|Year : 2015 | Volume
| Issue : 7 | Page : 190-193
Long noncoding ribonucleic acids maternally expressed gene 3 inhibits lung cancer tumor progression through downregulation of MYC
L Yan-hua, L Xiang-lei, L Hong, W Jian-jun
Department of Oncology, Huaihe Hospital of Henan University, Kaifeng, China
|Date of Web Publication||20-Jul-2016|
Department of Oncology, Huaihe Hospital of Henan University, Kaifeng
Source of Support: None, Conflict of Interest: None
OBJECTIVE: Long noncoding ribonucleic acids (RNAs) nowadays emerge as important biomarkers or potential therapeutic targets discussed in human cancers. Among them, maternally expressed gene 3 (MEG3) is known to be decreased in a variety of malignancies. MATERIALS AND METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect the expression of MEG3 in forty pairs of lung cancer (LC) tissues. Overexpression of MEG3 was carried out, and we determined its effect on cell proliferation, apoptosis, and migration evaluated by cell counting kit-8, flow cytometric, and transwell analysis. Messenger RNA and protein expression of MYC were determined by qRT-PCR and western blot, respectively. RESULTS: The expression of MEG3 was downregulated in LC tissues. Forced expression of MEG3 led to reduced abilities of cell proliferation and elevated apoptosis rate. It also slightly inhibited cell migration capacity in vitro. In addition, MYC was inhibited by MEG3 overexpression at both transcriptional and translational levels. CONCLUSION: Our findings revealed MEG3 could regulate LC progression and serve as an important target for LC treatment.
Keywords: Lung cancer, maternally expressed gene 3, MYC, tumor progression
|How to cite this article:|
Yan-hua L, Xiang-lei L, Hong L, Jian-jun W. Long noncoding ribonucleic acids maternally expressed gene 3 inhibits lung cancer tumor progression through downregulation of MYC. Indian J Cancer 2015;52, Suppl S3:190-3
|How to cite this URL:|
Yan-hua L, Xiang-lei L, Hong L, Jian-jun W. Long noncoding ribonucleic acids maternally expressed gene 3 inhibits lung cancer tumor progression through downregulation of MYC. Indian J Cancer [serial online] 2015 [cited 2019 Aug 22];52, Suppl S3:190-3. Available from: http://www.indianjcancer.com/text.asp?2015/52/7/190/186579
| » Introduction|| |
Lung cancer (LC), consisting of nonsmall cell LC (NSCLC) and small cell LC, is one of the most frequent neoplasms and the leading cause of cancer-related deaths all around the world. , Specifically, NSCLC accounts for nearly 85% of all LC cases.  Although advances have been made recently in the clinical as well as experimental oncology, the current prognosis for LC patients is still unsatisfactory, with a 5-year survival rate of only 11%.  Hence, digging out more about the underlined mechanisms of LC pathogenesis and developing other efficient strategies are essential to better early-stage diagnosis, treatment, and prevention of this disease. The finding of noncoding ribonucleic acids (RNAs) involved in LC progression provides new insights into the biology of LC.
High-throughput transcriptome gene expression analysis revealed that more than 90% of the total mammalian genome can be transcribed and may yield many short or long noncoding RNAs (lncRNAs) with limited or no protein-coding capacity, ,, among which microRNAs have been intensively studied and function as crucial tumor suppressors or oncogenes involved in multiple cellular processes. ,,, lncRNAs are over 200 nucleotides in length and are less well characterized in cancer research. To date, more than 3000 lncRNAs have been discovered; however, functions for only 1% of them have been interpreted. Not until recently, it is frequently discussed the roles of lncRNAs in malignancies: Regulation of cell apoptosis, proliferation, survival, and metastasis. ,,
Maternally expressed gene 3 (MEG3), an lncRNA, is widely expressed in multiple normal tissues, especially in brain and pituitary and is thought to be a tumor suppressor in thyroid carcinoma,  colorectal cancer,  gastric cancer,  and cervical cancer.  However, it was less investigated in LC. Lu et al. proved for the first time that MEG3 could inhibit NSCLC cell proliferation and induce apoptosis by affecting p53 expression.  Recently, other two groups almost simultaneously proved that MEG3 contributes to cisplatin resistance of LC. , However, still little is known about the expression and mechanisms of MEG3 in LC progression. Studying the functions of MEG3 might open new avenues into the biology of LC and provide novel possibilities for LC intervention.
| » Materials and Methods|| |
Lung cancer tissues
LCs and their adjacent normal tissue were obtained between 2012 and 2015 from forty NSCLC patients undergoing surgery at Huaihe Hospital of Henan University. Tissue samples were immediately snap-frozen in liquid nitrogen, and stored in −80°C until RNA extraction. The use of the tissue samples for all experiments was approved by all patients and by the Ethics Committee of the Institution.
