- Email: [email protected]
Long Noncoding RNA MALAT-1 is a New Potential Therapeutic Target for Castration Resistant Prostate Cancer
Long Noncoding RNA MALAT-1 is a New Potential Therapeutic Target for Castration Resistant Prostate Cancer Shancheng Ren,* Yawei Liu,* Weidong Xu,* Yi Sun, Ji Lu, Fubo Wang, Min Wei, Jian Shen, Jianguo Hou, Xu Gao, Chuanliang Xu, Jiaoti Huang, Yi Zhao and Yinghao Sun† From the Department of Urology, Shanghai Changhai Hospital, Second Military Medical University (SR, WX, YS, JL, FW, MW, JS, JH, XG, CX, YS), Shanghai and Health Division of Guard Bureau, General Staff Department of Chinese People’s Liberation Army (YL) and Bioinformatics Research Group, Key Laboratory of Intelligent Information Processing, Advanced Computer Research Center, Institute of Computing Technology, Chinese Academy of Sciences (YZ), Beijing, People’s Republic of China, and Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California-Los Angeles (JH), Los Angeles, California
Abbreviations and Acronyms CCK-8 ¼ Cell Counting Kit-8 CRPC ¼ castration resistant prostate cancer LNCaP-AI ¼ LNCaP-androgen independent subtype lncRNA ¼ long noncoding RNA M5 ¼ MALAT-1 siRNA-5 M9 ¼ MALAT-1 siRNA-9 MALAT-1 ¼ metastasis associated in lung adenocarcinoma transcript 1 PBS ¼ phosphate buffered saline PCR ¼ polymerase chain reaction PSA ¼ prostate specific antigen RT-PCR ¼ reverse transcriptase-PCR Accepted for publication July 1, 2013. Study received Shanghai Changhai Hospital institutional review board approval. Supported by National Basic Research Program of China 2012CB518306, National Natural Science Foundation of China 81101946, the Prostate Cancer Foundation Young Investigator Award and Shanghai Pujiang Program 12PJD008. * Equal study contribution. † Correspondence: Department of Urology, Changhai Hospital, No.168, Changhai Rd., Shanghai, People’s Republic of China (telephone: þ86 21 81873409; FAX: þ86 21 35030006; e-mail: [email protected]).
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Purpose: To understand the role of MALAT-1 in prostate cancer we evaluated its expression in prostate cancer tissues and cell lines. We also studied the therapeutic effects of MALAT-1 silencing on castration resistant prostate cancer cells in vitro and in vivo. Materials and Methods: Quantitative reverse transcriptase-polymerase chain reaction was used to detect MALAT-1 expression in prostate cancer tissues and cell lines. siRNA against MALAT-1 was designed and the silencing effect was examined by quantitative reverse transcriptase-polymerase chain reaction. The biological effects of MALAT-1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays as well as cell migration by in vitro scratch assay, cell invasion by TranswellÒ invasion assay and cell cycle by flow cytometry. We further investigated the effect of therapeutic siRNA targeting MALAT-1 on castration resistant prostate cancer in vivo. Results: MALAT-1 was up-regulated in human prostate cancer tissues and cell lines. Higher MALAT-1 expression correlated with high Gleason score, prostate specific antigen, tumor stage and castration resistant prostate cancer. MALAT-1 down-regulation by siRNA inhibited prostate cancer cell growth, invasion and migration, and induced castration resistant prostate cancer cell cycle arrest in the G0/G1 phases. Importantly, intratumor delivery of therapeutic siRNA targeting MALAT-1 elicited delayed tumor growth and reduced metastasis of prostate cancer xenografts in castrated male nude mice, followed by the concomitant prolongation of survival of tumor bearing mice. Conclusions: MALAT-1 may be needed to maintain prostate tumorigenicity and it is involved in prostate cancer progression. Thus, MALAT-1 may serve as a potential therapeutic target for castration resistant prostate cancer. Key Words: prostate; prostatic neoplasms; MALAT1 long non-coding RNA, human; RNA, small interfering; antiandrogens
PROSTATE cancer is the most prevalent malignancy in developed countries with an estimated 648,400 new cases and 80,900 new deaths in 2010.1 In
developing countries its incidence is also increasing. The biochemical recurrence rate is high despite initial definitive treatment for localized
0022-5347/13/1906-2278/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.07.001 Vol. 190, 2278-2287, December 2013 Printed in U.S.A.
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prostate cancer.2 Approximately 30% of patients experience prostate cancer recurrence after primary therapy.3 Androgen deprivation therapy is generally used to treat recurrent disease because at this time point prostate cancer progression is androgen dependent.4 However, in those patients CRPC may develop, for which the clinical outcomes of current anticancer treatments are not durable.5 Thus, exploring possible new targets holds great promise for CRPC management. The human genome is transcribed to produce protein coding and noncoding RNAs. Since protein coding genes have been the focus of most research, the functional role of lncRNAs has long been underestimated.6 In recent years they have been emerging as important regulatory factors in mammalian genomics.7 More than 90% of human genome transcripts, including lncRNAs, do not code for proteins.8,9 A number of reports in the last 2 years identified thousands of actively expressed lncRNA transcripts with distinct properties for cell development and differentiation.10e12 They have important roles during cell development and their deregulation is involved in various types of cancers, including prostate cancer, hepatocellular carcinoma, nonsmall cell lung cancer, leukemia, colon carcinoma and breast cancer.13e17 PCA3, also known as DD3, is one such lncRNA that has been extensively studied as a prostate cancer specific biomarker in body fluids.18 Thus, elucidating the roles of lncRNAs in tumors holds great promise for the early detection, prevention and treatment of tumors. One of the long noncoding RNAs is MALAT-1. It was described as a regulator of metastasis and motility, and expression is associated with nonsmall cell lung cancer metastasis. It is a noncoding RNA of more than 8,000 nt derived from chromosome 11q13.19 MALAT-1 was originally found to be over expressed in patients at high risk for nonsmall cell lung cancer metastasis.19 It was later noted to also be over expressed in many other human solid tumors, such as hepatocellular cell carcinoma, lung adenocarcinoma, endometrial stromal sarcoma and colorectal cancer.20e23 However, to our knowledge its role in prostate cancer remains to be elucidated. We evaluated MALAT-1 expression in prostate cancer tissues and cell lines, and explored the effects of MALAT-1 silencing on prostate tumorigenesis. We found that MALAT-1 is over expressed in prostate cancer tissues compared to adjacent normal tissues. Suppressing expression leads to reduced cell growth, invasion, migration and colony formation, and increased rates of apoptosis and cell cycle arrest in prostate cancer cells. We further noted that intratumor delivery of MALAT-1 siRNA can suppress CRPC growth and metastasis in vivo, and prolong the survival of tumor bearing animals.
