Asian Journal of Andrology (2017) 19, 453–457 www.asiaandro.com; www.ajandrology.com
Open Access
Prostate Disease
ORIGINAL ARTICLE
PlncRNA‑1 induces apoptosis through the Her‑2 pathway in prostate cancer cells Qing Yang1,*, Zi‑Lian Cui2,3,*, Qin Wang4, Xun‑Bo Jin3, Yong Zhao3, Mu‑Wen Wang3, Wei Song3, Hua‑Wei Qu3, Wei‑Ting Kang3 To determine whether PlncRNA‑1 induces apoptosis in prostate cancer cells through the Her‑2 pathway. The expression of PlncRNA‑1, Her‑2, and related cyclin proteins in 23 cases of prostate cancer and adjacent normal tissues was analyzed and compared. LNCaP cells were divided into a control group and an LNCaP‑PlncRNA‑1‑siRNA experimental group. Normal prostate RWPE‑1 cells were divided into an RWPE‑1 control group and an RWPE‑1‑PlncRNA‑1 experimental group. After PlncRNA‑1 silencing and overexpression, changes in Her‑2 and cyclinD1 expression levels were detected both in vivo and in vitro. In prostate cancer tissues, Her‑2 and PlncRNA‑1 were highly expressed and significantly correlated. In LNCaP cells, the expression of Her‑2 and cyclinD1 decreased following the downregulation of PlncRNA‑1 as assessed by real‑time PCR and Western blotting. In RWPE‑1 cells, the expression of Her‑2 and cyclinD1 increased following PlncRNA‑1 overexpression. Flow cytometry revealed that the proportion of LNCaP cells in G2/M phase was significantly increased after PlncRNA‑1 silencing and that the proportion of RWPE‑1 cells in G2/M phase was significantly decreased after PlncRNA‑1 overexpression. Furthermore, animal experiments validated these results. In conclusion, in prostate cancer, PlncRNA‑1 regulates the cell cycle and cyclinD1 levels and can also regulate proliferation and apoptosis in prostate cancer cells through the Her‑2 pathway. Asian Journal of Andrology (2017) 19, 453–457; doi: 10.4103/1008-682X.178849; published online: 27 May 2016 Keywords: apoptosis; cell cycle; Her‑2; LNCaP; long noncoding RNA; PlncRNA‑1; prostate cancer
INTRODUCTION Prostate cancer (PCa) is one of the most common malignant tumors of the male genitourinary system. In 2015, the American Cancer Society reported an estimated 220 800 new prostate cancer cases, accounting for 26% of cancers in men and making PCa the most prevalent cancer among males. PCa also has the second highest mortality rate among cancers, with an estimated 27 540 PCa‑attributed deaths.1 Previously, we found that the long noncoding RNA (lncRNA), PlncRNA‑1, is frequently overexpressed in PCa cell lines and tissues according to RNA‑seq and real‑time PCR analyses. We found that PlncRNA‑1 plays an important role in the pathogenesis of PCa. In addition to reciprocally regulating the androgen receptor (AR), PlncRNA‑1 knockdown induces tumor cell apoptosis.2 In the present study, we found that PlncRNA‑1 regulates the expression of Her‑2 during apoptosis. Thus, we propose that PlncRNA‑1‑induced apoptosis may act through the Her‑2 signaling pathway. Her‑2 (also known as ErbB‑2) is a 185‑kDa cell membrane receptor encoded by the proto‑oncogene ErbB‑2 and is a member of the epidermal growth factor receptor (EGFR) family. This protein family consists of ErbB‑1 (also known as EGFR, Her‑1), ErbB‑2, ErbB‑3 (Her‑3), and ErbB‑4 (Her‑4). Her‑2 p185 protein is composed of an extracellular domain, a transmembrane domain, and an intracellular domain that possesses endogenous tyrosine kinase activity. In normal
human tissues, the Her‑2/neu protein is absent or expressed at trace levels, whereas in breast, ovarian, lung, stomach, liver, prostate, and other malignant tumors, the Her‑2/neu protein is highly expressed.3–5 Tumor cells expressing high levels of Her‑2 exhibit increased proliferation and cell malignancy, while differentiation and apoptosis are inhibited. Her‑2 p185 protein promotes tumor cell growth through multiple pathways and molecules.6 The expression profile of Her‑2 is closely related to patient prognosis: patients with high Her‑2 expression levels are prone to tumor metastases and short survival times.7 Because Her‑2 expression significantly differs between normal and tumor cells, the Her‑2 protein has become an ideal target for immunotherapy.8,9 PlncRNA‑1 was first identified in PCa tissues; however, its role in the development of PCa has not been fully elucidated.2 This study confirms that PlncRNA‑1 and Her‑2 expression levels are highly correlated and that tumor cell apoptosis was induced following the downregulation of PlncRNA‑1, which also triggered a decrease in Her‑2 protein expression. These discoveries will provide a new theoretical basis for therapeutic interventions for the prevention and treatment of PCa. MATERIALS AND METHODS Samples, cells, and antibodies Frozen PCa tissue was obtained with informed consent from patients
