Epigenetically silenced LINC02381 functions as a tumor suppressor by regulating PI3K-Akt signaling pathway

doi:10.1016/j.biochi.2020.02.009

Biochimie

Volumes 171–172, April–May 2020, Pages 63-71

https://doi.org/10.1016/j.biochi.2020.02.009 Get rights and content

Highlights

•
LINC02381 is down-regulated in the CRC tissues.
•
De-methylating and chemotherapy agents affects LINC02381 expression.
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LINC02381 regulates CRC cell proliferation and apoptosis.
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LINC02381 may regulate colorectal cells phenotype via the PI3K signaling pathway.

Abstract

Recent advances have revealed that lncRNAs play important roles in tumorigenesis. However, only a small number of functional lncRNAs have been well characterized, particularly in colorectal cancer. Therefore, more extensive studies are needed to identify and characterize these lncRNAs to better understand cancer progression. In the present study, using available RNA-seq data, we found that LINC02381 (NR_026656.1) differentially expresses in CRC tissues compared to normal pairs. Consistently, RT-qPCR results showed that LINC02381 was down regulated in CRC tissues and also in different cancerous cell lines. CRC cells treatment with de-methylating and chemotherapy agents indicated that DNA methylation of LINC02381 may be responsible for the transcriptional silencing of LINC02381 in colorectal cancer cells. Then, the functional changes of the cells in response to LINC02381 alteration were assessed and the data indicated that LINC02381 up-regulation suppressed cell viability and proliferation while increasing the apoptosis in CRC-originated cell lines. Mechanistically, LINC02381 overexpression was increased PTEN protein levels but decreased phospho-Akt levels. Finally, we proposed a hypothesized model for PI3K signaling regulation by LINC02381. Altogether, the result of this study suggests that LINC02381 might have suppressive effects on human colorectal cancer tumorigenesis partly by regulating PI3K signaling pathway.

Introduction

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related death worldwide with high mortality both in men and women [1,2]. The patient survival rate have improved in recent years, however, due to recurrence and metastasis, prognosis of patients with CRC remains poor [2,3]. Therefore, it’s helpful to find new molecular strategies to improve patient survival.

The data adopted from genomics consortiums, such as ENCODE and FANTOM, shows that most of the human genome is transcribed as noncoding RNAs [4,5]. However, as a big part of noncoding RNAs, long noncoding RNAs (lncRNAs) function in numerous cancers still remains to be clarified. LncRNAs play pivotal roles in a wide variety of biological processes, including differentiation, proliferation and cell death [6,7]. Numerous studies have reported alteration of lncRNAs expression patterns in different cancers, including colorectal cancer (CRC) [[7], [8], [9]]. Down-regulation of some lncRNAs is a common event in progression of cancers that suggests possible tumor suppressive role of these molecules.

The PTEN/PI3K/Akt/mTOR signaling pathway plays a crucial role in the regulation of cancer growth, apoptosis and survival and is frequently deregulated in many cancers [10,11]. PTEN, as a tumor suppressor gene, suppresses the activation of Akt and negatively regulates PI3K signaling pathway [12]. It has been demonstrated that numerous miRNAs, including miR-21, miR-27a, miR-130b, miR-221 and miR-454, can activate PI3K signaling by targeting PTEN transcripts [[13], [14], [15], [16], [17]]. Therefore, identifying the lncRNAs that sponge these PTEN-inhibitory miRNAs may be beneficial to decipher the gene network involved in cancer progression.

In the present study, we aimed to investigate the role of LINC02381 (synonyms: NR_026656.1, LOC400043), an lncRNA located at 12q13.13 region, in human CRC. We found that lncRNA LINC02381 regulated cancer cell proliferation and apoptosis in colorectal cancer partly by regulating the PTEN-mediated PI3K/Akt signaling pathway. The data suggested that LINC02381 might have suppressive effects on human colorectal cancer tumorigenesis.

