MiR-338 suppresses cell proliferation and invasion by targeting CTBP2 in glioma

Abstract

BACKGROUND:

Previous studies indicated that microRNA-338-5p (miR-338-5p) functions as tumor suppressor in some cancer types including glioma. However, the clinical significance and biological function of miR-338-5p in glioma still need to be explored.

METHODS:

We used quantitative real time PCR (qRT-PCR) to detect the miR-338-5p expression in the 44 cases of glioma tissues and adjacent normal tissues. In vitro, CCK8 cell proliferation, cell colony formation, transwell invasion assay and flow cytometry analysis were performed to explore the effects of miR-338-5p on cell proliferation, cell invasion and cell cycle distribution. Dual luciferase assay, qRT-PCR and western blot analysis were applied to validate CTBP2 was a direct target of miR-338-5p in glioma cells.

RESULTS:

we demonstrated that miR-338-5p was significantly lower expression in 44 glioma patients, compared with adjacent normal tissues. MiR-338-5p expression was significantly correlated with glioma grades and Karnofsky Performance Status in patients. We then validated that increased miR-338-5p significantly inhibited the cell proliferation, cell invasion and epithelial-mesenchymal transition (EMT) in vitro. Moreover, Dual luciferase assay results indicated that CTBP2 was direct target of miR-338-5p in glioma cells. Meanwhile, CTBP2 silencing can rescued the phenotype changes induced by miR-338-5p inhibitor on cell proliferation and invasion in glioma.

CONCLUSION:

Our results suggested that miR-338-5p acts as tumor suppressor and could be a potential therapeutic target for glioma.

1. Introduction

Gliomas are the most common types of the central nervous system primary tumors and accounting for more than 50% of all primary human brain tumors [1, 2]. One of the most effective methods for tumor treatment is radiotherapy. Despite some others advances in early stage diagnosis, and larger development of surgery or chemotherapy, the over survival rate of glioma patients remains poor [3, 4]. Thus, to identify the potential molecular mechanism involving in glioma procession and investigate innovative therapeutic strategies is urgently needed.

MicroRNAs (miRNAs) are a series of endogenous and highly conserved non-coding RNAs and about 20–23 nucleotides in length [5]. Previous studies had showed that aberrant expression of microRNA-338 in some cancer types. Xu et al. verify that miR-338-3p sensitizes HCC cells to sorafenib in vitro and in a HCC subcutaneous nude mice tumor model by inhibiting HIF-1 $\alpha$ [6]. MiR-338-3p is negatively associtated with advanced TNM stage and local invasion and lower expression of miR-338-3p was associated with unfavorable outcome in disease-free survival (DFS) [7]. Wang et al. reveals that miR-338-3p is down-regulated in hepatocellular carcinoma tissues and inhibited cell proliferation by target forkhead box P4 (FOXP4) [8]. In gastric cancer, miR-338-3p is significantly down-regulated and inhibits cell proliferation, migration and invasion partially by suppressing ADAM17 expression [9]. In glioma, miR-338-5p sensitizes glioblastoma cells to radiation through regulation of genes involved in DNA damage response [10]. Mir-338-3p inhibits malignant biological behaviors of glioma cells by targeting MACC1 gene [11]. However, the clinical significance and biological function of of miR-338-5p in glioma still need to be explored.

In the study, we verified that miR-338-5p was significantly lower expression in glioma. Increased miR-338-5p inhibited the cell proliferation and invasion by targeting CTBP2. Thus, our results suggested that miR-338-5p acts as tumor suppressor and could be a potential therapeutic target for glioma.

Table 1

Correction between miR-338-5p expression and clinical characteristics

Clinical	Patients	MiR-338-5p expression		$p$ -value
characteristics	number	Low	High
		( $n=$ 23)	( $n=$ 21)
Sex				0.123
Female	24	10	14
Male	20	13	7
Age				0.460
$⩽$ 60	29	14	15
$>$ 60	15	9	6
Tumor size				0.490
$<$ 3 cm	27	13	14
$>$ 3 cm	17	10	7
Tumor location
Parenchyma	32	18	14	0.388
Ventricle	12	5	7
Karnofsky Performance Status				0.007 ${}^{**}$
$⩽$ 80	22	16	6
$>$ 80	22	7	15
WHO grade				0.006 ${}^{**}$
I–II	24	8	16
III–IV	20	15	5
Resection range				0.820
Total resection	28	15	13
Local resection	16	8	8

${}^{**}$ $P<$ 0.05.

