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 » Introduction
 » Methods
 » Results
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 » Conclusion
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ORIGINAL ARTICLE
Year : 2014  |  Volume : 51  |  Issue : 7  |  Page : 99-102
 

Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer


Tianjin Key Labotatory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China

Date of Web Publication27-Mar-2015

Correspondence Address:
J You
Tianjin Key Labotatory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052
China
Q Zhou
Tianjin Key Labotatory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052
China
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Source of Support: This study was partly supported by the grants from the Key Project from National Natural Science Foundation of China (No. 81000950), National 863 Program (No. 2012AA02A502, 2012AA02A201), National 973 Program (No. 2010CB529405)., Conflict of Interest: None


DOI: 10.4103/0019-509X.154092

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 » Abstract 

Background: Nonsmall cell lung cancer (NSCLC) is the major cause of cancer death worldwide. Increasing evidence shows that noncoding RNAs (ncRNAs) are widely involved in the development and progression of NSCLC. ncRNA small nucleolar RNA host gene 1 (SNHG1) has not been studied in cancer, especially its role in lung cancer remains unknown. Our studies were designed to investigate the expression and biological significance of SNHG1 in lung cancer. SNHG1 may be a novel ncRNA in early diagnosis in lung cancer. Methods: Noncoding RNA SNHG1 expression in 7 lung cancer cell lines was measured by quantitative real-time polymerase chain reaction. RNA interference approaches were used to find the biological functions of SNHG1. The effect of SNHG1 on proliferation was evaluated by cell count and crystal violet stains. Results: Noncoding RNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells. In addition, in vitro assays our results indicated that knockdown of SNHG1 inhibited cell proliferation. Conclusions: Our data indicated that ncRNA SNHG1 is significantly upregulated in NSCLC cell lines and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.


Keywords: Nonsmall cell lung cancer, proliferation, small nucleolar RNA host gene 1


How to cite this article:
You J, Fang N, Gu J, Zhang Y, Li X, Zu L, Zhou Q. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer. Indian J Cancer 2014;51, Suppl S3:99-102

How to cite this URL:
You J, Fang N, Gu J, Zhang Y, Li X, Zu L, Zhou Q. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer. Indian J Cancer [serial online] 2014 [cited 2020 Aug 9];51, Suppl S3:99-102. Available from: http://www.indianjcancer.com/text.asp?2014/51/7/99/154092

§You J and Fang N contributed equally to this work



 » Introduction Top


Lung cancer is the most common cause of cancer death, and the majority of lung cancer cases are nonsmall cell lung cancer (NSCLC), which accounts for approximately 80% of lung cancer. [1],[2] Though the enormous improvements made in chemotherapy and radiotherapy over the past few decades, the prognosis of lung cancer is unfavorable. [3] The 5-year overall survival rate is still <15%. [3] Therefore, it is urgent to reveal new prognostic markers for early diagnosis and therapeutic strategies to advantage treatment of NSCLC.

Noncoding RNAs (ncRNAs) are generally regarded as untranslated regulatory RNA molecules. In humans, nonprotein coding sequences comprise at least 98% of the total genome. Similar to the gene/protein regulators, recent evidence demonstrates that ncRNAs also have functional importance, and one ncRNA plays an important role to mediate one or more genes to affect in development and diseases. In addition to well-known ncRNAs, such as ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), the remaining ncRNAs can be broadly classified as small (<200 nt) and a large class of long (>200 nt) ncRNAs (lncRNAs). [4],[5],[6],[7],[8],[9] Small RNAs contain microRNAs (miRNAs), piwi-interacting RNAs, small nuclear RNAs, small nucleolar RNAs (snoRNAs), and promoter-associated small RNAs.

Small nucleolar RNAs may be divided into two classes: The H/ACA and the C/D box, regulating pseudouridylation and 2'O-methylation of specific sites, respectively. [10],[11] The methylation reaction is especially guided by an extensive region (10-21 nt) of complementarity between the C/D box snoRNA and rRNA sequences flanking the modification site. [12],[13],[14],[15] In mammals, snoRNAs are transcribed by the RNA polymerase II which is localized within the introns of small nucleolar RNA host genes (SNHG1). These host genes are also transcribed for either protein-coding or noncoding mRNAs [16] which often contain a 5' terminal oligopyrimidine (5' TOP) sequence responsible for their translational upregulation in response to growth factors or other conditions requiring increased protein synthesis; [17] however, the accurate function of 5' TOP motif with respect to snoRNA synthesis is unknown. [18] Moreover, the process of three-dimensional maps of the modified nucleotides in the ribosomes of  Escherichia More Details coli and yeast has found that rRNA modifications exist in conserved and functionally important regions for subunit-subunit and nascent protein interactions, for tRNA and mRNA binding, but not in those interacting with proteins. [19],[20] This correlation suggests that modifications affect both the structure and the function of the ribosome. [21] Thus, there is evidence that posttranscriptional rRNA modifications, containing pseudouridylation and methylation, affect ribosomal function [22],[23],[24] and that alterations in this modification pattern might be involved in human diseases, such as ribosomopathies and tumorigenesis. [25],[26]