Section snippets

Specimens and cell lines

U87, A172, LNCaP, 1321, SKNMC, MCF7, SK-BR-3 and 5637 cell lines (obtained from the Pasteur Institute, Iran), were cultured in RPMI-1640 media (Gibco), supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 mg/mL streptomycin (Sigma), followed by incubation in 37 °C with 5% CO2. Fibroblast, ACHN, Hek293, SW480, HCT-116, SKBR3, HepG2, Huh7, AGS, A549 and HT29 cell lines (obtained from the Pasteur Institute, Iran) were cultured in DMEM (Gibco)

LINC02381 is downregulated in cancerous cells

To evaluate LINC02381 expression in cancer, we first analyzed the expression of LINC02381 in different cancer and normal cell lines using quantitative RT-PCR (RT-qPCR). The results indicated that LINC02381 expression level in different cancer-originated human cell lines (including colorectal cancer originated cell lines; SW480 and HCT-116) has been significantly reduced, compared with the fibroblast and HEK293t normal cells (Fig. 1A). Consistently, COAD (Colorectal Adenomacarcinoma) TCGA data

Discussion

Colorectal cancer (CRC) is one of the common lethal disease [1]. Despite the great progress in cancer treatment and diagnosis, patients 5 years’ survival rates is low [2,3]. The stage of colorectal cancer at diagnosis has a major impact on five-year survival rates [29]. Therefore, a detailed understanding of colorectal cancer development and progression need to be explored to improve the cancer diagnosis patient’s survival.

A large body of evidence has demonstrated lncRNAs play key roles in the

Author contributions

M. J, B. S, S. H and M. S designed the experiments; M. J and B. S completed all the experiments; M. J and B. S wrote the first draft of manuscript; S. H and M. S revised its final version.

Financial support

This study was financially supported by the Research Administration of Tarbiat Modares University, Tehran, Iran.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors, hereby, appreciate and thank Tarbiat Modares University for financial and technical supports.

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These reports suggest lncRNAs as key molecules in glioma progression. Here, we found LINC02381 an interesting lncRNA because its role varies with cancer types, as it can accelerate tumorigenecity in cervical cancer and osteosarcoma (Bian et al., 2021; Chen et al., 2020), but can prevent tumorigenecity in gastric and colon cancer (Jafarzadeh and Soltani, 2020; Jafarzadeh et al., 2020). For now, there isn’t any report about LINC02381 function in glioma, but we discovered a predicted relation between LINC02381 and glioma on a bioinformatics website lncRNADisease, suggesting that LINC02381 might be implicated in glioma.
Glioma, featured with high incidence and low survival rate, is the most common type of primary brain tumor, severely affecting human life worldwide. LINC02381 is an interesting lncRNA functioning as oncogenic lncRNA in some cancers but as tumor-suppressor in others, but no report demonstrates its association with and function in glioma. Intriguingly, we found in a bioinformatics website LncRNADisease that LINC02381 was closely related to malignant glioma, so this study aimed to figure out the expression and function of LINC02381 in glioma. By RT-qPCR, we confirmed LINC02381 upregulation in glioma cells. Functional experiments demonstrated that LINC02381 knockdown repressed glioma cell proliferation and induced apoptosis. Boinformatics tools and RT-qPCR revealed the positive correlation between LINC02381 and CBX5 in glioma cells. More importantly, we confirmed that LINC02381 could interact and work synergistically with CEBPβ to bind to CBX5 promoter and activate CBX5 transcriptionally. Additionally, rescue experiments indicated that CBX5 up-regulation reversed the decline in cell proliferation and the augment in cell apoptosis caused by LINC02381 knockdown. To conclude, LINC02381 could facilitate CBX5 transcription via interaction with CEBPβ, thus exerting its oncogenic role in glioma cells, which could contribute to better understanding of glioma.
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A recent report has also linked HMS to noncancer diseases such as rheumatoid arthritis, wherein it was shown that HMS overexpression enhanced cell proliferation of fibroblast-like synoviocytes by sponging miR-590-5p and activating the MAPK signaling pathway (53). Contrary to the above reports, one group has reported that ectopic expression of HMS in colorectal and gastric cancer cells decreased cell viability and colony formation capability and proposed that HMS has tumor-suppressive effects on human colorectal tumorigenesis (54, 55). While this could indicate that HMS has a cancer-specific role, pleiotropic effects resulting from any experimental alterations have to be ruled out.
Long noncoding RNAs (lncRNAs) have been reported to drive key cancer pathways but the functions of majority of lncRNAs are unknown making a case for comprehensive functional evaluation of lncRNAs. With an aim to identify lncRNAs dysregulated in human cancers, we analyzed the cancer patient database of lung adenocarcinoma (LUAD), which revealed an upregulated lncRNA, LINC02381 (renamed HOXC10 mRNA stabilizing factor or HMS in this study), whose depletion results in proliferation defects and inhibition of colony formation of human cancer cells. In order to identify the binding targets of HMS, we screened for cis-genes and discovered that HOXC10, an oncogene, is downregulated in the absence of HMS. Depletion of HMS does not affect the HOXC10 promoter activity but inhibits the HOXC10 3′-UTR-linked luciferase reporter activity. Since lncRNAs have been known to associate with RNA-binding proteins (RBPs) to stabilize mRNA transcripts, we screened for different RBPs and discovered that HuR, an ELAV family protein, stabilizes HOXC10 mRNA. Using RNA pull-down and deletion mapping experiments, we show that HuR physically interacts with the cytosine-rich stretch of HMS and HOXC10 3′-UTR to stabilize HOXC10 mRNA. HOXC10 is overexpressed in many human cancers, and our discovery highlights that lncRNA HMS sustains the HOXC10 mRNA levels to maintain the invasive phenotypes of cancer cells.
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The PI3K/AKT pathway is an intracellular signaling pathway with an indispensable impact on cell cycle control. This pathway is functionally related with cell proliferation, cell survival, metabolism, and quiescence. The crucial role of this pathway in the development of cancer has offered this pathway as a target of novel anti-cancer treatments. Recent researches have demonstrated the role of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in controlling the PI3K/AKT pathway. Some miRNAs such as miR-155-5p, miR-328-3p, miR-125b-5p, miR-126, miR-331-3p and miR-16 inactivate this pathway, while miR-182, miR-106a, miR-193, miR-214, miR-106b, miR-93, miR-21 and miR-103/107 enhance activity of this pathway. Expression levels of PI3K/AKT-associated miRNAs could be used to envisage the survival of cancer patients. Numerous lncRNAs such as GAS5, FER1L4, LINC00628, PICART1, LOC101928316, ADAMTS9-AS2, SLC25A5-AS1, MEG3, AB073614 and SNHG6 interplay with this pathway. Identification of the impact of miRNAs and lncRNAs in the control of the activity of PI3K/AKT pathway would enhance the efficacy of targeted therapies against this pathway. Moreover, each of the mentioned miRNAs and lncRNAs could be used as a putative therapeutic candidate for the interfering with the carcinogenesis. In the current study, we review the role of miRNAs and lncRNAs in controlling the PI3K/AKT pathway and their contribution to carcinogenesis.
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Research paperEpigenetically silenced LINC02381 functions as a tumor suppressor by regulating PI3K-Akt signaling pathway