Figure 1.

MiR-338-5p was down-regulated in the glioma tissues. (A) The expression level of MiR-338-5p was determined by using qRT-PCR assays in 44 cases of primary glioma tissues and noncancerous tissues. (B)–(C) The association between expression of miR-338-5p and Karnofsky Performance Status (KPS) or WHO grade was shown (D)–(E) CCK8 assays were performed to detect cell proliferation ability in U87 or T98G cells after up-regulation of miR-338-5p at 48 h and 72 h. All experiments were performed at least independently three times, error bars represent SD. ${}^{**}$ $P<$ 0.05.

Figure 2.

MiR-338-5p inhibited cell proliferation in U87 and T98G cells. (A)–(B) Cell colony formation assay was used to evaluated cell proliferation after transfected miR-NC or miR-338 mimic into U87 or T98G cells. (C)–(D) Cell cycle distribution (G0/G1, G2/M, S phase) was assessed by flow cytometry analysis after transfected miR-NC or miR-338 mimic into U87 or T98G cells. All experiments were performed at least independently three times, error bars represent SD. ${}^{**}$ $P<$ 0.05.

Figure 3.

MiR-338-5p inhibited cell invasion and EMT phenomenon in U87 and T98G cells. (A)–(B) Transwell cell invasion assay and cell invasion number was shown after transfected miR-NC or miR-338 mimic into U87 or T98G cells. (C)–(D) The EMT related transcription factor Snail and markers E-cadherin, Vimentin and Fibronectin expression were shown by western blot after transfected miR-NC or miR-338 mimic into U87 or T98G cells. All experiments were performed at least independently three times, error bars represent SD. ${}^{**}$ $P<$ 0.05.

Figure 4.

MiR-338-5p inhibited cell proliferation by targeting CTBP2 in U87 and T98G cells. (A) Illustration of the putative predicted miR-338-5p binding sites in the CTBP2 3’UTR region. (B) Luciferase reporter assays were performed after co-transfecting with pmiR-CTBP2-wt or pmiR-CTBP2-mut and miR-338-5p mimic or miR-NC into HEK 293 cell. (C)–(D) CTBP2 mRNA and protein expression was determined in U87 cells by qRT-PCR or western blots analysis after transfected with miR-338-5p mimic, miR-338-5p inhibitor or miR-NC at 48 h. (E)–(F) CTBP2 mRNA and protein expression was detected in T98G cells by qRT-PCR or western blots analysis after transfected with miR-338-5p mimic, miR-338-5p inhibitor or miR-NC at 48 h. All experiments were performed at least independently three times, error bars represent SD. ${}^{**}$ $P<$ 0.05.

Figure 5.

CTBP2 mediated the inhibition of cell proliferation and invasion induced by miR-338-5p in gliomas cells. (A) CCK8 assays were performed to detect cell proliferation ability in U87 cells after cell were transfected with miR-NC, si-CTBP2 or miR-338-5p inhibitor and si-CTBP2. (B)–(C) Transwell cell invasion assay were performed to detect cell invasion ability in U87 cells after cell were transfected with miR-NC, si-CTBP2 or miR-338-5p inhibitor and si-CTBP2. All experiments were performed at least independently three times, error bars represent SD. ${}^{**}$ $P<$ 0.05. ## $P>$ 0.05.

2. Methods

2.1 Patient tissue samples

A total of 44 cases tumor tissues and paired adjacent noncancerous tissues from primary glioma patients were collected between January 2012 and July 2016 at China-Japan Union Hospital, Jilin University. All the patients were pathologically confirmed as primary glioma by two pathologists. After surgery, the tumor tissues and paired adjacent noncancerous tissues were collected immediately and stored at $-$ 80 ${}^{\circ }$ C for further analysis. Written informed consent was obtained from all the patients according to the guidelines approved by the Ethics Committee of the China-Japan Union Hospital, Jilin University.