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MATERIALS AND METHODS Cell Culture and Patient Samples We maintained 22RV1 cells in RPMI medium 1640 (GibcoÒ) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 25 mM HEPES. LNCaP-AI cells were maintained in phenol red-free RPMI medium 1640 supplemented with 10% charcoal stripped fetal bovine serum, 300 mg/l L-glutamine, 2,000 mg/l glucose and 2,000 mg/l NaHCO3. CRPC samples were obtained from patients treated with transurethral prostate resection due to urinary obstruction. Hematoxylin and eosin slides of frozen human primary prostate cancer tissues with matched frozen normal tissues were examined by the study pathologist to confirm the histological diagnosis and ensure that selected tumor tissues had high density cancer foci (greater than 80%) and normal tissues had no tumor contamination. Written informed consent for research using these tumor samples was obtained from all patients. The study protocol was approved by the Shanghai Changhai Hospital institutional review board.
Quantitative RT-PCR Analysis Total RNA was extracted from prostate cell lines using TRIzolÒ reagent and reverse transcribed using the PrimeScriptÔ RT Reagent Kit according to manufacturer instructions. Real-time quantitative PCR was used to detect MALAT-1 expression. For quantitative RT-PCR 10 ng cDNA were amplified in a 20 ml reaction containing SYBRÒ Premix Ex TaqÔ using a 2-step procedure. Melt curve analysis was enabled at the end of amplification. The relative expression of MALAT-1 was normalized to b-actin using the comparative threshold count method. The primers were 50 -CTTCCCT AGGGGATTTCAGG-30 and 50 -GCCCACAGGAACAAGTC CTA-30 for MALAT-1, and 50 -CGCGAGAAGATGCCCAG ATC-30 and 50 -TCACCGGAGTCCATCACGA-30 for b-actin. All experiments were done in triplicate.
siRNA Transfection We seeded 22RV1 and LNCaP-AI cells at a density of 8 104 per well in a 6-well CostarÒ 3516 culture plate with RPMI-1640 medium containing 10% fetal bovine serum or phenol red-free RPMI-1640 medium containing 10% charcoal stripped fetal bovine serum. At the same time the cells were transfected with MALAT-1 siRNA packaged by RNAiMAXÔ Transfection Reagent according to manufacturer instructions. At 48 hours after transfection the cells were harvested for further evaluation. The siRNAs used in this study were MALAT-1 siRNA M5 sense 50 -GGGCUUCUCUUAACAUUUATT-30 and antisense 50 -UAAAUGUUAAGAGAAGCCCTT-30 , MALAT-1 siRNA M9 sense 50 -GGGCAAAUAUUGGCAAUUATT-30 and antisense 50 -UAAUUGCCAAUAUUUGCCCTT-30 , and control sense 50 -CCUACGCCACCAAUUUCGU-30 and antisense 50 -ACGAAAUUGGUGGCGUAGG-30 .
Assays Cell proliferation. We evaluated 22RV1 and LNCaP-AI cell proliferation using the CCK-8 assay according to manufacturer instructions. This assay is based on cleavage of the tetrazolium salt WST-8 by mitochondrial
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dehydrogenase in viable cells. We incubated 2 103 cells per well with 100 ml culture medium in 96-multiwell plates. Cells were cultured for 1, 2, 3, 4 or 5 days before adding 10 ml CCK-8 (5 mg/ml) to the culture medium in each well. After 1-hour incubation at 37C we measured the absorbance at 450 nm of each well with a THERMOmaxÔ microplate reader. Each experiment was repeated 3 times and data represent the mean of all measurements. Cell colony formation. We seeded 22RV1 and LNCaP-AI cells in 6-well plates at a density of 200 per well and cultured them at 37C for 7 days. Medium was replaced every 2 to 3 days. Cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with Giemsa stain (SigmaÒ) for 10 minutes and washed 3 times with double distilled H2O. Plates were photographed with a digital camera. Each experiment was performed in triplicate and repeated 3 times.
Cell Cycle Analysis Cells were harvested 2 days after transfection by centrifugation at 1,200 rpm for 5 minutes. Pellets were sequentially washed with cold PBS, fixed in 70% cold ethanol, centrifuged at 1,500 rpm for 5 minutes and resuspended in PBS. Ribonuclease was added and the cells were incubated at 37C for 30 minutes. The cells were then filtered through a 400 mesh membrane, stained with propidium iodide in the dark at 4C for 30 minutes and analyzed using the BD FACSAriaÔ II and FlowJo (Tree Star, Ashland, Oregon). Each experiment was done in triplicate.
Further Assays
TUNEL. We used the TUNEL method to label the 30 end of apoptotic cell fragmented DNA. Cells treated as indicated were fixed with 4% paraformaldehyde phosphate buffered saline, rinsed with PBS and permeabilized by 0.1% Triton X-100 for 2 minutes on ice. TUNEL was done using the One Step TUNEL Apoptosis Assay Kit (C1088, Beyotime Institute of Biotechnology, Haimen, People’s Republic of China) for 1 hour at 37C. Fluorescein isothiocyanate labeled, TUNEL positive cells were imaged under fluorescent microscopy using 488 nm excitation and 530 nm emission wavelengths. Cells with green fluorescence were considered apoptotic cells. Migration. We seeded 22RV1 and LNCaP-AI cells transfected with MALAT-1 siRNA or scrambled siRNAs, or blank 22RV1 and LNCaP-AI cells in 6-well plates. Cell growth was allowed to continue until confluence was reached. The cell monolayer was then scratched with a 10 ml pipette tip and dislodged cells were washed away with PBS. Cell incubation continued under standard conditions and the degree of cell migration into the scraped area was documented every 24 hours.24 The exact migration distance was measured in mm using a screen from each side of the scratch at 3 spots within the same scratch. Invasion. Cell invasion assay was done using Boyden chambers containing Transwell membrane filter inserts with an 8 mm pore size. For the invasion assay the Transwell membrane was coated with BD BiocoatÔ MatrigelÔ. We seeded 3 104 22RV1 and LNCaP-AI cells on the
upper chamber of the Boyden chambers. After 36 hours of culture at 37C we removed the upper cell layer before visualization. Cells were stained with crystal violet and counted under a microscope in 5 predetermined fields at 100 using National Institutes of Health ImageJ.
