and apoptosis needs to be further investigated. Autophagy activity was determined with acridine orange (AO) (D) and monodansylcadaverine (MDC) staining (E) or immunofluorescence staining for LC3 puncta (F and G). Data were presented as mean ± SEM of three independent experiments (N = 5), and the difference of the samples between the two groups were analyzed with independent sample t-test. Compared with 0 h, *p < .05, **p < .01 autophagy, which can be inhibited to restore cell viability. To further demonstrate whether silencing RHPN1-AS1 triggered autophagy inhibited or promoted apoptosis, PC3 and DU145 cells in which autophagy was suppressed by 3MA with silencing or not silencing RHPN1-AS1 underwent a flow cytometry apoptosis assay. The results showed that 3MA-mediated autophagy inhibition moderately eliminated silencing RHPN1-AS1 induced apoptosis, which was consistent with the reduced cleaved-caspase-3 and cleaved-PARP ( Figure 3E). One-way analysis of variance (ANOVA) followed by Duncan test was used to evaluate multigroup comparisons of the means. *p < .05, **p < .01 Subsequently, the effect of inhibition of apoptosis on autophagy was determined. We observed that z-VAD-fmk had a minimal effect on the number of AO-staining red acidic vesicles induced by silencing RHPN1-AS1, in accordance with the expression of LC3-II ( Figure 3B, E). However, after pretreatment with z-VAD-fmk, a portion of the cell vitality was successfully restored and apoptosis was nearly abrogated in RHPN1-AS1 silenced PC3 and DU145 cells ( Figure 3A,C,D). Downregulated cleaved-caspase-3 and cleaved-PARP further demonstrated that z-VAD-fmk completely relieved silencing RHPN1-AS1 mediated apoptosis ( Figure 3F). Those indicated that silencing RHPN1-AS1 induced apoptosis is caspase-dependent.

| Overexpression of miR-7-5p inhibits prostate cancer cell proliferation and promotes autophagy and apoptosis
To explain the role of miR-7-5p in PCa cells autophagy and apoptosis, we transfected PC3 and DU145 cells with miR-7-5p mimic or NC mimic, and then observed the effects of miR-7-5p on PC3 cells. The results showed that miR-7-5p expression levels were markedly upregulated in PC3 and DU145 cells transfected with miR-7-5p mimic ( Figure 5A). Moreover, we found that overexpression of miR-7-5p sig- The expression of miR-7-5p was detected with RT-qPCR in prostate cancer tissues and adjacent normal tissues, N = 21. PC3 and DU145 cells were transfected with RHPN1-AS1 overexpression vector (RHPN1-AS1) or sh-RHPN1-AS1 or their control for 48 h, and then the expression of RHPN1-AS1 (E) and miR-7-5p (F) were detected with RT-qPCR. Data were presented as mean ± SEM of three independent experiments (N = 5), and the difference of the samples between the two groups were analyzed with independent sample t-test, and One-way analysis of variance (ANOVA) followed by Duncan test was used to evaluate multigroup comparisons of the means. *p < .05, **p < .01 autophagosomes revealed a smooth double-membrane containing cellular material, and intense electron-dense lysosomal structures were clearly observed in miR-7-5p overexpressed PC3 cells compared to cells transfected with NC mimic ( Figure 5E). In addition, we observed the same changes in miR-7-5p overexpressed DU145 cells ( Figure 5E). Therefore, our data revealed that miR-7-5p participated in F I G U R E 5 Overexpression of miR-7-5p inhibits prostate cancer cell proliferation and promotes autophagy and apoptosis. PC3 and DU145 cells were transfected with miR-7-5p mimic or NC mimic for 48 h, and then the cells were collected. (A) The expression of miR-7-5p was detected with RT-qPCR. (B) Cell viability was detected with CCK-8 assay. (C and D) Cell apoptosis was detected with flow cytometry assay. (E) Transmission electron microscope images show the ultrastructure of PC3 and DU145 cells. Data were presented as mean ± SEM of three independent experiments (N = 5), and the difference of the samples between the two groups were analyzed with independent sample t-test, and One-way analysis of variance (ANOVA) was used to evaluate multigroup comparisons of the means. Compared with NC mimic, *p < .05, **p < .01 the progression of PCa by inhibiting cell proliferation and promoting autophagy and apoptosis.

