The Role of mir-152 and DNMT1 in Gastric Cancer Cell Proliferation and Invasion

Gastric cancer is the fourth most common human malignant disease and the second leading cause of cancer related mortality worldwide on the basis of the most recent statistics, with particularly high incidences and mortality rates in eastern Asia [1-4]. Gastric cancer is a multifactorial disease. Genetic predisposition and environmental factors have been recognized to be involved in gastric cancer initiation and progress. Large scale multidisciplinary investigations on gastric cancer have indicated that gastric cancer is a multiple gene disease and a series of molecules are involved in gastric cancer carcinogenesis [5,6]. Nevertheless, relatively little is known about how specific molecules are involved in the initiation and progression of gastric cancer. Consequently, elucidation of the molecular mechanisms of the carcinogenesis of gastric cancer will be benefit for enhancing prevention, detection and treatment of this disease.

MicroRNAs (miRNAs) constitute a dominating class of small RNAs in most somatic tissues. In RNA silencing, miRNA functions play as a guide to induce translational repression, mRNA deadenylation and mRNA decays by base pairing with its target mRNAs [7]. miRNA - binding sites are usually located in the 3 Untranslated Region (UTR) of mRNAs [8]. More than 60% of human protein-coding genes contain at least one conserved miRNA - binding site, and considering that numerous non-conserved sites also exist, most protein-coding genes may be under the control of miRNAs [9]. A lot of study suggested that miRNA plays crucial roles in many biological processes such as development, inflammation, differentiation, cell proliferation, apoptosis and invasion [10-13]. Thus, their dysregulation is often associated with human diseases, including cancer and neurodevelopmental disorders [14,15]. 

Emerging evidences have demonstrated that miRNA are deregulated in various cancers including breast cancer, gallbladder cancer, bladder cancer, cutaneous squamous cell carcinoma, laryngeal cancer, rhabdomyosarcoma and GC [16-21]. miRNA can act as a tumor suppressor gene or oncogene in the initiation and development of tumors [12,22,23]. Recently, increasing studies have demonstrated that miR-152 are decreased in various tumor types, indicating that they have the potential to act as tumor-suppressor miRNAs [24-26]. miR-152 plays an important role in various tumors [27-29]. However, the expression and functional role of miR-152 in GC is still unknown. In this study, we studied the relation between miR-152 and DNMT1 in GC cell proliferation and invasion to show the role of miR-152 and DNMT1 in the development of GC.

Tissue samples

In Situ Hybridization (ISH)

Immunohistochemistry

Evaluation of staining:

Cell lines cultured and transfection

Quantitative real-time RT-PCR

Construction of Vectors

Dual-luciferase reporter assay

Western blot

Cell growth and colony formation

Cell invasion

Statistical analysis

miR-152 expression was down regulated in the GC cell and tissue

Figure 1A: miR-152 expression was downregulated in the GC cell and tissue.

Detection of miR-152 by in situ hybridization in serial sections from gastric cancer tissue and its matched non-tumor adjacent tissue. Images are overlay images with brown color representing miR-152 expression. Data are presented as mean ± SD from at least three separate experiments. The miR-152 expression in non-tumor tissue (A), Lower expression in GC tissue (B) higher expression in GC tissue (C) compared to non-tumor tissue.

Clinicao pathologic features of gastric cancer patient are listed in table 1.

Parameter	N (%)	miR-152		X2	P
		Lower	Higher
Gender
Male	63(78.75)	53(66.25)	10(12.5)	3.156	0.093
Female	17(21.25)	11(13.75)	6(7.45)
Age (years)
≤ 60	28(35)	23(28.75)	5(6.25)	4.515	0.034
> 60	52(65)	41(51.25)	11(13.75)
Tumor location
Proximal	42(52.5)	33(41.25)	9(11.25)	0.965	0.617
Middle	21(26.25)	16(20)	5(6.25)
Distal third	17(21.25)	15(18.75)	2(2.5)
Tumor size (cm)
≤ 3 cm	41(51.25)	29(36.25)	12(15)	3.405	0.065
> 3 cm	39(48.75)	35(43.75)	4(5)
Pathological differentiation
High	37(46.25)	31(38.75)	6(7.5)	1.036	0.596
Moderate	36(45)	27(33.75)	9(11.25)
Poor	7(8.75)	6(7.5)	1(1.25)
Depth of invasion
Mucosa and muscular	18(22.5)	10(12.5)	8(10)	8.674	0.003
Submucosa and serosa	62(77.5)	54(67.5)	8(10)
Lymph node metastasis
No	30(37.5)	14(17.5)	16(20)	33.333	0
Yes	50(62.5)	50(62.5)	0
TNM stage
I+II	35(43.75)	19(23.75)	16(20)	25.714	0
III+IV	45(56.25)	45(56.25)	0

Figure 1B: miR-152 expression was downregulated in the GC cell and tissue.

miR-152 expression was significantly downregulated in GC tissues compared with the paired non-tumor tissues.

