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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 575-580
High expression of lncRNA SNHG1 in prostate cancer patients and inhibition of SNHG1 suppresses cell proliferation and promotes apoptosis


1 Department of Urology, The Second Affiliated Hospital, University of South China; Department of Urology, The Third People's Hospital of Yongzhou City, Hunan, China
2 Department of Urinary Surgery, Hospital of Xi'an, Xi'an China, China
3 Department of Urinary Surgery, Tongren Hospital of Shanhai, Shanghai, China
4 Department of Surgery, Bao Ji Tr aditional Chinese Medicine Hospital of Baoji, Baoji, Shannxi, China
5 Department of Urology, The Second Affiliated Hospital, University of South China, Hunan, China

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Date of Submission06-Aug-2019
Date of Decision25-Feb-2020
Date of Acceptance11-Mar-2020
Date of Web Publication28-Oct-2020
 

   Abstract 


Objective: This study aimed to investigate the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in prostate cancer (PCa) patients and to assess the effects of SNHG1 on PCa cell proliferation and apoptosis. Materials and Methods: A total of 134 PCa patients were randomly included from patients who underwent surgical resection at our hospital from October 2015 to December 2016. The SNHG1 expression levels in PCa tissues and paired adjacent non-cancerous tissues were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The association of the SNHG1 expression with clinical-pathological features of PCa patients was summarized and evaluated. A short interfering (si) RNA targeting SNHG1 and pcDNA3.1-SNHG1 were transfected into PC3 and DU145 PCa cell lines, and transfection efficiency was verified by qRT-PCR. Cell proliferation and apoptosis were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, respectively. Results: The SNHG1 expression was significantly upregulated in PCa tumor tissues compared with paired adjacent non-cancerous tissues. The SNHG1 expression was obviously associated with the TNM stage, Gleason Score, lymph node invasion, and long-term metastasis mortality rate. Silencing of SNHG1 inhibited cell proliferation and promoted apoptosis in PC3 and DU145 PCa cell lines in vitro, while overexpression of SNHG1 led to opposite results. Conclusion: LncRNA SNHG1 was upregulated and associated with aggressive malignant behavior in PCa progression. SNHG1 might serve as a potential prognostic biomarker and potential therapeutic target for PCa.

Keywords: Apoptosis, long non-coding RNA, proliferation, prostate cancer, SNHG1

How to cite this article:
Tang Q, Li Z, Han W, Cheng S, Wang Y. High expression of lncRNA SNHG1 in prostate cancer patients and inhibition of SNHG1 suppresses cell proliferation and promotes apoptosis. Indian J Pathol Microbiol 2020;63:575-80

How to cite this URL:
Tang Q, Li Z, Han W, Cheng S, Wang Y. High expression of lncRNA SNHG1 in prostate cancer patients and inhibition of SNHG1 suppresses cell proliferation and promotes apoptosis. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 24];63:575-80. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/575/299317





   Highlights Top


  • SNHG1 is significantly upregulated in prostate cancer
  • SNHG1 is associated with tumor progression and prognosis
  • si-SNHG1 inhibits PCa cell proliferation and promotes apoptosis in vitro
  • SNHG1 is a potential prognostic biomarker for PCa patients.



   Introduction Top


Prostate cancer (PCa) is considered one of the most prevalent malignant cancers occurring in males worldwide, leading to an increasing quantity of morbidity and lethality with poor outcomes for patients in recent years.[1],[2],[3] The latest data indicate that the incidence rate of PCa in China has also increased rapidly with rapid development of social economy and changes in dietary habits and the occurrence.[4] Nevertheless, castration-resistant PCa remains the leading cause of mortality. Currently, chemotherapy, radiotherapy, and surgery remain the primary treatment methods for PCa but are often associated with side effects, potential bleeding, and high recurrence rates.[5],[6] Therefore, it is urgent to elucidate detailed underlying molecular mechanisms and discover novel biomarker and molecular targets for PCa early diagnosis and treatment.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are longer than 200 nucleotides without evident protein-coding function.[7],[8] Emerging evidence has revealed that abnormal expression of lncRNAs has been explored in a variety of human diseases including cancers.[9] As oncogenes or tumor suppressors, lncRNA is now known to be involved in tumor progression, invasion, proliferation, and metastasis.[10],[11] Therefore, lncRNAs may serve as novel biomarkers for cancer diagnosis and molecular therapy.