This study was undertaken to examine the molecular developments by which the IR exposure may elicit bystander responses. Our goal was to examine ncRNA expression in lung cancer cells. Changes in ncRNA levels have been linked to many human diseases. Few reports have demonstrated that SNHG1 have been found in cancer, especially in lung cancer. SNHG1 may be the therapeutic target in lung cancer.


 » Methods Top


Cell lines and cell culture

Seven cell lines of NSCLC (NL9980, L9981, A549, H1299, H460, SK-MES-1, YTMLC-90) and a normal human bronchial epithelial cell (BEAS-2B). All cells were obtained from our institute. All cells were maintained in the Dulbecco's modified Eagle's medium supplemented with 2 mmol/L L-alanyl-L-glutamine solution, 1.1% antibiotic antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum, at 37°C, 5% CO2 incubator.

Cell transfection

The sequences of SNHG1-specific siRNAs (si-SNHG1-126, si-SNHG1-468, si-SNHG1-862) were 5'- CUUAAAGUGUUAGCAGACATT-3', 5'- GGACAACCUAGCUGUUGAATT-3', 5'- GACCUAAGCUUGUUGCCAAUTT-3', respectively. Human NSCLC H1299 cells were transfected with either 50 nmol si-SNHG1 or si-NC using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's instruction. After 48 h, cells transfected with siRNA were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) to determine the transfection efficiency.

Cell crystal violet stains assay

The transfected cells were cultured in each six-well plate. After fixation in ice-cold 70% methanol for 10 min, 0.1% crystal violet was added into the culture for 5 min. Washing 3 times with phosphate buffered saline (PBS), the cell morphology was examined under the microscope.

Cell count assay

Each cell line was cultured in a 10 cm plate and divided equally into a 6-well plate. After 24 h, they were transfected with SNHG1-expressing siRNA or control NC following the manufacturer's instructions (Life). After transfection for 24 h, cells with 5 × 10 4 /well were seeded in 12-well plates. Total cells were microscopically observed and counted using Vi-CELL analyzer (BECKMAN COULTER, USA) after transfection for 48 h, 72 h, and 96 h.

Real-time quantitative polymerase chain reaction

Total RNA isolated using the mirVana Kit (Applied Biosystems, CA) was subsequently reverse transcribed to cDNA with the stem-loop reverse transcription primer for miRNA detection. Reverse transcription of SNHG1 and internal control U6a was performed using Reverse Transcriptase M-MLV (Takara, Japan). RT-PCR was conducted using SYBR® Premix Ex Taq™ (Takara, Japan) following the manual. The reactions were placed in a 96-well plate, or 384-well plate using a preheated real-time instrument (ABI 7500/7900HT, invitrogen, USA), respectively. Three independent experiments were performed to analyze relative gene expressions, and each sample was tested in triplicate. Ct values were used to calculate the expression of RNA levels. The amount of target gene expression (2 -ΔΔCt ) was normalized using U6a reference.

Statistical analysis

Statistical analyses were carried out using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Results are expressed as mean values ± standard deviations for the indicated number of independent measurements.