2.2 Cell culture and cell transfection

Two human glioma cell lines (U87 and T98G) were obtained from the Shanghai Institute of Life Sciences Cell Resource Center (Shanghai, China). All the cell lines were cultured in DMEM medium (Gibco, USA) and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). The cells were cultured at 37 ${}^{\circ }$ C and 5% CO ${}_2$ . Cell transfections with miR-338-5p mimic, miR-338-5p inhibitor or miR negtive control (NC) (Genomeditech, Shanghai, China) were performed with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

2.3 Cell proliferation assay

Cell proliferation assay were assessed using the Cell Counting Kit-8 assays. Briefly, The 200 $\mu$ L of cells transfected with miR-338-5p NC, miR-338-5p inhibitor or miR-338-5p mimic were seeded on a 96-well plate and the cell concentration was 2 $\times$ 10 ${}^3$ cells/per well. Then, cells were incubated at 37 ${}^{\circ }$ C and cell proliferation ability was measured at 0, 24, 48 and 72 h, the optical density was measured at 450 nm using a microtiter plate reader. For cell colony formation assay, cells were transfected with miR-338-5p NC or miR-338-5p mimic and cultured at 37 ${}^{\circ }$ C for 24 hours. 300 cells/well was seeded on the 12 well plates and cultured at 37 ${}^{\circ }$ C for two weeks. The cell colonies were stained with 1% crystal violet and were counted.

2.4 Cell invasion assay

Cells transfected with miR-338-5p NC or miR-338-5p were resuspended at a density of 1 $\times$ 10 ${}^5$ /ml and then seeded on the chamber in a 8 $\mu$ m transwell chamber (Corning Costar Corp., Cambridge, MA, USA) that was coated with Matrigel (Clontech, Mountain View, CA, USA). The lower chamber was added with 500 $\mu$ l DMEM supplemented with 10% FBS. After 48 hours incubation, cells on the lower were fixed with methanol and stained with 0.1% crystal violet. Invasion cells were calculated in five random fields under the microscope.

2.5 Quantitative real time PCR (qRT-PCR)

Total RNA from tissues and cells that were transfected with miR-338-5p NC, miR-338-5p inhibitor or miR-338-5p mimic was extracted using the Trizol ${}^{\mathrm{\circledR }}$ reagent according to manufacturer’s instructions. The 1 $\mu$ g of total RNA was reversed to cDNA using M-MLV reverse transcriptase (TAKARA). Real-time PCR was performed using SYBR Premix EX Taq (TAKARA). The qRT-PCR assay was performed following procedures: 95 ${}^{\circ }$ C at 5 min, followed by 40 cycles of 95 ${}^{\circ }$ C 5 s, 60 ${}^{\circ }$ C 30 s. GAPDH or U6 was used as an internal control to normalize CTBP2 mRNA expression and miR-338-5p expression. Fold change of mRNA was determined using 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }}$ CT method. Primer sequences for qRT-PCR wasmiR-338-5p-F:5’-ATCCAGTGCGTGTCGTG-3’, miR-338-5p-R:5’-TGCTAACAATATCCTGGTG-3’.

2.6 Western blot analysis

Protein was isolated from cells transfected with miR-338-5p mimic, miR-338-5p inhibitor or miR-338-5p NC and lysed using RIPA buffer (Sigma). The protein centrifuged at 14,000 g for 10 min. Supernatants were obtained and the equal amounts protein were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels. Then, the protein was transferred to PVDF membrane and was blocked with 5% non-fat milk, and incubated at 4 ${}^{\circ }$ C overnight. The membrane was incubated with CTBP2 antibody (1:1000, Santa Cruz, CA, USA), E-cadherin antibody (1:1000, Santa Cruz, CA, USA),Vimentin antibody (1:1000, Santa Cruz, CA, USA), Snail antibody (1:500, Santa Cruz, CA, USA), Fibronectin antibody (1:1000, Santa Cruz, CA, USA) and GAPDH (1:1000, CST, USA). The blot was visualized by enhanced chemiluminescence (Sigma) and analyzed using the Image J software (NIH, Bethesda, USA).