Cell cultures and cell transfection
Human LC cell lines H1299 was provided by American Type Culture Collection (Manassas, VA, USA) and was maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen, Life Technologies, Germany) with 10% fetal bovine serum (FBS). The same cells were transfected with MEG3 constructs or control plasmids at a final concentration using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's instructions.
The coding sequence of MEG3 was cloned into pCDNA3.1 (Invitrogen) vector. Ectopic expression of MEG3 was performed using the pCDNA-MEG3 transfection and empty pCDNA control vector was used as a control.
Quantitative reverse transcription-polymerase chain reaction
Total RNA was extracted from forty pairs of NSCLC tissue samples using the TRIzol reagent, and the reverse transcription reactions were carried out by M-MLV Reverse Transcriptase kit. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using a standard protocol from the SYBR Green PCR kit (Toyobo, Osaka, Japan) on ABI 7500 (Applied Biosystems, US). β-actin was used as internal control for lncRNAs. ΔCt values were normalized to β -actin levels. Each sample was analyzed in triplicate.
Western blot assay and antibodies
Cells protein lysates were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to 0.22-μm nitrocellulose membranes, and incubated with specific antibodies. β-actin antibody was purchased from sigma and was used as a control; anti-MYC (1:1000) was purchased from Santa Cruz.
Cell proliferation assay
H1299 cells were incubated in 10% cell counting kit-8 (Dojindo, Japan) diluted in DMEM medium at 37°C until visual color conversion occurred. Proliferation rates were determined at 24, 48, and 72 h after transfection. The absorbance of each well was measured with a microplate reader set at OD450. All experiments were performed in triplicate.
Cell apoptosis assay
To detect the apoptosis of H1299 cells, flow cytometric analysis was applied with Annexin-V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions. Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells, and then the relative ratio of apoptotic cells was compared with control transfection group.
Transwell chamber system (Applied Biosystems, USA) was used to investigate the effects of MEG3 on cellular migration. The 1 × 10 4 cells were plated onto the upper chamber, and 600 μl DMEM medium without FBS was added in the bottom chamber. Twenty-four hours later, the cells at the bottom membrane were fixed, stained, and counted under a microscope.
Statistical analysis was performed using SPSS version 21.0 software (Chicago, IL, USA). The t-test was performed to explore the changes between the MEG3 overexpression and control. P < 0.05 was considered to be statistically significant.
| » Results|| |
The expression level of maternally expressed gene 3 was decreased in lung cancer patients
Although the expression of MEG3 has been shown to be downregulated in NSCLC tissues previously,  it still needs to be confirmed finely. To address more about its expression and function in LC cells, qRT-PCR was firstly performed in our study to investigate the altered expression level of MEG3 in LC tissues collected from the clinical LC patients. The paired normal adjacent tissues were also collected and were used as the controls. β-actin was used as the internal controls. The results came out that in forty cases of cancerous LC tissues, MEG3 expression was significantly decreased than the levels of matching normal specimens [Figure 1]. This finding was consistent with the former study  and indicated that MEG3 might function as a tumor suppressor in LC cells.
|Figure 1: The expression of maternally expressed gene 3 was downregulated in lung cancer tissues. The expression of the level of maternally expressed gene 3 was detected in forty lung cancer patients by quantitative reverse transcription-polymerase chain reaction. Data were presented as fold change of lung cancer tissues relative to adjacent normal tissues|
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Effect of maternally expressed gene 3 on malignant phenotypes of H1299 cells
To study the role of MEG3 in LC cell growth, we next analyzed the effect of MEG3 on H1299 cells. Forced expression of MEG3 was achieved by pcDNA-MEG3 plasmid transfected into the H1299 cells. Left panel of [Figure 2]a observed the expression effect of MEG3 after transfection of indicated plasmids. As shown in [Figure 3]a, overexpression of MEG3 in H1299 significantly inhibited cell proliferation since 48 h after transfection. We also found that its overexpression led to obvious induction of apoptosis [Figure 3]b, indicating apoptosis was a contributing factor to cell growth inhibition of MEG3. Since migration and invasion of tumor cells are a significant aspect of tumor progression, we further determined whether MEG3 might also regulate LC cell migration. We performed the transwell method to evaluate the migration capacity of H1299 cells after MEG3 overexpression. As shown in [Figure 3]c, MEG3 slightly suppressed cell migration capacity as well.
