MiR-143 inhibits cell proliferation and invasion by targeting DNMT3A in gastric cancer

Abstract

Increasing evidence has suggested that MircroRNAs (miRNAs) dysregulated in pathogenesis and tumorigenicity in human cancers including gastric cancer (GC). MiR-143 had been reported to function as tumor suppressor in GC progression, however, the underlying function of miR-143 in GC still need to be well known. In the study, we revealed that miR-143 was significantly down-regulated in GC cell lines. Upregulation of miR-143 inhibited cell proliferation, invasion, S phase cell proportion and cell cycle related protein levels of Cyclin D1, CDK4 and CDK6 in GC. Furthermore, luciferase reporter assays demonstrated that DNMT3A was a direct target of miR-143 and Upregulation of miR-143 inhibited the DNMT3A mRNA and protein expression levels in GC cells. Moreover, we demonstrated that DNMT3A knockdown rescued the promoting effect of miR-143 inhibitor on cell proliferation in GC. Thus, these results demonstrated that miR-143 targeted DNMT3A in GC cells and inhibit GC tumorigenesis and progression, which may provide a novel therapeutic target of GC.

Instruction

Gastric cancer (GC) is most common malignant cancers and the second leading cause of cancer-associated mortality in the world.^1,2 In 2012, an estimated 951,600 gastric cancer cases and 723,100 deaths occurred in the world. Among both males and females, highest gastric cancer incidence and mortality cases are found in Eastern and Western Asia, Latin America, and some former Soviet European countries.³ In spite of some advances in chemotherapy and surgery have developed, the prognosis for GC patients who are diagnosed at advanced stage have a median overall survival of ≤12 months.⁴ Thus, exploring the underlying mechanism involved in gastric cancer carcinogenesis is urgently needed.

Increasing evidences have demonstrated that microRNAs (miRNAs) play critical functional roles in human cancer development and progression.⁵ MiRNAs are reported to be dysregulated in tumors and function either as oncogenes or tumor suppressors.⁶ MiR-143 had been reported to involve in the tumorigenesis and invasion, metastasis of various cancer types as a tumor suppressors. Wang et al.⁷ showed that miR-143 targeted CTGF and exerted tumor-suppressing functions in epithelial ovarian cancer to inhibit cell proliferation, migration, and invasion. MiR-143 inhibited esophageal squamous cell carcinoma cell proliferation and invasion by targeting Stat3.⁸ in the GC cells, downregulation of microRNA-143 was founded to enhance the higher sensitivity to 5-fluorouracil following the transfection with miR-143.⁹ Wu et al.¹⁰ reported that microRNA-143 inhibited gastric cancer cell growth and induced cell apoptosis by targeting COX-2. However, the functional role and the mechanism underlying gastric carcinogenesis for miR-143 still remain unclear. We here further explored the regulatory role of miR-143 in GC.

In the study, we demonstrated that miR-143 was downregulated in GC cells lines, and upregulation of miR-143 inhibited cell proliferation, invasion and cell cycle progression in GC. Moreover, we found that upregulation of miR-143 inhibited GC cell proliferation by targeting DNMT3A. Thus, our results indicated that miR-143 acted as a tumor suppressor by targeting DNMT3A in GC cells. miR-143 may be used as a potential target for GC treatment.

Materials and methods

Cell culture

The human gastric cell lines (MKN28, MKN-45, BGC-823, SGC-7901 and MGC803) and a normal gastric epithelial cell line GES-1 were obtained from Shanghai Institute for Biological Sciences, Chinese Academy of Science. Cells were cultured in RPMI 1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C.

Cell transfection

The miR-143 mimic, miR-143 inhibitor and negative control (NC) were designed and synthesized by GenePharma (GenePharma, China). Cells were transfected using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Cell proliferation analysis

Cell proliferation was measured using the CCK-8 Kit (Dojindo Laboratories, Japan) according to manufacturer protocols. Briefly, the 3 × 10³ cells/well transfected with miR-143 mimic, miR-143 inhibitor or miR-NC were seeded on 6-well plates and cultured for 24 h. In indicated time 1, 2 and 3 and 4 days, 10 µl of the CCK-8 solution was added to each well and cultured for 2 h, then the absorbance of the plate was measured at 450 nm under a microplate reader (Molecular Devices, Menlo Park, CA).