Animal Treatment One week after castration male Sprague DawleyÒ athymic nude mice were injected subcutaneously with 2 106 22RV1 cells suspended in 0.1 ml Matrigel. Mice were randomly selected for treatment with MALAT-1 siRNA (6) or as controls (6) when tumor volume reached 200 to 500 mm3. The tumor was then injected once every 3 days for 3 weeks. Tumor size was measured 3 times each week and calculated using the formula, p/6 ab2, where a represents the longest tumor dimension and b represents tumor width. For survival analysis mice were sacrificed after a moribund state was noted, as defined by evident, persistent shivering, extreme prostration, labored breathing, greater than 20% of body weight loss and/or a tumor greater than 2 cm with diabrosis. After sacrifice the lymph nodes and other main organs, including the prostate, bone, liver, kidney, lung, gastrointestinal tract and brain, were examined macroscopically and microscopically for tumor cells. Additional groups of mice received the treatment described but were sacrificed 3 weeks after treatment for histological TUNEL and immunohistochemical evaluation, and MALAT-1 expression analysis. All animal procedures were done in accordance with institutional animal welfare guidelines.
Statistical Analysis Data are shown as the mean SD. Data were analyzed using the Student t-test with SPSSÒ 11.0 and PrismÒ 5.0. Statistical significance was considered at p <0.05.
RESULTS MALAT-1 Over expression in prostate cancer tissues and cell lines.
To study what percent of tumor samples had higher MALAT-1 expression vs adjacent normal samples we first examined MALAT-1 expression in 23 pairs of prostate cancer and adjacent normal tissues by quantitative RT-PCR (supplementary fig. 1, http://jurology.com/). MALAT-1 expression was separately normalized to adjacent normal tissue. MALAT-1 expression that was increased 1.5-fold or greater in cancer was considered over expression. MALAT-1 was over expressed in 18 of 23 samples (78%) (fig. 1, A). Average MALAT-1 expression was 7.5-fold higher in prostate cancer than in normal tissue (p <0.01). MALAT-1 expression was also significantly higher in CRPC specimens relative to primary prostate cancer specimens (p ¼ 0.006, fig. 1, B), suggesting that MALAT-1 may have a role in CRPC development. We further studied the association of MALAT-1 tumor expression with PSA, Gleason score and
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Figure 1. Quantitative RT-PCR of MALAT-1 expression. A, MALAT-1 was over expressed in prostate cancer vs adjacent normal tissue. B, MALAT-1 was significantly greater in 9 CRPCs than in 12 primary prostate cancers (p ¼ 0.006). C, MALAT-1 in tumor vs PSA, Gleason score and TNM stage.
TNM stage. In general, higher MALAT-1 tumor expression significantly correlated with higher Gleason score, TNM stage and PSA (supplementary table, http://jurology.com/, and fig. 1, C ). MALAT-1 was most highly expressed in patients with Gleason score 8 or greater, PSA greater than 20 ng/ml and locally advanced prostate cancer (T3 þ T4), suggesting that MALAT-1 is associated with prostate cancer aggressiveness and progression. We next performed quantitative RT-PCR to explore MALAT-1 expression in the prostate cell lines C4-2, PC-3, DU-145, LNCaP, 22RV1 and LNCaP-AI, and the normal prostate epithelial cell line PWR1-E. MALAT-1 expression was significantly greater in prostate cancer cell lines than in PWR1-E (fig. 2, A). Of the prostate cancer cell lines
22RV1 and LNCaP-AI showed the highest MALAT1 expression. Thus, we chose these 2 cell lines for further study. We then designed 4 MALAT-1 siRNAs. After transfecting the 2 cell lines with these siRNAs we performed quantitative RT-PCR to determine their efficacy to silence MALAT-1 expression. M5 and M9 showed the highest efficacy with statistical significance (fig. 2, B and C ). Thus, M5 and M9 were chosen for further functional studies. Suppression inhibited human prostate cancer cell growth and colony formation. To investigate the
effects of MALAT-1 suppression on human prostate cancer cell proliferation we transfected 22RV1 and LNCaP-AI cells with M5 and M9. Two, 3, 4, 5 and
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Figure 2. Quantitative RT-PCR of MALAT-1 expression. A, MALAT-1 was over expressed in prostate cancer cell lines vs normal prostate epithelial cell line PWR1-E. B and C, M5 and M9 significantly inhibited MALAT-1 expression. B, 22RV1 cells. C, LNCaP-AI cells. MOCK, sham treated. NC, negative control. KD, knockdown.
6 days after transfection prostate cancer cell proliferation was measured by the CCK-8 assay. Cell proliferation was significantly decreased after MALAT-1 siRNA transfection compared with that in siRNA control transfected and sham treated cells (fig. 3). However, we observed no inhibitory effect of MALAT-1 siRNA in the normal prostate cell line PWR1-E, indicating that the functional effects of MALAT-1 siRNA were limited to MALAT-1 over expressing cells or cancer cells (supplementary fig. 2, http://jurology.com/). Cell colony formation assay showed that MALAT-1 silencing drastically decreased 22RV1 and LNCaP-AI cell proliferation capacity compared with that of sham treated and control cells (fig. 4). These results suggest that MALAT-1 is critical for prostate cancer cell proliferation. MALAT-1 Silencing in Prostate Cancer Induced cell cycle arrest. At 48 hours after MALAT-1 siRNA transfection flow cytometry analysis was performed in all 3 groups of 22RV1 and LNCaP-AI
cells. In the 22RV1 and LNCaP-AI cell lines an increased proportion of G0/G1 phase cells were observed among MALAT-1 knockdown cells compared with that of the sham treated and control groups (fig. 5 and supplementary fig. 3, http:// jurology.com/). Thus, MALAT-1 silencing caused cell cycle arrest in the 22RV1 and LNCaP-AI cell lines. Notably, we also observed a slight increase of G2/M cells in 22RV1 but not in LNCaP-AI upon MALAT-1 knockdown. This suggests that MALAT-1 regulation of the cell cycle may be cell context dependent, for example according to PTEN/P53 status in each cell line examined. Associated with decreased cell migration and invasiveness. In vitro scratch assay was performed
to evaluate the influence of MALAT-1 on cell migration. We seeded the same number of cells in plates before transfection with MALAT-1 siRNAs or scrambled siRNAs. Results were determined at 48 hours. Microscopic analysis of 22RV1 and LNCaP-AI cells revealed that MALAT-1 siRNA
Figure 3. MALAT-1 knockdown attenuated prostate cancer cell growth potential in vitro, as assessed by CCK-8 assay of MALAT-1 downregulation effect on proliferation in 6-day period. Data are shown as mean SD. A, 22RV1 cells. B, LNCaP-AI cells. NC, negative control. MOCK, transfected with transfection reagent. KD, knockdown. OD, optical density.