Small nucleolar RNA host gene 1 (SNHG1), a new-found lncRNA located at chromosome 11q12.3, has been demonstrated to be upregulated and plays a tumor-promoting role in several cancers, such as hepatocellular carcinoma,[12],[13] lung cancer,[14] esophageal carcinoma,[15] colorectal cancer,[16],[17] and prostate cancer.[18] Li et al.[18] confirmed that SNHG1 was overexpressed in prostate tumor tissues and negatively regulated miR-199a-3p to enhance CDK7 expression and promote cell proliferation. However, the role of SNHG1 in PCa patients is not yet clear, especially for the correlation between SNHG1 and clinicopathological features or prognosis.

In this study, our results showed that SNHG1 was upregulated in PCa. Furthermore, SNHG1 expression was correlated with the TNM stage, Gleason Score, lymph node invasion, long-term metastasis, and death. Subsequently, we demonstrated that knockdown of SNHG1 inhibited cell proliferation and promoted apoptosis in PC3 and DU145 PCa cell lines in vitro. Our study uncovered that SNHG1 plays an important role in PCa tumorigenesis and suggested the potential value of SNHG1 in clinical diagnosis and treatment of PCa.


   Materials and Methods Top


Patients and tissue specimens

PCa tissues and paired adjacent non-cancerous tissues were obtained from 134 patients who underwent surgical resection at our hospital from October 2015 to December 2016. All patients were diagnosed and confirmed as PCa by histological analysis confirmed by a pathologist. None of the patients received preoperative chemotherapy before surgery. Demographic and clinical data including age, gender, TNM stage, Gleason Score, lymph node invasion, long-term metastasis, and mortality of the patients during hospitalization were collected. Tissue specimens were immediately kept in RNA keeper tissue stabilizer (Vazyme) after surgery and then stored at -80°C until RNA extraction. Written informed consent was obtained from all patients. All experimental procedures were approved by the Ethics Board of the Bao Ji Traditional Chinese Medicine Hospital of Baoji.

Cell lines and culture

PCa cell lines, DU145 and PC3, were purchased from American Type Culture Collection (ATCC, Rockville, MD). PCa cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin (Sigma-Aldrich Co, USA). Cell lines were maintained in a 5% CO2 humidified atmosphere at 37°C.

Transfection

Small interfering RNA (siRNA) targeting SNHG1 (si-SNHG1), pcDNA3.1-SNHG1, and corresponding negative controls (NCs) were specifically synthesized by GenePharma Co., Ltd. (Shanghai, China). The sequences included as following: si-SNHG1: 5'-GAGAGC TCTGTT GTTGCAA TGTTCA-3'; si-NC: 5'-GAGTCTCGTTG CGTTGTAATG ATCA-3'. Cells were seeded in 6-well plates in antibiotic-free media overnight, and then transfections were performed using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Cells were incubated in the media containing the transfection mixture. All transfection experiments were performed with 50 nM of each siRNA.

RNA extraction and real-time quantitative PCR

Total RNA was isolated from tissues and cultured cells using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quality and quantity were evaluated with the NanoDrop 1000 (ThermoFisher, Santa Clara, USA). The first strand cDNA was synthesized using a reverse transcriptase Kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's guide. qRT-PCR (quantitive real-time PCR) was performed in triplicate with LightCycler2.0 (BIO-RAD) on a LightCycler 480 System (Roche Diagnostics, Thebarton, Australia). Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The primers are below: SNHG1: Forward: 5'-GGGGTACCG TTCTCATTTTTCTACT GCTCGTG-3' and reverse: 5'-CGGGATCCATG TAATCAATCATTTTAT TATTTTCATC-3'. GAPDH: Forward: 5'-CGCTGAGTACGT CGTGGAGT-3'; reverse: 5'-CGTCAAAGGTGG AGGAGTGG -3'. The real-time PCRs were performed in triplicate and fold changes were calculated by the relative quantification (2–ΔΔCt) method.

MTT Assay

The PC3 cells were seeded at a density of 5 × 103 cells/well (DU145 cell, 3 × 103 cells/well) into a 96-well culture plate and cultured in an incubator for different lengths of time. Cell viability was measured by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Life Technologies, Waltham, MA, USA) as follows: 100 μL MTT-medium mixed solution (0.5 mg/ml) was added to each well and incubated with cells at 37°C for 4 h. The MTT solution was then discarded and 200 μL dimethyl sulfoxide (DMSO) was added to dissolve the formazan sediment. Finally, the optical density was detected using a microplate reader (Molecular Devices) at an absorption wavelength of 490 nm.