| EGFR is a target of miR-7-5p and is regulated by miR-7-5p
Biological prediction website starBase v3.0 (http://starbase.sysu.edu. cn/index.php) was used to predict the potential target genes of miR-7-5p. The prediction results identified that there was a specific binding region between the 3 0 UTR of EGFR and miR-7-5p sequences, indicating EGFR may be a target gene of miR-7-5p ( Figure 6A). The dualluciferase reporter gene assay was performed to verify the binding relationship between EGFR and miR-7-5p. The results showed that the luciferase activity of the EGFR wild type was significantly inhibited by miR-7-5p mimic, but the luciferase activity of the EGFR mutant was not inhibited ( Figure 6B). Then, we overexpressed miR-7-5p or inhibited miR-7-5p in PC3 and DU145 cells, and the results F I G U R E 6 EGFR is a target of miR-7-5p and is regulated by miR-7-5p. (A) The binding target of EGFR and miR-7-5p was predicted by StarBase. (B) The luciferase activity was measured with dual luciferase reporter gene assay. PC3 and DU145 cells were transfected with miR-7-5p mimic or miR-7-5p inhibitor or their control for 48 h, and then the cells were collected. (C) The expression of miR-7-5p was detected with RT-qPCR. (D) The protein expression levels of EGFR were measured with Western blotting. (E) PC3 and DU145 cells were transfected with RHPN1-AS1 overexpression vector (RHPN1-AS1) and/or miR-7-5p mimic or their control for 48 h, and then the protein expression levels of EGFR were measured with Western blotting. Data were presented as mean ± SEM of three independent experiments (N = 5), and the difference of the samples between the two groups were analyzed with independent sample t-test, and One-way analysis of variance (ANOVA) followed by Duncan test was used to evaluate multigroup comparisons of the means. *p < .05, **p < .01 showed that overexpression of miR-7-5p promoted expression of miR-7-5 and inhibited EGFR expression ( Figure 6C,D). Subsequently, we overexpressed RHPN1-AS1 and/or miR-7-5p in PC3 and DU145 cells, and the results showed that overexpression of RHPN1-AS1 promoted EGFR expression, whereas miR-7-5p overexpression reversed the promoting effect of RHPN1-AS1 on EGFR expression ( Figure 6E).
The above results indicated that RHPN1-AS1 acted as a ceRNA of miR-7-5p to upregulate EGFR expression.

| Silencing EGFR counteracts the inhibitory effect of RHPN1-AS1 on prostate cancer cell apoptosis
To further determine whether RHPN1-AS1 inhibited apoptosis and autophagy in PCa cells through upregulating EGFR expression, we performed overexpression of RHPN1-AS1 and/or silencing EGFR in PC3 and DU145 cells and observed that overexpression of RHPN1-AS1 increased the expression levels of RHPN1-AS1 and EGFR ( Figure 8A,B,C). Moreover, we found that overexpression of RHPN1-AS1 decreased the expression of autophagy relative protein Beclin-1 and Atg12, increased p62 expression, and reduced LC3-I to LC3-II conversion, silencing EGFR reversed the effects of RHPN1-AS1 on autophagy relative protein expression ( Figure 7D,E).
Then, we detected the apoptotic relative protein expression and cell apoptosis in PC3 and DU145 cells transfected with RHPN1-AS1 and/or si-EGFR. The results showed that apoptotic relative protein cleaved-caspase-3, cleaved-PARP and Bax expression were markedly reduced, and Bcl-2 expression was increased in RHPN1-AS1 overexpressed PC3 and DU145 cells. Silencing EGFR promoted the protein expression of cleaved-caspase-3, cleaved-PARP and Bax and inhibited Bcl-2 protein expression, while the apoptotic relative protein expression returned to control levels in PC3 and DU145 cells co-transfected with RHPN1-AS1 and si-EGFR ( Figure 7F,G). In addition, we observed that overexpression of RHPN1-AS1 inhibited cell apoptosis, and silencing EGFR induced apoptosis in PC3 and DU145 cells ( Figure 7H, I). From the results above, it could be concluded that silencing RHPN1-AS1 induced autophagy and apoptosis in PCa cells through upregulating EGFR expression.