|Figure 2: MYC was inhibited by maternally expressed gene 3 overexpression at both transcriptional and translational levels. (a) Quantitative reverse transcription-polymerase chain reaction analysis of MYC after pCDNA-maternally expressed gene 3 or control vector transfected into H1299 cells. (b) Western blot analysis of MYC after pCDNA-maternally expressed gene 3 or control vector transfected into H1299 cells. **P < 0.01|
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|Figure 3: Overexpression of maternally expressed gene 3 leads to reduced abilities of cell proliferation, migration, and induces apoptosis. (a) At 24 h after transfection, cell counting kit-8 assay was performed to determine the proliferation of H1299 cells. (b) H1299 cells were transfected with control vector or pCDNA-maternally expressed gene 3. Forty-eight hours after transfection, cells were stained and analyzed by flow cytometry. (c) Transwell analysis of H1299 cells after treatment with pcDNA-maternally expressed gene 3 or control vector. The data are presented as the mean ± standard deviation, and the error bars represent the standard deviation obtained from three independent experiments. **P < 0.01|
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Maternally expressed gene 3 suppressed MYC expression
Although previous studies have demonstrated that MEG3 could regulate p53 activation, thus to control tumor cell transformation, , we considered if other mechanisms existed to explain the roles of MEG3 in LC cells. We investigated whether the well-known oncogene, MYC might be regulated by MEG3, thus to explain the tumor suppressor activity of MEG3 in the LC cells. qRT-PCR revealed a significant overexpression of MEG3 after pcDNA-MEG3 transfection [Figure 2]a, left panel]. Meanwhile, right panel of [Figure 2]a and b revealed that in the MEG3-overexpressed cells, MYC was significantly downregulated than the control cells. Hence, our results provided novel clues to explain the tumor suppressive role of MEG3 in LC cells.
| » Discussion|| |
In our study, we demonstrated that lncRNA MEG3 was downregulated in clinical LC tissues, which was consistent with the former study in NSCLC.  Therefore, our study once again proofed the abnormal expression of MEG3 in LC cells. In vitro functional analysis showed that forced expression of MEG3 contributed to reduced cell proliferation ability and enhanced cell apoptosis rate. However, the impact of its overexpression on LC cell migration was merely indicating its influence on other aspects of tumor progression, including cell proliferation and apoptosis, was specifically, which might be interpreted by its targeting molecules in these aspects of cellular processes. Importantly, we found that MEG3 could negatively modulate the expression of MYC, and this regulation might be used to explain the tumor suppressive roles of MEG3 in LC cells. Although evidence limited, we still provide crucial and novel clues for understanding the function of MEG3 in LC.
Genome-wide surveys have shown that 90% of the genome is actively transcribed, yielding many short- and long-ncRNAs, which are with limited or no protein-coding capacity. ,, Although initially seemed as transcriptional noise, recent understanding of these ncRNAs of the human genome reveals crucial functions in various cellular development and human diseases. Among them, the aberrant expression and regulation of lncRNAs were recently thought as important modulators in tumor progression. Nevertheless, the molecular basis is still not well known. Therefore, the relationship between proteins and specific lncRNAs is a hot topic in the field of tumor biology, in which lncRNAs may represent as the missing clues of the well-known oncogenic or tumor suppressor network.
Our study highlights the regulation of the famous oncogene MYC by lncRNA MEG3 and provides another mechanism to explain the role of MEG3 in LC cells. However, is this regulation direct, how MEG3 suppresses its expression are questions need to be further answered. Furthermore, could restore of MYC reverses the effect of MEG3 in H1299 cells? This point needs to be quickly characterized. In this study, we found a slightly impact of MEG3 on LC cell migration, could downregulation of MYC be responsible for this phenotype? We observed all above the MEG3-induced phenotypes in H1299 cells. Whether it was the same in other subtypes of LC cell lines? In the following research, we will focus more about the indicated questions more seriously.
Taken together, our findings in this study proved once again the significant downregulation of lncRNA MEG3 in LC tissues, and showed its tumor suppressor role in H1299 cells by inhibiting cell growth, slightly inhibiting cell migration and inducing apoptosis. More importantly, we showed the negative regulation of MYC expression by MEG3, which might be responsible for the functions of MEG3 in the tested cells.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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