2.7 Luciferase reporter assay

3’UTR of CTBP2 Wild type (WT) and mutant type (MUT) containing the miR-338-5p target sequence was constructed by Biotech Co, Beijing, China and inserted it into pmir-reporter vector (Promega, Madison, WI). HEK-293T cells were co-transfected with miR-338-5p mimic or miR-NC and reporter plasmids by Lipofectamine 3000. Lucferase activity was detected by the dual-luciferase reporter assay system at 48 h (Promega, USA).

2.8 Statistical analysis

The data was analyzed using SPSS software version 20.0 (SPSS, Inc., Chicago, IL, USA) and results were presented as the mean $\pm$ standard deviation (SD). The comparisons between groups were performed by one-way analysis of variance, t-test and $\chi$ 2 test, as appropriate. $P<$ 0.05 was considered as statistically significant difference.

3. Results

3.1 MiR-338-5p is down-regulated in glioma tissues

To determine the role of miR-338-5p in glioma tissues, the qRT-PCR assay was performed. The results confirmed that miR-338-5p expression was dramatically lower in the glioma tissues compared with adjacent normal tissues (Fig. 1A). We then detected the association between miR-338-5p expression and clinicopathological parameters in the 44 cases of patients. The results showed that miR-338-5p expression was correlated with Karnofsky Performance Status (KPS) ( $P=$ 0.007, Fig. 1B, Table 1) and WHO grade ( $P=$ 0.006, Fig. 1C, Table 1) in glioma patients.

3.2 MiR-338-5p suppresses the cell proliferation and cell cycle in glioma

Subsequently, to investigate the effects of miR-338-5p on cell proliferation in vitro, we performed the CCK8 cell proliferation, cell colony formation and flow cytometry analysis in U87 and T98G cells. The present results by CCK8 assay verified that miR-338-5p mimic inhibited cell proliferation ability, compared with the miR-NC group in U87 and T98G cells (Fig. 1D–E). Cell colonies number was significantly decreased in miR-338-5p mimic group, compared with miR-NC group in U87 and T98G cells (Fig. 2A–B). The flow cytometry analysis results showed that miR-338-5p mimic decreased the S phase cell number and increased the G0/G1 phase cell number, compared with the miR-NC group in U87 and T98G cells (Fig. 2C–D).Thus, these results indicated that increased miR-338-5p expression inhibited the cell proliferation in glioma.

3.3 MiR-338-5p inhibits cell invasion and epithelial to mesenchymal transition (EMT) in glioma

We identified whether miR-338-5p affected the cell invasion and epithelial to mesenchymal transition process, the cell were transfected with miR-338-5p mimic or miR-NC in U87 and T98G cells. The results showed that cell invasion ability was significantly inhibited and cell invasive number was notably reduced in miR-338-5p mimic group, compared with miR-NC group in U87 and T98G cells (Fig. 3A–B). Moreover, western blot analysis results verified that transcription factor Snail and EMT maker Vimentin and Fibronectin expression were down-regulated, whereas, E-cadherin expression was up-regulated in miR-338-5p mimic group, compared with miR-NC group in U87 and T98G cells (Fig. 3C–D).Thus, these results showed miR-338-5p could suppress cell invasion and epithelial to mesenchymal transition process in glioma.