Department of Breast and Thyroid Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, PR China; 2Shandong University School of Medicine, Jinan, Shandong, PR China; 3Minimally Invasive Urology Center, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, PR China; 4 Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan, Shandong, PR China. * These two authors contributed equally to this article. Correspondence: Dr. ZL Cui ([email protected]) Received: 21 August 2015; Revised: 20 November 2015; Accepted: 26 January 2016 1
PlncRNA‑1 induces apoptosis through Her‑2 Q Yang et al 454
who underwent radical resections at the Shandong Provincial Hospital. Ethical consent was granted by the Committees for Ethical Review of Research involving Human Subjects of Shandong Provincial Hospital. LNCaP and RWPE‑1 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, at 37°C and 5% CO2. Mouse monoclonal Her‑2, cyclinD1, and β‑actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AC480 (BMS‑599626) was purchased from Selleck (Houston, TX, USA). Plasmid construction and transfection For overexpression analyses, cDNA encoding the complete open reading frame of PlncRNA‑1 was cloned into the pBabe vector to generate the PlncRNA‑1 expression plasmid. The expression plasmid was verified by sequencing both strands and was used to transfect RWPE‑1 cells to establish the PlncRNA‑1‑overexpression cell line. For PlncRNA‑1 interference, the control (pSuper) and pSuper‑shPlncRNA‑1s plasmids were purchased from Shanghai Genechem Co., LTD (Shanghai, China) and were used to transfect LNCaP cells to establish the PlncRNA‑1‑knockdown cell line. The transfection efficiency of PlncRNA‑1 was confirmed by quantitative reverse transcription PCR (qRT‑PCR) analysis. Western blotting Equal amounts of protein were separated on SDS polyacrylamide gels and were electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were immunoblotted overnight at 4°C with primary antibodies followed by the corresponding secondary antibodies. β‑actin was used as the loading control. Quantitative reverse‑transcription PCR RNA was extracted using TRIzol reagent according to the manufacturer’s recommended protocol (Invitrogen). qRT‑PCR was performed using the StepOne and StepOne Plus Real‑Time PCR Systems (Applied Biosystems, Foster City, CA, USA). GAPDH was used as a loading control. The experiments were repeated a minimum of 3 times. Flow cytometry analysis of the cell cycle To assay the cell cycle stage, exponentially growing cells were trypsinized, rinsed twice with ice‑cold phosphate‑buffered saline (PBS), and fixed in 75% ice‑cold ethanol. The fixed cells were washed with ice‑cold PBS and incubated at 37°C for 30 min in 1 ml of PBS containing 20 µg ml−1 RNase A (Fermentas) and stained with 20 µg ml−1 propidium iodide (Sigma‑Aldrich) for 10 min at room temperature. The DNA content was then determined by flow cytometry. The percentages of cells in the G0/G1, S, and G2/M phases were determined on a BD FACSCalibur (Becton Dickinson, NJ, USA), and data were analyzed with FlowJo software (Tree Star, Inc, OR, USA.). In vivo tumorigenesis assays In vivo tumorigenesis and metastasis assays were performed as described previously. Briefly, 1 × 106 cells were subcutaneously injected into the right flanks of nude mice. Tumor length (L) and width (W) were measured every 3 days, and tumor volume was calculated using the following equation: volume = (W2 × L)/2. After 4 weeks, the mice were killed, and the tumor volume and weight were measured. All animal experiments were performed with the approval of the Shandong Provincial Hospital’s Animal Care and Use Committee. Statistical analysis Experimental data are shown as the means ± standard deviation (s.d.). The results from different treatment groups were compared using two‑tailed Student’s t‑tests. Differences were considered to be Asian Journal of Andrology