Moreover, patients with the early stages (stage I and II) had a much higher miR-152 expression than patients with the advanced stages (stage III and IV) (Figure 1C).

Figure 1C: miR-152 expression was downregulated in the GC cell and tissue.

miR-152 level was significantly lower in patients with advanced stage (stage III and IV) than in patients with early stage (stage I and II). Patients were staged according to the 7th Edition of the AJCC Cancer’s TNM Classification.

Unfortunately, due to a lack of follow-up data, the correlation between miR-152 expression and prognosis could not be investigated. In addition, we also demonstrated that miR-152 expression was decreased in the GC cell lines compared with normal gastric mucosa cells line (GES-1) (Figure 1D).

Figure 1D: miR-152 expression was downregulated in the GC cell and tissue.

The expression of miR-152 was downregulated in the GC cell lines (MKN-42, AGS, MKN-28 and HGC-27) compared with normal gastric mucosa cells line (GES-1).
?- P < 0.05,?- P < 0.01,? - P < 0.001.

Among these GC cell lines, MKN-45 and HGC-27 cells displayed the most reduction (approximated 80%) in miR-152 expression compared with its expression in GES-1. Hence, further functional analyses on miR-152 were performed in MKN-45 and HGC-27 cells. Taken together, our data showed that miR-152 expression was downregulated in both human GC tissues and GC cell lines, and its downregulation was correlated with clinicopathological features of GC, which indicates that miR-152 may play a vital role in GC progression and development.

Differential expression of DNMT family members in gastric cancer

Figure 2: DNA methyltransferase (DNMT1) 1, 3a, 3b expression determined by immunohistochemistry in gastric cancer biopsies.

Gastric carcinomas that express negative DNMT1 (A), DNMT3a (B), DNMT3b (C) and positive DNMT1 (D), DNMT3a (E) and DNMT3b (F) are shown. Original magnification: × 200.

DNMT expression in non cancerous tissue was primarily focused on the proliferating zone of gastric mucosa. DNMTs perform DNA methylation in the nucleus; thus, nuclear staining was investigated. The positive expression rate of DNMT1 was higher than DNMT3a and DNMT3b in GC (Table 2).

Parameter	n	DNMT1	DNMT 3a	DNMT 3b
		Expression (%)	Expression (%)	Expression (%)
Gender
Male	63	46(57.5)	23(28.75)	25(31.25)
Female	17	9(11.25)	8(10)	7(8.75)
P		0.113	0.576	0.911
Age (years)
≤ 60	28	20(25)	12(15)	13(16.25)
> 60	52	35(43.75)	19(23.75)	19(23.75)
P-value		0.704	0.635	0.389
Tumor location
Proximal	42	30(37.5)	17(21.25)	18(22.5)
Middle	21	11(13.75)	8(10)	7(8.75)
Distal third	17	14(17.5)	6(7.5)	7(8.75)
P-value		1.121	0.931	0.763
Tumor size (cm)
≤ 3cm	41	26(32.5)	18(22.5)	16(20)
> 3cm	39	29(36.25)	13(16.25)	16(20)
P- value		0.291	0.332	0.855
Differentiation
High	37	29(36.25)	12(15)	15(18.75)
Moderate	36	22(27.5)	16(20)	14(17.5)
Poor	7	4(5)	3(3.75)	2(2.5)
P-value		0.222	0.559	0.809
Depth of invasion
Mucosa and muscular	18	8(10)	8(10)	6(7.5)
Submucosaand serosa	62	47(58.75)	23(28.75)	26(32.5)
P-value		0.011?	0.573	0.512
Lymph node metastasis
No	30	8(10)	12(15)	14(17.5)
Yes	50	47(58.75)	19(23.75)	18(22.5)
P-value		0.000?	0.859	0.346
TNM stage
I+II	35	15(18.75)	15(18.75)	17(21.25)
III+IV	45	40(50)	16(20)	15(18.75)
P-value		0.000?	0.506	0.168

DNMT1 expression was upregulated in the GC cell and tissues

Figure 3: DNMT1 expression was upregulated in the GC cell compared with GES-1.

A) The protein expression of DNMT1 was measured by western blot
(B) The mRNA expression of DNMT1 was measured by qRT-PCR.
?- P < 0.05, ? - P < 0.001.