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Figure 4. MALAT-1 knockdown resulted in lower prostate cancer cell colony formation efficiency, as assessed by colony formation assay. Number of colonies of knocked down M5 and M9 transfected cells (KD) was statistically less than in transfection reagent (MOCK ) and negative control (NC ) groups (p <0.05). A, 22RV1 cells. B, LNCaP-AI cells.
transfected cells had the lowest migratory ability compared with sham treated and normal control cells (figs. 6 and 7). Considering that proliferation was strongly decreased in MALAT-1 knockdown cells, the MALAT-1 siRNA mediated reduced migration effect could have been partially due to the results of decreased proliferation. To evaluate the effects of MALAT-1 on tumor cell invasiveness we transfected prostate cancer cells with MALAT-1 siRNA and performed standard in vitro chamber assays using the Matrigel model. The number of cells that digested Matrigel
and penetrated the Transwell polycarbonate filter significantly decreased when MALAT-1 was silenced (fig. 8). These findings suggest that MALAT-1 has a key role in prostate cancer cell invasiveness.
Figure 5. MALAT-1 knockdown resulted in G0/G1 phase cell cycle arrest. Proportion of 22RV1 and LNCaP-AI cells in different cell cycle phases was quantified by propidium iodide staining, followed by flow cytometry for cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown.
Figure 6. Representative images show 22RV1 and LNCaP-AI cell migration inhibition by MALAT-1 silencing, as assessed by scratch assay at 0 and 48 hours (h). MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Reduced from 10.
Therapeutic MALAT-1 siRNA CRPC Suppressed Growth and Metastasis In Vivo, and Prolonged Tumor Bearing Mouse Survival We further investigated the effect of therapeutic MALAT-1 siRNA on CRPC growth in vivo. Castrated male nude mice bearing 22RV1 xenograft
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Figure 7. Percent quantification of inhibition of cellular migration by MALAT-1 silencing, as assessed by cell migration distance of scratch closure on scratch assay at 0 and 48 hours. A, 22RV1 cells. B, LNCaP-AI cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Single asterisk indicates p <0.05. Double asterisks indicate p <0.01.
tumors were randomly selected and treated with MALAT-1 or control siRNA once every 3 days for 3 weeks by intratumor injection. Mean 22RV1 tumor volume and body weight at baseline were similar in the 2 groups. MALAT-1 siRNA treatment significantly decreased tumor growth, as shown by strongly reduced tumor size and weight compared with control treatment (fig. 9, A and B). To confirm that MALAT-1 was knocked down in vivo by siRNA we measured MALAT-1 expression and found that it was significantly decreased in treated xenograft tumors (fig. 9, C ). The proliferation index Ki67 was also significantly decreased in MALAT-1 siRNA treated tumors and silencing MALAT-1 induced apoptosis in vivo (fig. 10). Importantly, MALAT-1 siRNA treatment led to a significant reduction in metastasis (fig. 9, D). In addition, MALAT-1 siRNA treatment significantly
prolonged the survival of tumor bearing mice (fig. 9, E ). Together these results indicate that targeting MALAT-1 through the intratumor administration of therapeutic siRNA retarded the growth and metastasis of established CRPC xenografts, and significantly extended animal survival.
DISCUSSION In this study MALAT-1 was over expressed in prostate cancer tissues compared with adjacent normal prostate tissues. MALAT-1 was also up-regulated in prostate cancer cells compared with normal prostate epithelial cells. Moreover, MALAT-1 up-regulation correlated with higher Gleason score, PSA and tumor stage. Importantly, MALAT-1 expression was significantly higher in CRPC than in primary prostate cancer. MALAT-1 knockdown resulted in
Figure 8. Boyden chamber assay revealed that MALAT-1 silencing inhibited cell invasion ability. Data are shown as mean SD. A, 22RV1 cells. B, LNCaP-AI cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Asterisk indicates p <0.05. Reduced from 10.
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Figure 9. Effect of therapeutic MALAT-1 siRNA on castration resistant 22RV1 xenograft growth in vivo. A and B, MALAT-1 siRNA significantly decreased tumor vs control (siCtrl ) treatment. A, size. B, weight. C, MALAT-1 expression was significantly reduced in vivo (p ¼ 0.03). D, MALAT-1 silencing significantly decreased prostate cancer metastasis. E, Kaplan-Meier graph shows that MALAT-1 therapeutic siRNA prolonged 22RV1 xenograft bearing mouse survival.
decreased cell growth, invasion and migration, and an increased apoptosis rate. Together these findings indicate that MALAT-1 is a key regulator of prostate cancer.
Several biological processes are important in tumorigenesis, such as angiogenesis, cell proliferation and motility, immune response inhibition and apoptosis.25 Emerging studies have proved that
Figure 10. MALAT-1 silencing. Immunohistochemistry revealed decreased Ki67 proliferation index. TUNEL assay showed apoptosis induced in vivo. Ctrl, control. Reduced from 20.
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lncRNAs have important roles in various physiological processes, including embryonic stem cell differentiation, keratinocyte differentiation and cancer.19e23 Recent evidence suggests that MALAT-1 has a pivotal role in the molecular events of tumorigenesis.26 Inspired by these findings, we explored its implications for prostate tumorigenesis. Based on the data presented in this study, we propose that increased MALAT-1 expression may contribute to increased tumor cell growth and invasion. Notably, MALAT-1 is over expressed in androgen dependent and CRPC cell lines, suggesting a consistent positive role for MALAT-1 in the whole process of prostate tumorigenesis and progression. Silencing MALAT-1 resulted in the inhibition of CRPC cell growth and metastasis in vivo. Thus, MALAT-1 targeted therapy may hold great promise for the future. lncRNAs regulate gene expression through various mechanisms, including transcription, posttranscriptional processing, chromatin modification, genomic imprinting and protein function regulation.27,28 Two alternative models of action were proposed for MALAT1, including gene
expression regulation and alternative splicing. Previous studies showed that MALAT-1 promotes cancer progression, mainly through the regulation of gene expression, for example the epithelialmesenchymal transition associated genes, ZEB1, ZEB2 and Slug, and the apoptosis related genes CASP3, CASP8, BAX, BCL2 and BCL2L1.29 Since knockdown MALAT-1 can inhibit prostate cancer migration and invasion, we hypothesized that these effects may be modulated by regulating epithelialmesenchymal transition associated genes.