 » Results Top


Noncoding RNA expression in lung cancer cells

Through this article, [27] we selected high expression of 20 ncRNAs [Table 1]. Furthermore, we detected the high expression of 20 ncRNAs in lung cancer cell lines (L9981, NL9980, H460, H1299, SK-MES-1, A549, YTMLC-9 and compared with a normal human bronchial epithelial cells, BEAS-2B cells). RT-PCR was performed on 7 cell lines in lung cancer (L9981, NL9980, H460, H1299, SK-MES-1, A549, YTMLC-9 cell lines) and a normal human bronchial epithelial cells (BEAS-2B cells), respectively. As shown in [Figure 1], it was high expression of 5 ncRNAs in lung cancer cell lines compared with normal human bronchial epithelial cells among the 20 ncRNAs. The 5 ncRNAs include LOC100505685, LOC101926959, SNHG1, MIR210HG, LINC00426. The expression of LOC100505685 was up-regulation in L9980, H1299, SK-MES-1, and YTMLC-90 compared with BEAS-2B cell lines. The expression of LOC101926959 was up-regulation in NL9980, L9981, and YTMLC-9 compared with BEAS-2B cell lines. The expression of SNHG1 was up-regulation in NL9980, L9981, H1299, H460, and YTMLC-9 compared with BEAS-2B cell lines. The expressions of MIR210HG were up-regulation in NL9980 and SK-MES-1 compared with BEAS-2B cell lines. The expressions of LINC00426 were up-regulation in L9981, SK-MES-1, and YTMLC-9 compared with BEAS-2B cell lines.
Figure 1: The expressions of 5 noncoding RNA were detected by real-time polymerase chain reaction

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The effect of small nucleolar RNA host gene 1 on cell clone formation

We performed crystal violet assay to measure the effect of SNHG1 on lung cancer cells. After transfection for 7 days, washing 3 times with PBS, the cells were stained immediately. We observed that the ratio of colony formation of si-SNHG1-862 was significant lower than that of control cells, which showed that SNHG1 indeed regulated clone formation ability of lung cancer cells [Figure 2].
Figure 2: H1299 cells were transfected with si-SNHG1-862, si-small nucleolar RNA host gene 1 (SNHG1)-126, si-SNHG1-468 or si-SNHG1- NC. (a) Real-time polymerase chain reaction of si-SNHG1 in H1299 cell line (*P < 0.01). (b) The colony formation of H1299 cell line

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The effect of small nucleolar RNA host gene 1 on the cell proliferation

The cell count assay was used to study cell proliferation. H1299 cells were transfected with si-SNHG1-RNA or negative control RNA prior to the proliferation assay. We observed that after interfering of SNHG1 expression, the proliferation of H1299 cells was significantly inhibited [Figure 3].
Figure 3: H1299 cells were transfected with si-small nucleolar RNA host gene 1 (SNHG1)-862, si-SNHG1-126, si-SNHG1-468 or si-SNHG1-NC for 24 h, 48 h, 72 h, 96 h and 120 h. The harvested cells were counted by Vi- CELL, respectively

Click here to view



 » Discussion Top


Lung cancer is the primary cause of cancer-related deaths worldwide, so finding new molecular targets for its diagnosis, prognosis, and treatment has the potential to improve the clinical strategies and outcomes of this disease. [15] In recent years, many studies have shown that the expression of ncRNAs is aberrant in human cancer. [16] Identification of tumor-associated ncRNAs is critical for understanding the roles of ncRNAs in tumorigenesis and may be important for novel therapeutic targets. [17] In the present study, our attention focused on the ncRNA SNHG1.

In this study, we characterized the function of SNHG1 in NSCLC development and progression. We found that SNHG1 was up-regulated in L9981, NL9980, H1299, H460, and YTMLC-9 cell lines than in human bronchial epithelial cells (BEAS-2B). These results indicated that SNHG1 expression plays an important role in tumorigenesis and progression of NSCLC. Hence, it is necessary to identify the biological function of SNHG1 in NSCLC cells. Then, knockdown of SNHG1 significantly inhibited proliferation of H1299 cells. Our data showed that forced down-regulation of SNHG1 inhibited cellular proliferation, suggesting that SNHG1 may play an oncogenic role in NSCLC.

In summary, the present study, for the 1 st time, suggested that the up-regulation of ncRNA SNHG1 expression was closely associated with occurrence and development of NSCLC. Moreover, we preliminarily suggested a role that ncRNA SNHG1 plays in cell proliferation in lung cancer cells. As a result, these results suggested that SNHG1 may become a novel promising reference for the prognosis and therapy for NSCLC.


 » Conclusion Top


This study elucidates a novel function of SNHG1. Our finding suggests that SNHG1 may be a novel tumor promoter ncRNA. SNHG1 enhanced proliferation in lung cancer cell additionally; SNHG1 may serve as a potential therapeutic candidate in the treatment of lung cancer.


 » Acknowledgments Top


This study was partly supported by the grants from the Key Project from National Natural Science Foundation of China (No. 81000950), National 863 Program (No. 2012AA02A502, 2012AA02A201), National 973 Program (No. 2010CB529405).

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]

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Medical Science Monitor. 2016; 22: 4820
[Pubmed] | [DOI]



 

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