3.4 MiR-338-5p inhibits cell proliferation and invasion by targeting CTBP2 in glioma

By searching for miRanda database, we found the possible complementary base pair between seed region of miR-338-5p and the 3’-UTR of CTBP2. We further constructed the 3’UTR of CTBP2 containing miR-338-5p binding sites and a luciferase reporter gene, then inserted it into a pmir-reporter vector to detect whether CTBP2 was a direct target of miR-338-5p (Fig. 4A). Luciferase reporter assays showed that luciferase activities were reduced approximately 35% by co-transfecting with miR-338-5p mimic and pmir-CTBP2-WT, but no change by co-transfecting with miR-338-5p mimic and pmir-CTBP2-MUT in HEK-293T cell (Fig. 4B). Furthermore, we detected whether miR-338-5p affected the mRNA and protein expression of CTBP2 in glioma cells. The results showed that the mRNA and protein expression of CTBP2 was inhibited by transfecting miR-338-5p mimic into U87 or T98G cells (Fig. 4C–D), Furthermore, we investigated whether miR-338-5p inhibitor affected the CTBP2 expression, the results confirmed that CTBP2 was increased by transfecting miR-338-5p inhibitor into U87 or T98G cells (Fig. 4E–F).

We further identified whether CTBP2 played any role in miR-338-3p mediated inhibition of glioma cell proliferation and invasion, we demonstrated that knockdown of CTBP2 can rescue the cell proliferation and invasion changes induced by miR-338-5p inhibitor in U87 and T98G cells (Fig. 5A–C). Thus, these results showed that CTBP2 was a direct target of miR-338-5p and CTBP2 mediated the inhibition of cell proliferation and invasion induced by miR-338-5p in gliomas cells.

4. Discussion

MicroRNAs (miRNAs) play essential roles in many physiological and pathological processes, and are related to cell proliferation, invasion and metastasis in some tumors [12]. MiR-338 had been found to function as a tumor suppressor in several tumors, such as, miR-338-3p inhibited cell epithelial-mesenchymal transition by targeting ZEB2 and MACC1/Met/Akt signaling in gastric cancer [13]. MiR-338-3p suppresses tumor progression through a PTEN-AKT axis by targeting P-REX2a in gastric cancer [14]. MiR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a [15]. In human colorectal carcinoma, miRNA-338-3p suppresses cell growth by targeting smoothened [16]. MiR-338-3p targets pyruvate kinase M2 and affects cell proliferation and metabolism of ovarian cancer [17]. Anticancer bioactive peptide-3 inhibits human gastric cancer growth by targeting miR-338-5p [18]. In our study, we found miR-338-3p was down-regulated in glioma tissues and associated with Karnofsky Performance Status (KPS) and WHO grade in glioma patients. Furthermore, we showed that increased miR-338-5p significantly inhibited the cell proliferation, cell colony formation and cell invasion abilities in gliomas cells. Additionally, we verified that increasing miR-338-5p inhibited cell EMT process by suppressing transcription factor Snail and EMT maker Vimentin and fibronectin expression, but up-regulating the E-cadherin expression. These results indicated that miR-338-5p function as a tumor suppressor in gliomas, which was consistent with previous study in other tumor progression [10].

We then investigated the underlying function of miR-338-5p in glioma. By online predicted software, we found that CTBP2 was a potential target of miR-338-5p, luciferase reporter assay results that CTBP2 was direct target of miR-338-5p. Moreover, miR-338-5p overexpression inhibited the expression level of CTBP2, while downregulated miR-338-5p enhanced the expression level of CTBP2 in glioma cells. CTBP2 was reported to act as a oncogene in some tumors, for example, increased CTBP2 expression in gliomas was significantly associated with poor prognosis in patients [19]. CTBP2 is an independent prognostic marker that promotes Gli1 induced epithelial-mesenchymal transition in hepatocellular carcinoma [20]. CTBP2 could promote cell proliferation through c-Myc signaling in prostate cancer [21]. In the study, we demonstrated that downregulation of CTBP2 can rescue the cell proliferation and invasion changes inducing by miR-338-5p inhibitor in gliomas cells. Thus, these above results showed that CTBP2 was a direct target of miR-338-5p and mediated the inhibition of cell proliferation and invasion induced by miR-338-5p in gliomas cells.

5. Conclusion

In conclusion, our results showed that miR-338-5p was downregulated in glioma tissues and inhibited cell proliferation and invasion. In additional, we confirmed that miR-338-5p inhibits cell proliferation and cell invasion capability by targeting CTBP2. Thus, these results indicated that miR-338-5p may be a potential target of treatment for glioma.

Conflict of interest

The authors declare that they have no competing interests.