CONCLUSIONS Our study shows that MALAT-1 is involved in prostate tumorigenesis and progression. MALAT-1 silencing inhibits cell growth, migration and invasion, and induces apoptosis and cell cycle arrest in CRPC cells. This suggests that MALAT-1 should be further evaluated as a promising therapeutic target for CRPC.
ACKNOWLEDGMENTS Bioneer China provided siRNA.
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Abbreviations and Acronyms CCK-8 ¼ Cell Counting Kit-8 CRPC ¼ castration resistant prostate cancer LNCaP-AI ¼ LNCaP-androgen independent subtype lncRNA ¼ long noncoding RNA M5 ¼ MALAT-1 siRNA-5 M9 ¼ MALAT-1 siRNA-9 MALAT-1 ¼ metastasis associated in lung adenocarcinoma transcript 1 PBS ¼ phosphate buffered saline PCR ¼ polymerase chain reaction PSA ¼ prostate specific antigen RT-PCR ¼ reverse transcriptase-PCR Accepted for publication July 1, 2013. Study received Shanghai Changhai Hospital institutional review board approval. Supported by National Basic Research Program of China 2012CB518306, National Natural Science Foundation of China 81101946, the Prostate Cancer Foundation Young Investigator Award and Shanghai Pujiang Program 12PJD008. * Equal study contribution. † Correspondence: Department of Urology, Changhai Hospital, No.168, Changhai Rd., Shanghai, People’s Republic of China (telephone: þ86 21 81873409; FAX: þ86 21 35030006; e-mail: [email protected]).
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Purpose: To understand the role of MALAT-1 in prostate cancer we evaluated its expression in prostate cancer tissues and cell lines. We also studied the therapeutic effects of MALAT-1 silencing on castration resistant prostate cancer cells in vitro and in vivo. Materials and Methods: Quantitative reverse transcriptase-polymerase chain reaction was used to detect MALAT-1 expression in prostate cancer tissues and cell lines. siRNA against MALAT-1 was designed and the silencing effect was examined by quantitative reverse transcriptase-polymerase chain reaction. The biological effects of MALAT-1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays as well as cell migration by in vitro scratch assay, cell invasion by TranswellÒ invasion assay and cell cycle by flow cytometry. We further investigated the effect of therapeutic siRNA targeting MALAT-1 on castration resistant prostate cancer in vivo. Results: MALAT-1 was up-regulated in human prostate cancer tissues and cell lines. Higher MALAT-1 expression correlated with high Gleason score, prostate specific antigen, tumor stage and castration resistant prostate cancer. MALAT-1 down-regulation by siRNA inhibited prostate cancer cell growth, invasion and migration, and induced castration resistant prostate cancer cell cycle arrest in the G0/G1 phases. Importantly, intratumor delivery of therapeutic siRNA targeting MALAT-1 elicited delayed tumor growth and reduced metastasis of prostate cancer xenografts in castrated male nude mice, followed by the concomitant prolongation of survival of tumor bearing mice. Conclusions: MALAT-1 may be needed to maintain prostate tumorigenicity and it is involved in prostate cancer progression. Thus, MALAT-1 may serve as a potential therapeutic target for castration resistant prostate cancer. Key Words: prostate; prostatic neoplasms; MALAT1 long non-coding RNA, human; RNA, small interfering; antiandrogens
PROSTATE cancer is the most prevalent malignancy in developed countries with an estimated 648,400 new cases and 80,900 new deaths in 2010.1 In
developing countries its incidence is also increasing. The biochemical recurrence rate is high despite initial definitive treatment for localized
0022-5347/13/1906-2278/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.07.001 Vol. 190, 2278-2287, December 2013 Printed in U.S.A.
RNA MALAT-1 IS POTENTIAL TARGET FOR CASTRATION RESISTANT PROSTATE CANCER
prostate cancer.2 Approximately 30% of patients experience prostate cancer recurrence after primary therapy.3 Androgen deprivation therapy is generally used to treat recurrent disease because at this time point prostate cancer progression is androgen dependent.4 However, in those patients CRPC may develop, for which the clinical outcomes of current anticancer treatments are not durable.5 Thus, exploring possible new targets holds great promise for CRPC management. The human genome is transcribed to produce protein coding and noncoding RNAs. Since protein coding genes have been the focus of most research, the functional role of lncRNAs has long been underestimated.6 In recent years they have been emerging as important regulatory factors in mammalian genomics.7 More than 90% of human genome transcripts, including lncRNAs, do not code for proteins.8,9 A number of reports in the last 2 years identified thousands of actively expressed lncRNA transcripts with distinct properties for cell development and differentiation.10e12 They have important roles during cell development and their deregulation is involved in various types of cancers, including prostate cancer, hepatocellular carcinoma, nonsmall cell lung cancer, leukemia, colon carcinoma and breast cancer.13e17 PCA3, also known as DD3, is one such lncRNA that has been extensively studied as a prostate cancer specific biomarker in body fluids.18 Thus, elucidating the roles of lncRNAs in tumors holds great promise for the early detection, prevention and treatment of tumors. One of the long noncoding RNAs is MALAT-1. It was described as a regulator of metastasis and motility, and expression is associated with nonsmall cell lung cancer metastasis. It is a noncoding RNA of more than 8,000 nt derived from chromosome 11q13.19 MALAT-1 was originally found to be over expressed in patients at high risk for nonsmall cell lung cancer metastasis.19 It was later noted to also be over expressed in many other human solid tumors, such as hepatocellular cell carcinoma, lung adenocarcinoma, endometrial stromal sarcoma and colorectal cancer.20e23 However, to our knowledge its role in prostate cancer remains to be elucidated. We evaluated MALAT-1 expression in prostate cancer tissues and cell lines, and explored the effects of MALAT-1 silencing on prostate tumorigenesis. We found that MALAT-1 is over expressed in prostate cancer tissues compared to adjacent normal tissues. Suppressing expression leads to reduced cell growth, invasion, migration and colony formation, and increased rates of apoptosis and cell cycle arrest in prostate cancer cells. We further noted that intratumor delivery of MALAT-1 siRNA can suppress CRPC growth and metastasis in vivo, and prolong the survival of tumor bearing animals.