Moreover, DNMT1 expression was upregulated in 55 cases (55/80; 68.75%) compared with the adjacent tissues (Table 2). There was a negative relationship between the expression level of miR-152 and DNMT1 in the GC tissues (R2 = 0.546, P < 0.001) (Figure 4).

Figure 4: There was negative relationship between the expression level of miR-152 and DNMT1 in the GC tissues.

miR-152 suppressed the GC cell proliferation, colony formation and invasion

Figure 5: miR-152 suppressed the GC cell proliferation, colony formation and invasion.

A) The expression of miR-152 expression was upregulated in the HGC-27 cell and MKN-45 cell after transfected with miR-152 mimic using qRT-PCR.
(B & C) Overexpression of miR-152 suppressed the MKN-45 and HGC-27 cell proliferation.
(D & E) Ectopic expression of miR-152 inhibited the MKN-45 and HGC-27 cell colony information. 
(F & G) miR-152 overexpression suppressed the MKN-45 and HGC-27 cell invasion.
?- P < 0.001, ?- P < 0.05.

DNMT1 was a direct target gene of miR-152 in GC cell

Figure 6: DNMT1 was a direct target gene of miR-152 in GC cell.

A) Bioinformatics assay with Target Scan algorithms predicted DNMT1as a hypothetical target gene of miR-152. 
(B & C) Luciferase activity assays of luciferase vectors with wild-type or mutant DNMT1 3?-UTR were performed after co-transfection with miR-152 mimic or mimic NC in the MKN-45 and HGC-27 cell. 
(D) Overexpression of miR-152 suppressed the DNMT1 protein expression in the MKN-45 and HGC-27 cell.
?- P < 0.05 miR-152 mimic and wild-type DNMT1 3?-UTR co-transfection group vs. other three groups.

DNMT1 rescued the effect of miR-152-mediated GC cell proliferation, colony formation and invasion

Figure 7: DNMT1 expression was upregulated in the pCDNADNMT1- WT 3?-UTR or pCDNA DNMT1- MT 3?-UTR treated GES cell.

A) The protein expression of DNMT1 was measured by western blot. 
(B) The mRNA expression of DNMT1 was measured by qRT-PCR. ? - P < 0.001.

We rescued DNMT1 expression in the miR-152 overexpressing MKN-45 and HGC-27 cell in the pCDNADNMT1- MT 3?-UTR treated group (Figures 8A & 8B). Furthermore, overexpression of miR-152 suppressed the DNMT1 protein expression in the MKN-45 and HGC-27 cell with wt DNMT1 3?-UTR. CCK8 analysis demonstrated that DNMT1 overexpression promoted the miR-152-overexpressing MKN-45 and HGC-27 cell proliferation (Figures 8C & 8D). Colony formation analysis showed that DNMT1 overexpression increased the miR-152 overexpressing MKN-45 and HGC-27 cell colony formation (Figures 8E & 8F). Invasion analysis demonstrated that ectopic expression of DNMT1 promoted the miR-152-overexpressing MKN-45 and HGC-27 cell invasion (Figures 8G & 8H).

Figure 8: DNMT1 rescued the effect of miR-152-mediated GC cell proliferation, colony formation and invasion.

(A & B) The expression of DNMT1 was decreased in the MKN-45 and HGC-27 cell after co-transfected with miR-152 mimic and pCDNADNMT1-wt3?-UTR,but which was upregulated after co-transfected with miR-152 mimic and pCDNADNMT1-mt3?-UTR, or miR-152 NC and pCDNADNMT1-wt3?-UTR,or miR-152 NC and pCDNADNMT1-mt3?-UTR.
(C & D) Overexpression of DNMTI promoted the miR-152 overexpressing MKN-45 and HGC-27 cell proliferation compared with the group of decreased expression of DNMT1.
(E & F) DNMT1 overexpression increased the miR-152 overexpressing MKN-45 and HGC-27 cell colony formation compared with the group of decreased expression of DNMT1. 
(G & H) Ectopic expression of DNMT1 promoted the miR-152-overexpressing MKN-45 and HGC-27 cell invasion compared with the group of decreased expression of DNMT1.
? – P < 0.001, co-transfected with miR-152 mimic and pCDNADNMT1-wt3?-UTR vs. Other three groups.