Quantitative RT-PCR analysis

Total RNA was extracted from gastric cancer cell lines using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was measured using absorbance at 260 nm and was reversed into cDNA using the miScript II RT Kit (Qiagen, Venlo, Limburg, Netherlands). Quantitative RT-PCR was performed using SYBR Premix Ex Taq kit (Takara, Dalian, China). The PCR reaction were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 33 s. GAPDH and U6 was used as the endogenous control. The relative mRNA expression was evaluated by the 2^−ΔΔCT method. The primer sequences used were as follows: miR-143: F: 5’-ACACTCCAGCTGGGTGAGATGAAGCACTGTAG-3’;R:5’-CTCAACTGGTGTCGTGGA-3’;GAPDH:F:5’-GGAAGGTGAAGGTCGGAGTC-3’;R:5’-GAAGATGGTGATGGGATTTC-3’.

Transwell invasion assay

MKN-45 and SGC-7901 cells invasion abilities were assessed using Transwell chambers. Briefly, cells transfected with miR-143 mimic or miR-143 NC and miR-143 inhibitor or miR-NC were incubated for 48 h at 37°C in DMEM. Then,the 5 × 10⁴ cells were seeded into the upper chamber added with free FBS DMEM, and the lower chamber was supplemented with DMEM supplemented with 10% FBS that functions as a chemo-attractant. The chambers were incubated at 37°C in 5% CO² for 24 h, the cells on the upper chamber were removed. Cells on the lower chamber were fixed with methanol and stained with 0.1% crystal violet. Stained cells were counted under a microscope. All assays were performed in triplicate.

Cell cycle analysis

For cell cycle analysis, After cells transfection at 48 h, MKN-45 and SGC-7901 cells were permeated with 75% ethanol for overnight at 4°C and stained with Propidium Iodide (PI) in the presence of 5 µg/ml RNase (Sigma) for 10 min. The cell cycle phase distribution (G0/G1, S and G2/M) was determined with DNA cell cycle analysis software (ModFit, Becton Dickinson).

Western-blot analysis

Total protein was extracted from MKN-45 and SGC-7901 cells using RIPA buffer (Sigma). The protein concentrations were determined by using a bicinchoninic acid protein assay kit (BCA) assay kit (Beyotime, Shanghai, China). The equal amounts of protein lysates (40 µg) was separated 10% SDS-PAGE gel and proteins were transferred to a PVDF membrane. Then the membrane was blocked using 0.5% skim milk. The membranes were incubated with antibody against DNMT3A (1:3000, Abcam, USA), Cyclin D1 (1:2000, Cell Signaling Technology, USA) CDK4 (1:1500, Beyotime, Shanghai, China), CDK6 (1:1000, Beyotime, Shanghai, China) and GAPDH (1:2000, Cell Signaling Technology) overnight at −4°C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Proteins blot were detected with chemiluminescent detection system (Beyotime, Shanghai, China) and exposure with an autoradiography film (Kodak, Shanghai, China).

Luciferase reporter assays

The mutant and wild 3′-UTR of DNMT3A which contains target region of miR-143 was constructed by GenePharma, Shanghai, China. Fragments were inserted between the XhoI and NotI sites in the psiCHECK™-2 Dual Luciferase miRNA target expression vector (Promega, USA). The MKN-45 and SGC-7901 cells were co-transfected with 3′-UTR of DNMT3A -WT or 3′-UTR of DNMT3A-MUT and miR-143 mimic or miR-NC using Lipofectamine 3000. After transfection at 48 h, luciferase and renilla signals were measured using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol.