The PI3K/AKT-mTOR pathway is downstream of the EGFR gene and plays a crucial role in regulating the progression of autophagy.
Here, we found that the phosphorylation levels of PI3K and AKT involved in autophagy were obviously upregulated in RHPN1-AS1 overexpressed PC3 and DU145 cells and markedly reduced in EGFR silenced PC3 and DU145 cells ( Figure 7J). These results uncovered that overexpression of RHPN1-AS1 activated the EGFR/AKT/mTOR pathway.
3.8 | RHPN1-AS1 inhibits prostate cancer cell apoptosis by activating the PI3K/AKT/mTOR pathway To further determine whether the PI3K/AKT/mTOR pathway was involved in cell apoptosis regulated by RHPN1-AS1, we pretreated PC3 and DU145 cells with GSK2126458 (a PI3K inhibitor) before overexpression of RHPN1-AS1. The results showed that the expression of RHPN1-AS1 and EGFR was markedly upregulated and miR-7-5p expression was decreased in RHPN1-AS1 overexpressed PC3 and DU145 cells, While GSK2126458 had no effect on expression of RHPN1-AS1, miR-7-5p and EGFR ( Figure 8A-C). Next, we observed that overexpression of RHPN1-AS1 promoted the phosphorylation of the PI3K/AKT/mTOR pathway key proteins PI3K, AKT and mTOR, inhibited autophagy relative protein Beclin-1 expression, and reduced LC3-I to LC3-II conversion in PC3 and DU145 cells by using Western blotting ( Figure 8D,E). Furthermore, we also found that the number of AO puncta formation in PC3 and DU145 cells were significantly reduced by overexpressing RHPN1-AS1, and GSK2126458 counteracted the effects of RHPN1-AS1 on AO puncta formation ( Figure 8F).
Then, we examined the expression of apoptotic relative proteins and the number of apoptotic cells in PC3 and DU145 cells by using Western blotting and flow cytometry, respectively, and found that overexpression of RHPN1-AS1 significantly decreased the expression of cleaved-caspase-3, and reduced the number of apoptotic cells in PC3 and DU145 cells, which was reversed by GSK2126458 ( Figure 8D,G, H). Hence, these data suggested that silencing RHPN1-AS1 sup- Long non-coding RNAs (lncRNAs) are important factors in human diseases and ontogeny. Aberrant expression of lncRNAs may lead to tumorigenesis and aggressive progression, making these molecules attractive therapeutic targets. 9,26 A key finding of a previous study was that RHPN1-AS1 was upregulated in PCa tissues. 10 In our current study, we investigated whether RHPN1-AS1 is a regulator of PCa tumorigenesis. Through a loss of function experiments, we determined that RHPN1-AS1 has a positive effect on PCa cell proliferation but a negative effect on PCa cell apoptosis. Therefore, the present study determined the oncogenic properties of RHPN1-AS1 in PCa tumorigenesis.
Related studies have revealed the interaction between lncRNAs, miRNAs and target genes in the mechanism study of tumorigenesis, and lncRNAs can function by acting as ceRNA of miRNAs to regulate downstream target genes. 27 For example, You et al. 28 found that DSCR8 competitively binds to Yin Yang 1 (YY1) with miR-3192-5p to promote the proliferation and migration and inhibit apoptosis in ovarian cancer cells, thereby promoting ovarian cancer progression. Fen et al. 16 found that RHPN1-AS1 reduced miR-596 expression by sponging it and enhanced insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) expression to accelerate proliferation and metastasis of hepatocellular carcinoma cells. Zhu et al. 9 found that DLX6-AS1 acted as a sponge for miR-497-5p to upregulated, miR-497-5p downstream target gene synuclein gamma (SNCG) promote prostate cancer tumor growth. In our study, we found that RHPN1-AS1 acted as a ceRNA of miR-7-5p to upregulate EGFR, and promoted PCa tumor progression.