There has been a rapid increase in the number of publications focusing on miR?152 in recent years, which revealed that the expression of miR?152 was inhibited in a variety of tumors, including ovarian cancer [25], endometrial cancer [26], liver cancer [29], non-small lung cancer [28], and colorectal cancer [27]. Here, we investigate the functions of miR-152 in GC and found that miR-152 expression was signifycantly downregulated in GC tissues and cell lines. On the other hand, overexpression of miR-152 significantly inhibited cell proliferation, colony formation and tumor angiogenesis in vitro studies. Su et al., demonstrated that restoration of miR-152 significantly reduced cell proliferation, colony formation, migration and invasion partially via targeting ADAM17 [30]. In addition, miR?152 is also able to target Wnt1 and MMP3 to inhibit cell proliferation in liver cancer cells, thus reducing glioma cell invasion and angiogenesis, respectively [31]. Our study also showed ectopic expression of miR-152 suppressed GC cell proliferation, colony formation and invasion. These findings suggest that miR?152 may potentially function as a tumor suppressor in human cancer. 

Aberrant DNA hypermethylation of tumor suppressor genes, Global DNA hypermethylation (GDM) and disruption of histone modification patterns are the three most important epigenetic changes contributing to the malignant phenotype [32]. In particular, DNA hypermethylation may be important in the initiation of multiple types of cancer [32,33]. Nakajima et al., also found that DNA methylation levels in the gastric mucosae correlate with gastric cancer risk levels [34]. Methylation of mammalian genomic DNA is catalyzed by DNMTs. The mammalian DNMT family includes three active members: DNMT1, DNMT3a and DNMT3b [35,36]. The human DNMT 1, 3a, and 3b coordinate mRNA expression in normal tissues and overexpression in tumors [37]. The expression levels of these DNMTs are reportedly elevated in human cancers [36-39]. There is considerable evidence indicating an up-regulation of DNMT1 in cancer [40,41]. In our study, the positive expression rate of DNMT1 was higher than DNMT3a and DNMT3b in GC. And DNMT1 expression was significantly related with depth of invasion, Lymph node metastasis and TNM stage of GC.

Recent studies have found that DNMT1 plays important roles in the GC development [42-44]. But there was no evidence to show the relation between DNA hypermethylation and miR-152 in GC. Here, we identified DNMT1 as a direct target gene of miR-152 in GC cell and DNMT1 expression was upregulated in GC tissues and cells. Furthermore, the expression level of miR-152 was negatively correlated with the expression level of DNMT1 in the GC tissues. DNMT1 overexpression rescued the effect of miR-152 mediated GC cell proliferation, colony formation and invasion. These observations are consistent with previous findings that miRNA-152 induces aberrant DNA methylation in hepatitis B virus - related hepatocellular carcinoma and endometrial cancer by targeting DNMT1 [24]. Our findings indicate that miR-152 directly interacted with the DNMT1 and the lower levels of miR-152 expression in GC cells may be one of the reasons for the abnormal expression of DNMT1, which leads to aberrant DNA methylation, contributing to GC tumorigenicity and development. 

This study was limited by its small number of patients and cancer-related genes. Thus, larger multi-gene studies are needed to validate our results. Further work is necessary to elucidate the exact function and interaction with other factors to develop strategies for early diagnosis, prevention and treatment of GC. On the other hand, some researches provided evidences that the down expression of miR-152 in NiS-transformed cells was regulated by DNMT1 via promoter hypermethylation, and miR-152 was linked to DNA methylation by directly targeting DNMT1 3′-UTR, revealing a crucial functional crosstalk between miR-152 and the DNMT1 via a double-negative feedback loop is involved in NiS-induced malignant transformation [45,46]. miR-152 is located at 17q21.32, and a typical CpG is land is around miR-152 promoter. Recently, relatively low expression of miR-152 was shown in gastrointestinal cancer and cholangiocarcinoma [47,48]. DNA hypermethylation and downregulation of miR-152 were frequently observed in endometrial cancer cell lines and primary tumors [26]. Aberrant DNA methylation of miR-152 CpG islands was reported to correlate with a poor clinical outcome in Mixed Lineage Leukemia (MLL) - rearranged acute lymphoblastic leukemia [49]. Therefore, the next study needs to identify the influence of DNMT1 on the expression of miR-152 in GC.

In conclusion, miR-152 maybe acts as a tumor suppressor miRNA in the development of GC through inhibiting DNMT1 expression. Therefore, restoration of miR-152 may be a potential therapeutic strategy for GC patients in the future.

Ethical approval The study protocol was approved by the Ethics Committee of Jinlin Hospital (Nanjing, China).

Wang ZK wrote the paper; Ye YQ and Wang SD designed and analyzed; Shi H and Wu L collected the specimen; Yuan BS and Wang H reviewed histopathology of specimen.

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