Statistic analysis

Statistic analysis was performed using SPSS 17 software (SPSS Inc., USA). All independent experiments were performed at least three times. The data are presented as mean ± SD. The differences between two groups were tested using a paired t-test. A P-value < 0.05 was considered statistically significant

Results

MiR-143 is decreased in GC cells and promoted the cell proliferation ability

In order to evaluate the potential role of miR-143 in GC, the expression levels of miR-143 in five gastric cancer cell lines and normal gastric epithelial cell line GES-1 were determined by qRT-PCR. As shown in Figure 1(a), the relative expression levels of miR-143 in GC cells were significantly down-regulated compared with that of the normal epithelial cell line GES-1. The effect of miR-143 on cell proliferation was analyzed by CCK8 assay at 1, 2, 3 and 4 days after transfected with miR-143 mimic or miR-143 inhibitor into MKN45 and SGC-7901 cells. The results showed that cell proliferation ability was inhibited when miR-143 mimic was transfected into MKN-45 and SGC-7901 cells, compared with miR-NC group (Figure 1(b) and (c)). However, the cell proliferation was promoted when miR-143 inhibitor was transfected into MKN-45 and SGC-7901 cells, compared with miR-NC group (Figure 1(d) and (e)). Thus, these finding indicated miR-143 inhibited the cell proliferation in GC.

Figure 1.

MiR-143 is lower expression in gastric cancer cell lines.(a) QRT-PCR assays was used to analyze miR-143 expression in the human gastric cell lines MKN28, MKN-45, BGC-823, SGC-7901 and MGC803 and a normal gastric epithelial cell line GES-1. The expression of miR-143 was normalized to U6. (b) and (c) CCK8 cell proliferation assays were used to evaluate the cell proliferation of gastric cancer in MKN-45 and SGC-7901 cells after transfection with the miR-143 mimic or miR-NC. (d) and (e) CCK8 cell proliferation assays were used to evaluate the cell proliferation of gastric cancer in MKN-45 and SGC-7901 cells after transfection with the miR-143 inhibitor or miR-NC. All data are mean ± SD of three independent assays, *P < 0.05.

[Figure omitted. See PDF]

MiR-143 inhibited GC cells invasion and cell cycle progression

Furthermore, we analyzed the cell invasion ability after miR-143 mimic or miR-143 inhibitor was transfected into MKN-45 or SGC-7901 cells. The results showed that increased miR-143 dramatically inhibited the cell invasion and invasive cell number, compared with miR-NC group in MKN-45 or SGC-7901 cells (Figure 2(a) and (b)). However, down-regulated miR-143 significantly promoted the cell invasion and invasive cell number, compared with miR-NC group in MKN-45 or SGC-7901 cells (Figure 2(c) and (d)). Furthermore, we examined the cell cycle progression by flow cytometry analysis. The results showed that G1 phase cell proportion was increased, but S phase cell proportion was decreased when cells were transfected with miR-143 mimic, compared with miR-NC group in MKN-45 and SGC-7901 cells (Figure 3(a) and (b)). The cell cycle related protein expression of Cyclin D1, CDK4 and CDK6 was reduced when cells were transfected with miR-143 mimic compared with miR-NC group in MKN-45 and SGC-7901 cells (Figure 3(c) and (d)). Thus, these results suggested that miR-143 suppressed cell invasion and cell cycle progression in GC.

Figure 2.

MiR-143 inhibited cell invasion capability in GC. (a) and (b)Transwell invasion assay was performed to detect the ability of cell invasion in MKN-45 and SGC-7901 cells after transfection with the miR-143 mimic or miR-NC. *P < 0.05. (c) and (d)Transwell invasion assay was performed to detect the ability of cell invasion in MKN-45 and SGC-7901 cells after transfection with the miR-143 inhibitor or miR-NC. All data are mean ± SD of three independent assays, *P < 0.05.

[Figure omitted. See PDF]

Figure 3.