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MATERIALS AND METHODS Cell Culture and Patient Samples We maintained 22RV1 cells in RPMI medium 1640 (GibcoÒ) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 25 mM HEPES. LNCaP-AI cells were maintained in phenol red-free RPMI medium 1640 supplemented with 10% charcoal stripped fetal bovine serum, 300 mg/l L-glutamine, 2,000 mg/l glucose and 2,000 mg/l NaHCO3. CRPC samples were obtained from patients treated with transurethral prostate resection due to urinary obstruction. Hematoxylin and eosin slides of frozen human primary prostate cancer tissues with matched frozen normal tissues were examined by the study pathologist to confirm the histological diagnosis and ensure that selected tumor tissues had high density cancer foci (greater than 80%) and normal tissues had no tumor contamination. Written informed consent for research using these tumor samples was obtained from all patients. The study protocol was approved by the Shanghai Changhai Hospital institutional review board.
Quantitative RT-PCR Analysis Total RNA was extracted from prostate cell lines using TRIzolÒ reagent and reverse transcribed using the PrimeScriptÔ RT Reagent Kit according to manufacturer instructions. Real-time quantitative PCR was used to detect MALAT-1 expression. For quantitative RT-PCR 10 ng cDNA were amplified in a 20 ml reaction containing SYBRÒ Premix Ex TaqÔ using a 2-step procedure. Melt curve analysis was enabled at the end of amplification. The relative expression of MALAT-1 was normalized to b-actin using the comparative threshold count method. The primers were 50 -CTTCCCT AGGGGATTTCAGG-30 and 50 -GCCCACAGGAACAAGTC CTA-30 for MALAT-1, and 50 -CGCGAGAAGATGCCCAG ATC-30 and 50 -TCACCGGAGTCCATCACGA-30 for b-actin. All experiments were done in triplicate.
siRNA Transfection We seeded 22RV1 and LNCaP-AI cells at a density of 8 104 per well in a 6-well CostarÒ 3516 culture plate with RPMI-1640 medium containing 10% fetal bovine serum or phenol red-free RPMI-1640 medium containing 10% charcoal stripped fetal bovine serum. At the same time the cells were transfected with MALAT-1 siRNA packaged by RNAiMAXÔ Transfection Reagent according to manufacturer instructions. At 48 hours after transfection the cells were harvested for further evaluation. The siRNAs used in this study were MALAT-1 siRNA M5 sense 50 -GGGCUUCUCUUAACAUUUATT-30 and antisense 50 -UAAAUGUUAAGAGAAGCCCTT-30 , MALAT-1 siRNA M9 sense 50 -GGGCAAAUAUUGGCAAUUATT-30 and antisense 50 -UAAUUGCCAAUAUUUGCCCTT-30 , and control sense 50 -CCUACGCCACCAAUUUCGU-30 and antisense 50 -ACGAAAUUGGUGGCGUAGG-30 .
Assays Cell proliferation. We evaluated 22RV1 and LNCaP-AI cell proliferation using the CCK-8 assay according to manufacturer instructions. This assay is based on cleavage of the tetrazolium salt WST-8 by mitochondrial
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dehydrogenase in viable cells. We incubated 2 103 cells per well with 100 ml culture medium in 96-multiwell plates. Cells were cultured for 1, 2, 3, 4 or 5 days before adding 10 ml CCK-8 (5 mg/ml) to the culture medium in each well. After 1-hour incubation at 37C we measured the absorbance at 450 nm of each well with a THERMOmaxÔ microplate reader. Each experiment was repeated 3 times and data represent the mean of all measurements. Cell colony formation. We seeded 22RV1 and LNCaP-AI cells in 6-well plates at a density of 200 per well and cultured them at 37C for 7 days. Medium was replaced every 2 to 3 days. Cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with Giemsa stain (SigmaÒ) for 10 minutes and washed 3 times with double distilled H2O. Plates were photographed with a digital camera. Each experiment was performed in triplicate and repeated 3 times.
Cell Cycle Analysis Cells were harvested 2 days after transfection by centrifugation at 1,200 rpm for 5 minutes. Pellets were sequentially washed with cold PBS, fixed in 70% cold ethanol, centrifuged at 1,500 rpm for 5 minutes and resuspended in PBS. Ribonuclease was added and the cells were incubated at 37C for 30 minutes. The cells were then filtered through a 400 mesh membrane, stained with propidium iodide in the dark at 4C for 30 minutes and analyzed using the BD FACSAriaÔ II and FlowJo (Tree Star, Ashland, Oregon). Each experiment was done in triplicate.
Further Assays
TUNEL. We used the TUNEL method to label the 30 end of apoptotic cell fragmented DNA. Cells treated as indicated were fixed with 4% paraformaldehyde phosphate buffered saline, rinsed with PBS and permeabilized by 0.1% Triton X-100 for 2 minutes on ice. TUNEL was done using the One Step TUNEL Apoptosis Assay Kit (C1088, Beyotime Institute of Biotechnology, Haimen, People’s Republic of China) for 1 hour at 37C. Fluorescein isothiocyanate labeled, TUNEL positive cells were imaged under fluorescent microscopy using 488 nm excitation and 530 nm emission wavelengths. Cells with green fluorescence were considered apoptotic cells. Migration. We seeded 22RV1 and LNCaP-AI cells transfected with MALAT-1 siRNA or scrambled siRNAs, or blank 22RV1 and LNCaP-AI cells in 6-well plates. Cell growth was allowed to continue until confluence was reached. The cell monolayer was then scratched with a 10 ml pipette tip and dislodged cells were washed away with PBS. Cell incubation continued under standard conditions and the degree of cell migration into the scraped area was documented every 24 hours.24 The exact migration distance was measured in mm using a screen from each side of the scratch at 3 spots within the same scratch. Invasion. Cell invasion assay was done using Boyden chambers containing Transwell membrane filter inserts with an 8 mm pore size. For the invasion assay the Transwell membrane was coated with BD BiocoatÔ MatrigelÔ. We seeded 3 104 22RV1 and LNCaP-AI cells on the
upper chamber of the Boyden chambers. After 36 hours of culture at 37C we removed the upper cell layer before visualization. Cells were stained with crystal violet and counted under a microscope in 5 predetermined fields at 100 using National Institutes of Health ImageJ.
Animal Treatment One week after castration male Sprague DawleyÒ athymic nude mice were injected subcutaneously with 2 106 22RV1 cells suspended in 0.1 ml Matrigel. Mice were randomly selected for treatment with MALAT-1 siRNA (6) or as controls (6) when tumor volume reached 200 to 500 mm3. The tumor was then injected once every 3 days for 3 weeks. Tumor size was measured 3 times each week and calculated using the formula, p/6 ab2, where a represents the longest tumor dimension and b represents tumor width. For survival analysis mice were sacrificed after a moribund state was noted, as defined by evident, persistent shivering, extreme prostration, labored breathing, greater than 20% of body weight loss and/or a tumor greater than 2 cm with diabrosis. After sacrifice the lymph nodes and other main organs, including the prostate, bone, liver, kidney, lung, gastrointestinal tract and brain, were examined macroscopically and microscopically for tumor cells. Additional groups of mice received the treatment described but were sacrificed 3 weeks after treatment for histological TUNEL and immunohistochemical evaluation, and MALAT-1 expression analysis. All animal procedures were done in accordance with institutional animal welfare guidelines.