Highlights

Abstract

Introduction

Section snippets

Specimens and cell lines

LINC02381 is downregulated in cancerous cells

Discussion

Author contributions

Financial support

Declaration of competing interest

Acknowledgements

Dev. Reprod. Biol.

Canc. Cell

Biochim. Biophys. Acta Rev. Canc

FEBS Lett.

Eur. J. Pharmacol.

Gastroenterology

Biochim. Biophys. Acta (BBA) - Mol. Basis Dis.

Worldwide burden of colorectal cancer: a review

Updates Surg.

Global patterns and trends in colorectal cancer incidence and mortality

Gut

Follow-up strategies for patients treated for non-metastatic colorectal cancer

Cochrane Database Syst. Rev.

The landscape of long noncoding RNAs in the human transcriptome

Nat. Genet.

Unique features of long non-coding RNA biogenesis and function

Nat. Rev. Genet.

The emerging role of lncRNAs in cancer

Nat. Med.

The bright side of dark matter: lncRNAs in cancer

J. Clin. Invest.

Role of PI3K/AKT/mTOR in cancer signaling

A pan-cancer proteogenomic atlas of PI3K/AKT/mTOR pathway alterations

Canc. Cell

MicroRNA-21 promotes cell proliferation, migration, and resistance to apoptosis through PTEN/PI3K/AKT signaling pathway in esophageal cancer

Tumor Biol.

MicroRNA-27a promotes tumorigenesis via targeting AKT in triple negative breast cancer

Mol. Med. Rep.

MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway

Sci. Rep.

MicroRNA-221 targets PTEN to reduce the sensitivity of cervical cancer cells to gefitinib through the PI3K/Akt signaling pathway

Tumor Biol.

DNA methylation of the first exon is tightly linked to transcriptional silencing

PloS One

Research paper
Epigenetically silenced LINC02381 functions as a tumor suppressor by regulating PI3K-Akt signaling pathway