significant at P
Open Access
Prostate Disease
ORIGINAL ARTICLE
PlncRNA‑1 induces apoptosis through the Her‑2 pathway in prostate cancer cells Qing Yang1,*, Zi‑Lian Cui2,3,*, Qin Wang4, Xun‑Bo Jin3, Yong Zhao3, Mu‑Wen Wang3, Wei Song3, Hua‑Wei Qu3, Wei‑Ting Kang3 To determine whether PlncRNA‑1 induces apoptosis in prostate cancer cells through the Her‑2 pathway. The expression of PlncRNA‑1, Her‑2, and related cyclin proteins in 23 cases of prostate cancer and adjacent normal tissues was analyzed and compared. LNCaP cells were divided into a control group and an LNCaP‑PlncRNA‑1‑siRNA experimental group. Normal prostate RWPE‑1 cells were divided into an RWPE‑1 control group and an RWPE‑1‑PlncRNA‑1 experimental group. After PlncRNA‑1 silencing and overexpression, changes in Her‑2 and cyclinD1 expression levels were detected both in vivo and in vitro. In prostate cancer tissues, Her‑2 and PlncRNA‑1 were highly expressed and significantly correlated. In LNCaP cells, the expression of Her‑2 and cyclinD1 decreased following the downregulation of PlncRNA‑1 as assessed by real‑time PCR and Western blotting. In RWPE‑1 cells, the expression of Her‑2 and cyclinD1 increased following PlncRNA‑1 overexpression. Flow cytometry revealed that the proportion of LNCaP cells in G2/M phase was significantly increased after PlncRNA‑1 silencing and that the proportion of RWPE‑1 cells in G2/M phase was significantly decreased after PlncRNA‑1 overexpression. Furthermore, animal experiments validated these results. In conclusion, in prostate cancer, PlncRNA‑1 regulates the cell cycle and cyclinD1 levels and can also regulate proliferation and apoptosis in prostate cancer cells through the Her‑2 pathway. Asian Journal of Andrology (2017) 19, 453–457; doi: 10.4103/1008-682X.178849; published online: 27 May 2016 Keywords: apoptosis; cell cycle; Her‑2; LNCaP; long noncoding RNA; PlncRNA‑1; prostate cancer
INTRODUCTION Prostate cancer (PCa) is one of the most common malignant tumors of the male genitourinary system. In 2015, the American Cancer Society reported an estimated 220 800 new prostate cancer cases, accounting for 26% of cancers in men and making PCa the most prevalent cancer among males. PCa also has the second highest mortality rate among cancers, with an estimated 27 540 PCa‑attributed deaths.1 Previously, we found that the long noncoding RNA (lncRNA), PlncRNA‑1, is frequently overexpressed in PCa cell lines and tissues according to RNA‑seq and real‑time PCR analyses. We found that PlncRNA‑1 plays an important role in the pathogenesis of PCa. In addition to reciprocally regulating the androgen receptor (AR), PlncRNA‑1 knockdown induces tumor cell apoptosis.2 In the present study, we found that PlncRNA‑1 regulates the expression of Her‑2 during apoptosis. Thus, we propose that PlncRNA‑1‑induced apoptosis may act through the Her‑2 signaling pathway. Her‑2 (also known as ErbB‑2) is a 185‑kDa cell membrane receptor encoded by the proto‑oncogene ErbB‑2 and is a member of the epidermal growth factor receptor (EGFR) family. This protein family consists of ErbB‑1 (also known as EGFR, Her‑1), ErbB‑2, ErbB‑3 (Her‑3), and ErbB‑4 (Her‑4). Her‑2 p185 protein is composed of an extracellular domain, a transmembrane domain, and an intracellular domain that possesses endogenous tyrosine kinase activity. In normal
human tissues, the Her‑2/neu protein is absent or expressed at trace levels, whereas in breast, ovarian, lung, stomach, liver, prostate, and other malignant tumors, the Her‑2/neu protein is highly expressed.3–5 Tumor cells expressing high levels of Her‑2 exhibit increased proliferation and cell malignancy, while differentiation and apoptosis are inhibited. Her‑2 p185 protein promotes tumor cell growth through multiple pathways and molecules.6 The expression profile of Her‑2 is closely related to patient prognosis: patients with high Her‑2 expression levels are prone to tumor metastases and short survival times.7 Because Her‑2 expression significantly differs between normal and tumor cells, the Her‑2 protein has become an ideal target for immunotherapy.8,9 PlncRNA‑1 was first identified in PCa tissues; however, its role in the development of PCa has not been fully elucidated.2 This study confirms that PlncRNA‑1 and Her‑2 expression levels are highly correlated and that tumor cell apoptosis was induced following the downregulation of PlncRNA‑1, which also triggered a decrease in Her‑2 protein expression. These discoveries will provide a new theoretical basis for therapeutic interventions for the prevention and treatment of PCa. MATERIALS AND METHODS Samples, cells, and antibodies Frozen PCa tissue was obtained with informed consent from patients