It is well known that aberrant proliferation and cell apoptosis are one of the hallmarks of cancer cells, and inhibition of proliferation or induction of cell apoptosis is a key to anti-tumor research. 29 Previous studies have found that the expression of RHPN1-AS1 in epithelial ovarian cancer (EOC) tissues were higher than that in para-cancerous control tissues. Overexpression of RHPN1-AS1 promoted proliferation and metastasis of EOC cells, knockdown of RHPN1-AS1 significantly inhibited the proliferation, migration and invasion of ovarian cancer cells. 11,30 In retinoblastoma (RB) cells, RHPN1-AS1 was One-way analysis of variance (ANOVA) followed by Duncan test was used to evaluate multigroup comparisons of the means. *p < .05, **p < .01 upregulated. Silencing RHPN1-AS1 inhibited the activity of RB cells and promoted apoptosis. 31 In our study, we found that expression of RHPN1-AS1 markedly upregulated in PCa tissues and cells, silencing RHPN1-AS1 inhibited proliferation and invasion, and induced apoptosis in PCa cells. Moreover, we found that overexpression of miR-7-5p or silencing EGFR induced PCa cell apoptosis. From this, we determined that RHPN1-AS1 promoted proliferation and invasion and inhibited apoptosis in PCa cells through acting as a ceRNA of miR-7-5p to upregulate EGFR.
In some cellular contexts, autophagy acts as a cell survival pathway that prevents tumor cells from inducing apoptosis in response to cancer therapy, whereas in other settings, prolonged stress and progressive autophagy may ultimately lead to cell death, either in concert with apoptosis, or as a backup mechanism. 20,21,32,33 TEM revealed that the accumulation of AO puncta, the formation of autophagosomes, and the increased LC3-I/LC3-II conversion all induced autophagy. 34 We further studied the effect of RHPN1-AS1 on the biological function of PCa cells, and on the basis of this study, we found that silencing RHPN1-AS1 induced apoptosis and autophagy in PCa cells. However, RHPN1-AS1 inhibition induced cell death was markedly restored by 3MA (an inhibitor of autophagy) or z-VAD-fmk (an irreversible pan-caspase inhibitor), indicating that autophagic cell F I G U R E 8 RHPN1-AS1 inhibits prostate cancer cell apoptosis through the PI3K/AKT/mTOR pathway. PC3 and DU145 cells were transfected with RHPN1-AS1 overexpression vector (RHPN1-AS1) or pcDNA3.1 empty vector for 48 h after 2 h of pretreatment with 3 nM GSK2126458 (an inhibitor of PI3K). The expression of RHPN1-AS1 (A) and miR-7-5p (B) was detected with RT-qPCR. (C) The protein expression levels of EGFR were measured with Western blotting. (D and E) The protein expression of the PI3K/AKT/mTOR pathway key proteins and autophagy relative proteins was detected with Western blotting. (F) Autophagy activity was determined with acridine orange (AO). (G and H) Cell apoptosis was detected with flow cytometry assay. Data were presented as mean ± SEM of three independent experiments (N = 5). One-way analysis of variance (ANOVA) followed by Duncan test was used to evaluate multigroup comparisons of the means. *p < .05, **p < .01 death induced by RHPN1-AS1 inhibition is a cause of apoptosis and is caspase dependent. This is consistent with previous reports that caspase pathway activation induces apoptosis in prostate cancer cells. 35,36 Recently, cytotoxic autophagy was also observed in cervical cancer cells. 37 To better understand the underlying mechanism by which RHPN1-AS1 inhibition induces autophagy, we explored the downstream pathways.
EGFR is the key mediator of cell proliferation, differentiation and transformation. Transactivation of EGFR is of great significance for tumor cell growth. 38

ACKNOWLEDGMENT
We appreciate all members involved in this study.

CONFLICT OF INTEREST
There are no conflicts of interest of any kind in this study.

CONSENT TO PARTICIPATE
Each participant signed written informed consent before the study.