TUNEL assay

The apoptosis of PCa cells was determined by the terminal deoxyribonucleotidyl transferase-mediated terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick end labeling (TUNEL) system (Promega, Madison, WI, USA). TUNEL staining was performed according to the manufacturer's instructions. In brief, cultured PCa cells on cover slides were fixed with 4% paraformaldehyde for 25 min, pretreated with protease K (10 mg/mL, Sigma, USA) for 20 min, and blocked with 3% bovine serum albumin for 20 min at room temperature. The slides were then incubated with TUNEL reaction mixture for 1 h at 37°C. A light microscopy (Olympus, Japan) was used to image the TUNEL-positive cells (apoptotic cells).

Statistical analysis

All experiments were repeated at least three times and results were expressed as means ± standard deviation (SD). Statistical package for the social sciences 18.0 (SPSS, Inc., Chicago, IL, USA) software was used to perform statistical analyses. Chi-square test was used to compare the counting materials and rates. Continuous data were compared using the two-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by the Tukey posthoc test. For survival analysis, all course death for patients was recorded and the follow-up lasted for 1 year through clinical visit or telephone. Kaplan–Meier curve was performed for analysis of 1-year mortality. P < 0.05 was considered statistically significant.


   Results Top


LncRNA SNHG1 expression was upregulated in PCa tissues

To investigate the role of SNHG1 in PCa development, we detected the expression of SNHG1 in 134 pairs of PCa tumor tissues and paired adjacent non-cancerous tissues by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that the expression level of SNHG1 in PCa tumor tissues was significantly increased compared with matched adjacent non-cancerous tissues [Figure 1]. This result suggested that SNHG1 might act as an oncogene in PCa.
Figure 1: LncRNA SNHG1 expression was upregulated in PCa tissues. The levels of SNHG1 in clinical tissue specimens (n = 134) were assessed by qRT-PCR, using GAPDH as a normalization control. ***P < 0.001 compare to control group

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Correlation between lncRNA SNHG1 expression and clinicopathological features of PCa patients

To further explore the oncogenic function of SNHG1 in PCa patients, the association between the clinicopathological characteristics of PCa patients and SNHG1 expression was analyzed [Table 1]. According to the mean value of SNHG1 expression level, all patients were divided into high SNHG1 expression group (n = 78) and low SNHG1 expression group (n = 56). The relationship between the SNHG1 expression level and the survival of PCa patients was detected. Results of Kaplan–Meier analyses indicated patients with high levels of SNHG1 had significantly worse overall survival compared with the low SNHG1 group (P < 0.05; [Figure 2]). In addition, the SNHG1 expression was positively corrected with TNM stage (P = 0.034), Gleason score (P = 0.008), lymph node invasion (P = 0.028), long-term metastasis (P = 0.016), and 1-year mortality (P = 0.027) in PCa patients. However, there were no obvious correlations between SNHG1 expression and age or gender.
Table 1: Clinical characteristics in patients with different expression of SNHG1

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Figure 2: Kaplan–Meier analysis and log-rank test presented the 1-year survival rate of PCa patients with high expression or low expression levels. Data are presented as the mean ± SD

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Silencing of lncRNA SNHG1 inhibits PCa cell proliferation in vitro

To determine whether SNHG1 affects PCa cell proliferation in vitro, we overexpressed or knockdown the expressions of SNHG1 transfection with si-SNHG1 or pcDNA3.1-SNHG1 in PC3 and DU145 cells, respectively [Figure 3]a. MTT assayresults indicated that SNHG1 knockdown (si-SNHG1) inhibited the proliferation vitality, while overexpression of SNHG1 promoted the cell proliferation in both PC3 and DU145 cells at 24 (P < 0.05), 48 (P < 0.05), and 72 (P < 0.05) hours after transfection compared with negative control (NC) [Figure 3]b.
Figure 3: Silencing of lncRNA SNHG1 inhibits cell proliferation in PCa cells. (a) qRT-PCR confirmed the transfection efficiency after transfection with si-SNHG1, pcDNA3.1-SNHG1 or NC in PC3 and DU145 cells. (b) MTT assay showed the proliferation vitality of PC3 and DU145 cells transfected with si-SNHG1, pcDNA3.1-SNHG1 or NC. Data are presented as the mean ± SD for three replicate determination. *P < 0.05, **P < 0.01, ***P < 0.001 compare to the control group

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Silencing of lncRNA SNHG1 induces PCa cell apoptosis in vitro