Statistical Analysis Data are shown as the mean SD. Data were analyzed using the Student t-test with SPSSÒ 11.0 and PrismÒ 5.0. Statistical significance was considered at p <0.05.
RESULTS MALAT-1 Over expression in prostate cancer tissues and cell lines.
To study what percent of tumor samples had higher MALAT-1 expression vs adjacent normal samples we first examined MALAT-1 expression in 23 pairs of prostate cancer and adjacent normal tissues by quantitative RT-PCR (supplementary fig. 1, http://jurology.com/). MALAT-1 expression was separately normalized to adjacent normal tissue. MALAT-1 expression that was increased 1.5-fold or greater in cancer was considered over expression. MALAT-1 was over expressed in 18 of 23 samples (78%) (fig. 1, A). Average MALAT-1 expression was 7.5-fold higher in prostate cancer than in normal tissue (p <0.01). MALAT-1 expression was also significantly higher in CRPC specimens relative to primary prostate cancer specimens (p ¼ 0.006, fig. 1, B), suggesting that MALAT-1 may have a role in CRPC development. We further studied the association of MALAT-1 tumor expression with PSA, Gleason score and
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Figure 1. Quantitative RT-PCR of MALAT-1 expression. A, MALAT-1 was over expressed in prostate cancer vs adjacent normal tissue. B, MALAT-1 was significantly greater in 9 CRPCs than in 12 primary prostate cancers (p ¼ 0.006). C, MALAT-1 in tumor vs PSA, Gleason score and TNM stage.
TNM stage. In general, higher MALAT-1 tumor expression significantly correlated with higher Gleason score, TNM stage and PSA (supplementary table, http://jurology.com/, and fig. 1, C ). MALAT-1 was most highly expressed in patients with Gleason score 8 or greater, PSA greater than 20 ng/ml and locally advanced prostate cancer (T3 þ T4), suggesting that MALAT-1 is associated with prostate cancer aggressiveness and progression. We next performed quantitative RT-PCR to explore MALAT-1 expression in the prostate cell lines C4-2, PC-3, DU-145, LNCaP, 22RV1 and LNCaP-AI, and the normal prostate epithelial cell line PWR1-E. MALAT-1 expression was significantly greater in prostate cancer cell lines than in PWR1-E (fig. 2, A). Of the prostate cancer cell lines
22RV1 and LNCaP-AI showed the highest MALAT1 expression. Thus, we chose these 2 cell lines for further study. We then designed 4 MALAT-1 siRNAs. After transfecting the 2 cell lines with these siRNAs we performed quantitative RT-PCR to determine their efficacy to silence MALAT-1 expression. M5 and M9 showed the highest efficacy with statistical significance (fig. 2, B and C ). Thus, M5 and M9 were chosen for further functional studies. Suppression inhibited human prostate cancer cell growth and colony formation. To investigate the
effects of MALAT-1 suppression on human prostate cancer cell proliferation we transfected 22RV1 and LNCaP-AI cells with M5 and M9. Two, 3, 4, 5 and
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Figure 2. Quantitative RT-PCR of MALAT-1 expression. A, MALAT-1 was over expressed in prostate cancer cell lines vs normal prostate epithelial cell line PWR1-E. B and C, M5 and M9 significantly inhibited MALAT-1 expression. B, 22RV1 cells. C, LNCaP-AI cells. MOCK, sham treated. NC, negative control. KD, knockdown.
6 days after transfection prostate cancer cell proliferation was measured by the CCK-8 assay. Cell proliferation was significantly decreased after MALAT-1 siRNA transfection compared with that in siRNA control transfected and sham treated cells (fig. 3). However, we observed no inhibitory effect of MALAT-1 siRNA in the normal prostate cell line PWR1-E, indicating that the functional effects of MALAT-1 siRNA were limited to MALAT-1 over expressing cells or cancer cells (supplementary fig. 2, http://jurology.com/). Cell colony formation assay showed that MALAT-1 silencing drastically decreased 22RV1 and LNCaP-AI cell proliferation capacity compared with that of sham treated and control cells (fig. 4). These results suggest that MALAT-1 is critical for prostate cancer cell proliferation. MALAT-1 Silencing in Prostate Cancer Induced cell cycle arrest. At 48 hours after MALAT-1 siRNA transfection flow cytometry analysis was performed in all 3 groups of 22RV1 and LNCaP-AI
cells. In the 22RV1 and LNCaP-AI cell lines an increased proportion of G0/G1 phase cells were observed among MALAT-1 knockdown cells compared with that of the sham treated and control groups (fig. 5 and supplementary fig. 3, http:// jurology.com/). Thus, MALAT-1 silencing caused cell cycle arrest in the 22RV1 and LNCaP-AI cell lines. Notably, we also observed a slight increase of G2/M cells in 22RV1 but not in LNCaP-AI upon MALAT-1 knockdown. This suggests that MALAT-1 regulation of the cell cycle may be cell context dependent, for example according to PTEN/P53 status in each cell line examined. Associated with decreased cell migration and invasiveness. In vitro scratch assay was performed
to evaluate the influence of MALAT-1 on cell migration. We seeded the same number of cells in plates before transfection with MALAT-1 siRNAs or scrambled siRNAs. Results were determined at 48 hours. Microscopic analysis of 22RV1 and LNCaP-AI cells revealed that MALAT-1 siRNA
Figure 3. MALAT-1 knockdown attenuated prostate cancer cell growth potential in vitro, as assessed by CCK-8 assay of MALAT-1 downregulation effect on proliferation in 6-day period. Data are shown as mean SD. A, 22RV1 cells. B, LNCaP-AI cells. NC, negative control. MOCK, transfected with transfection reagent. KD, knockdown. OD, optical density.