Department of Breast and Thyroid Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, PR China; 2Shandong University School of Medicine, Jinan, Shandong, PR China; 3Minimally Invasive Urology Center, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, PR China; 4 Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan, Shandong, PR China. * These two authors contributed equally to this article. Correspondence: Dr. ZL Cui ([email protected]) Received: 21 August 2015; Revised: 20 November 2015; Accepted: 26 January 2016 1
PlncRNA‑1 induces apoptosis through Her‑2 Q Yang et al 454
who underwent radical resections at the Shandong Provincial Hospital. Ethical consent was granted by the Committees for Ethical Review of Research involving Human Subjects of Shandong Provincial Hospital. LNCaP and RWPE‑1 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, at 37°C and 5% CO2. Mouse monoclonal Her‑2, cyclinD1, and β‑actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AC480 (BMS‑599626) was purchased from Selleck (Houston, TX, USA). Plasmid construction and transfection For overexpression analyses, cDNA encoding the complete open reading frame of PlncRNA‑1 was cloned into the pBabe vector to generate the PlncRNA‑1 expression plasmid. The expression plasmid was verified by sequencing both strands and was used to transfect RWPE‑1 cells to establish the PlncRNA‑1‑overexpression cell line. For PlncRNA‑1 interference, the control (pSuper) and pSuper‑shPlncRNA‑1s plasmids were purchased from Shanghai Genechem Co., LTD (Shanghai, China) and were used to transfect LNCaP cells to establish the PlncRNA‑1‑knockdown cell line. The transfection efficiency of PlncRNA‑1 was confirmed by quantitative reverse transcription PCR (qRT‑PCR) analysis. Western blotting Equal amounts of protein were separated on SDS polyacrylamide gels and were electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were immunoblotted overnight at 4°C with primary antibodies followed by the corresponding secondary antibodies. β‑actin was used as the loading control. Quantitative reverse‑transcription PCR RNA was extracted using TRIzol reagent according to the manufacturer’s recommended protocol (Invitrogen). qRT‑PCR was performed using the StepOne and StepOne Plus Real‑Time PCR Systems (Applied Biosystems, Foster City, CA, USA). GAPDH was used as a loading control. The experiments were repeated a minimum of 3 times. Flow cytometry analysis of the cell cycle To assay the cell cycle stage, exponentially growing cells were trypsinized, rinsed twice with ice‑cold phosphate‑buffered saline (PBS), and fixed in 75% ice‑cold ethanol. The fixed cells were washed with ice‑cold PBS and incubated at 37°C for 30 min in 1 ml of PBS containing 20 µg ml−1 RNase A (Fermentas) and stained with 20 µg ml−1 propidium iodide (Sigma‑Aldrich) for 10 min at room temperature. The DNA content was then determined by flow cytometry. The percentages of cells in the G0/G1, S, and G2/M phases were determined on a BD FACSCalibur (Becton Dickinson, NJ, USA), and data were analyzed with FlowJo software (Tree Star, Inc, OR, USA.). In vivo tumorigenesis assays In vivo tumorigenesis and metastasis assays were performed as described previously. Briefly, 1 × 106 cells were subcutaneously injected into the right flanks of nude mice. Tumor length (L) and width (W) were measured every 3 days, and tumor volume was calculated using the following equation: volume = (W2 × L)/2. After 4 weeks, the mice were killed, and the tumor volume and weight were measured. All animal experiments were performed with the approval of the Shandong Provincial Hospital’s Animal Care and Use Committee. Statistical analysis Experimental data are shown as the means ± standard deviation (s.d.). The results from different treatment groups were compared using two‑tailed Student’s t‑tests. Differences were considered to be Asian Journal of Andrology
significant at P