To further investigate whether the effect of SNHG1 on colorectal cancer cell proliferation reflected cell apoptosis, we performed TUNEL staining assays in PC3 and DU145 cell lines. The results showed that PC3 and DU145 cells transfected with SNHG1 siRNA had obvious higher apoptotic rates in comparison with control cells [Figure 4]a. Quantitative results of TUNEL assays are shown in [Figure 4]b.
Figure 4: Silencing of lncRNA SNHG1 induces cell apoptosis in PCa cells. (a) TUNEL staining assays were performed to analyze cell apoptosis after lncRNA SNHG1 knockdown or overexpression. The images of TUNEL positive cells were captured by a fluorescence microscope (200×). (b) Quantitative results of the TUNEL assay were analyzed. Representative images and data based on three independent experiments and data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compare to the control group

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   Discussion Top


PCa is a common malignant tumor in men worldwide, leading to serious outcomes including difficulty in urinating, blood in the urine, and death.[19] Despite multiple measures of treatment for PCa cancer, it is also urgent to identify new biomarkers and therapies to enhance treatment outcomes. Currently, more and more emerging lncRNAs have been confirmed to participate in PCa progression, including SNHG1.[18] Up to now, few studies focused on the correlation between SNHG1 and clinicopathological features or prognosis. Hence, we performed a series of validation and functional experiments to investigate the role of SNHG1 in the PCa process.

LncRNA SNHG1, a new lncRNA located on chromosome 11 and containing 11 exons, could be identified as a potential prognostic marker and therapeutic target in various cancers. Zhanget al. showed that increased expression of lncRNA SNHG1 predicted a poor prognosis and exacerbated hepatocellular carcinoma by suppressing miR-195 in hepatocellular carcinoma.[12],[13] Cuiet al. revealed that upregulated lncRNA SNHG1 contributed to the progression of non-small cell lung cancer through inhibition of miR-101-3p and activation of the Wnt/beta-catenin signaling pathway.[14] Yan et al. suggested that lncRNA SNHG1 promoted the expression of proto-oncogene CST3 through sponging for miR-338 in primary esophageal cancer cells.[15] Furthermore, SNHG1 was also found to be involved in colorectal cancer and neuroblastoma.[16],[17],[20] By using qRT-PCR, we found that SNHG1 expression was obviously upregulated in PCa tumor tissues in comparison with matched adjacent non-cancerous tissues, which was consistent with those of previous studies of other tumors[12],[13],[14],[15],[16],[17],[18],[20] suggesting that SNHG1 might play an important role in the development of PCa.

In the study, we investigated the clinical significance of SNHG1 in PCa patients for the first time. We also found that the relative expression level of SNHG1 was associated with TNM stage, Gleason score, lymph node invasion, long-term metastasis, and death, which is similar to findings in other tumors.[13],[20] However, SNHG1 expression was not correlated with patients' age, gender, and other clinicopathological features. In addition, SNHG1 overexpression was associated with lower overall survival rates and could be an independent prognostic factor in PCa patients. These results indicated that SNHG1 expression was an independent prognostic factor for PCa patients. Taken together, these findings supported our previous hypothesis that SNHG1 might play an important role in the development and progression of PCa.

Based on clinicopathological data of SNHG1, we suppose SNHG1 might also regulate the growth and apoptosis of PCa cells. To further understand the underlying mechanism of SNHG1 in PCa progression, experiments in vitro were conducted. Knockdown of SNHG1 significantly decreased proliferation and increased apoptosis of both PC3 and DU145 cells compared with the control group, which indicated that downregulation of SNHG1 could suppress the development of PCa. In summary, these results indicated that SNHG1 could function as an oncogenic gene or risk factor through regulating cell proliferation and apoptosis in the PCa tumorigenesis, which is consistent with existing literature.[18] This study also has some limitations. First, the cases included were limited and the follow-up only lasted for 1 year, which might bring study bias; second, the downstream signaling pathways of SNHG1 were not clear yet; third, the effects of SNHG1 on cell invasion and migration, as well as chemoresistance were not clear. Further studies are required to overcome these limitations.

In conclusion, lncRNA SNHG1 was upregulated in PCa tissues and predicted a poor prognosis in PCa patients. The downregulation of SNHG1 inhibited PCa cells proliferation and promoted apoptosis. Thus, SNHG1 may serve as a novel predictor of PCa prognosis and potential therapeutic target for PCa treatment.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Yi Wang
The Second Affiliated Hospital, University of South China, Hunan, 421001
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_612_19

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