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Figure 4. MALAT-1 knockdown resulted in lower prostate cancer cell colony formation efficiency, as assessed by colony formation assay. Number of colonies of knocked down M5 and M9 transfected cells (KD) was statistically less than in transfection reagent (MOCK ) and negative control (NC ) groups (p <0.05). A, 22RV1 cells. B, LNCaP-AI cells.
transfected cells had the lowest migratory ability compared with sham treated and normal control cells (figs. 6 and 7). Considering that proliferation was strongly decreased in MALAT-1 knockdown cells, the MALAT-1 siRNA mediated reduced migration effect could have been partially due to the results of decreased proliferation. To evaluate the effects of MALAT-1 on tumor cell invasiveness we transfected prostate cancer cells with MALAT-1 siRNA and performed standard in vitro chamber assays using the Matrigel model. The number of cells that digested Matrigel
and penetrated the Transwell polycarbonate filter significantly decreased when MALAT-1 was silenced (fig. 8). These findings suggest that MALAT-1 has a key role in prostate cancer cell invasiveness.
Figure 5. MALAT-1 knockdown resulted in G0/G1 phase cell cycle arrest. Proportion of 22RV1 and LNCaP-AI cells in different cell cycle phases was quantified by propidium iodide staining, followed by flow cytometry for cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown.
Figure 6. Representative images show 22RV1 and LNCaP-AI cell migration inhibition by MALAT-1 silencing, as assessed by scratch assay at 0 and 48 hours (h). MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Reduced from 10.
Therapeutic MALAT-1 siRNA CRPC Suppressed Growth and Metastasis In Vivo, and Prolonged Tumor Bearing Mouse Survival We further investigated the effect of therapeutic MALAT-1 siRNA on CRPC growth in vivo. Castrated male nude mice bearing 22RV1 xenograft
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Figure 7. Percent quantification of inhibition of cellular migration by MALAT-1 silencing, as assessed by cell migration distance of scratch closure on scratch assay at 0 and 48 hours. A, 22RV1 cells. B, LNCaP-AI cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Single asterisk indicates p <0.05. Double asterisks indicate p <0.01.
tumors were randomly selected and treated with MALAT-1 or control siRNA once every 3 days for 3 weeks by intratumor injection. Mean 22RV1 tumor volume and body weight at baseline were similar in the 2 groups. MALAT-1 siRNA treatment significantly decreased tumor growth, as shown by strongly reduced tumor size and weight compared with control treatment (fig. 9, A and B). To confirm that MALAT-1 was knocked down in vivo by siRNA we measured MALAT-1 expression and found that it was significantly decreased in treated xenograft tumors (fig. 9, C ). The proliferation index Ki67 was also significantly decreased in MALAT-1 siRNA treated tumors and silencing MALAT-1 induced apoptosis in vivo (fig. 10). Importantly, MALAT-1 siRNA treatment led to a significant reduction in metastasis (fig. 9, D). In addition, MALAT-1 siRNA treatment significantly
prolonged the survival of tumor bearing mice (fig. 9, E ). Together these results indicate that targeting MALAT-1 through the intratumor administration of therapeutic siRNA retarded the growth and metastasis of established CRPC xenografts, and significantly extended animal survival.
DISCUSSION In this study MALAT-1 was over expressed in prostate cancer tissues compared with adjacent normal prostate tissues. MALAT-1 was also up-regulated in prostate cancer cells compared with normal prostate epithelial cells. Moreover, MALAT-1 up-regulation correlated with higher Gleason score, PSA and tumor stage. Importantly, MALAT-1 expression was significantly higher in CRPC than in primary prostate cancer. MALAT-1 knockdown resulted in
Figure 8. Boyden chamber assay revealed that MALAT-1 silencing inhibited cell invasion ability. Data are shown as mean SD. A, 22RV1 cells. B, LNCaP-AI cells. MOCK, transfected with transfection reagent. NC, negative control. KD, knockdown. Asterisk indicates p <0.05. Reduced from 10.
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Figure 9. Effect of therapeutic MALAT-1 siRNA on castration resistant 22RV1 xenograft growth in vivo. A and B, MALAT-1 siRNA significantly decreased tumor vs control (siCtrl ) treatment. A, size. B, weight. C, MALAT-1 expression was significantly reduced in vivo (p ¼ 0.03). D, MALAT-1 silencing significantly decreased prostate cancer metastasis. E, Kaplan-Meier graph shows that MALAT-1 therapeutic siRNA prolonged 22RV1 xenograft bearing mouse survival.
decreased cell growth, invasion and migration, and an increased apoptosis rate. Together these findings indicate that MALAT-1 is a key regulator of prostate cancer.
Several biological processes are important in tumorigenesis, such as angiogenesis, cell proliferation and motility, immune response inhibition and apoptosis.25 Emerging studies have proved that
Figure 10. MALAT-1 silencing. Immunohistochemistry revealed decreased Ki67 proliferation index. TUNEL assay showed apoptosis induced in vivo. Ctrl, control. Reduced from 20.
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lncRNAs have important roles in various physiological processes, including embryonic stem cell differentiation, keratinocyte differentiation and cancer.19e23 Recent evidence suggests that MALAT-1 has a pivotal role in the molecular events of tumorigenesis.26 Inspired by these findings, we explored its implications for prostate tumorigenesis. Based on the data presented in this study, we propose that increased MALAT-1 expression may contribute to increased tumor cell growth and invasion. Notably, MALAT-1 is over expressed in androgen dependent and CRPC cell lines, suggesting a consistent positive role for MALAT-1 in the whole process of prostate tumorigenesis and progression. Silencing MALAT-1 resulted in the inhibition of CRPC cell growth and metastasis in vivo. Thus, MALAT-1 targeted therapy may hold great promise for the future. lncRNAs regulate gene expression through various mechanisms, including transcription, posttranscriptional processing, chromatin modification, genomic imprinting and protein function regulation.27,28 Two alternative models of action were proposed for MALAT1, including gene
expression regulation and alternative splicing. Previous studies showed that MALAT-1 promotes cancer progression, mainly through the regulation of gene expression, for example the epithelialmesenchymal transition associated genes, ZEB1, ZEB2 and Slug, and the apoptosis related genes CASP3, CASP8, BAX, BCL2 and BCL2L1.29 Since knockdown MALAT-1 can inhibit prostate cancer migration and invasion, we hypothesized that these effects may be modulated by regulating epithelialmesenchymal transition associated genes.
CONCLUSIONS Our study shows that MALAT-1 is involved in prostate tumorigenesis and progression. MALAT-1 silencing inhibits cell growth, migration and invasion, and induces apoptosis and cell cycle arrest in CRPC cells. This suggests that MALAT-1 should be further evaluated as a promising therapeutic target for CRPC.
ACKNOWLEDGMENTS Bioneer China provided siRNA.
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