MiR-143 inhibited the cell cycle progression in GC. (a) and (b)The cell cycle phase distribution (G0-G1, S and G2-M) was determined by flow cytometry analysis in MKN-45 and SGC-7901 cells after transfection with the miR-143 mimic or miR-NC. (c) and (d)The cell cycle related protein expression of CyclinD1, CDK4 and CDK6 were determined by western-blot analysis in MKN-45 and SGC-7901 cells after transfection with the miR-143 mimic or miR-NC. All data are mean ± SD of three independent experiments, *P < 0.05.

[Figure omitted. See PDF]

DNMT3A was a direct target of miR-143 in GC cells

To identify potential target genes of miR-143, we applied algorithms that predicted the mRNA targets of miRNAs by miRanda (www.mircrorna.org). We found that DNMT3A represented a miR-143 recognition sites in its 3’-UTRs. Furthermore, we tested whether DNMT3A is a direct target of miR-143, the plasmid psicheck2 containing the wild-type or mut-type 3’-UTR region of DNMT3A vector were constructed (Figure 4(a)). The MKN-45 and SGC-7901 cells were co-transfected with miR-143 mimic or miR-NC and the wild-type or mut-type 3’-UTR region of DNMT3A vector. The results showed that luciferase activity was markedly decreased by about 35% or 33% in the psicheck 2 containing the wild-type vector, whereas no change in the co-transfected with miR-143 mimic and psicheck 2 containing mut-type 3’-UTR region of DNMT3A (Figure 4(b) and (c)). To further confirmed the association between miR-143 and DNMT3A in GC cells, the results showed that the DNMT3A mRNA expression was markedly inhibited by transfecting miR-143 mimic, but was increased by transfecting miR-143 inhibitor into MKN-45 and SGC-7901 cells (Figure 4(d) and (e)). Besides, the protein expression of DNMT3A was markedly decreased by transfecting miR-143 mimic, but was increased by transfecting miR-143 inhibitor into MKN-45 and SGC-7901 cells (Figure 4(f) and (g)). Thus, these results suggested that DNMT3A is direct target of miR-143 and miR-143 suppressed DNMT3A expression in GC cells.

Figure 4.

DNMT3A was a direct target of miR-143 in GC cells. (a) DNMT3A was predicted as a potential target of miR-143 by online software miRanda. (b) and (c)The relative luciferase activity in MKN-45 or SGC-7901 cells was detected after the psicheck2-wt or mut DNMT3A 3′UTR genes were co-transfected with miR-NC or miR-143 mimic. (d) and (e)The relative DNMT3A mRNA expression in MKN-45 or SGC-7901 cells was detected after cells were transfected with miR-143 mimic miR-143 inhibitor or miR-NC. (e) and (f)The relative DNMT3A protein expression in MKN-45 or SGC-7901 cells was detected after cells were transfected with miR-143 mimic and miR-NC or miR-143 inhibitor and miR-NC. All data are mean ± SD of three independent experiments, * P < 0.05,**P < 0.01.

[Figure omitted. See PDF]

DNMT3A is involved in miR-143-mediated suppression of cell proliferation

To further determine the association between miR-143 and DNMT3A in GC cell proliferation, the rescue experiments were performed by co-transfecting with miR-143 inhibitor and si-DNMT3A oligos into MKN-45 and SGC-7901 cells. The results showed that cell proliferation was promoted when miR-143 inhibitor was transfected into MKN45 and SGC-7901 cells, but was partially reversed by co-transfecting with miR-143 inhibitor and si-DNMT3A (Figure 5(a) and (b)). Thus, DNMT3A was involved in miR-143-mediated suppression of cell proliferation.

Figure 5.

DNMT3A was involved in miR-143-mediated suppression of cell proliferation capability. (a) CCK8 assays were used to evaluate the cell proliferation capability of gastric cancer in MKN-45 cells after transfection with the miR-NC, miR-143 inhibitor or miR-143 inhibitor+si-DNMT3A. (b) CCK8 assays were used to evaluate the cell proliferation capability of gastric cancer in SGC-7901 cells after transfection with the miR-NC, miR-143 inhibitor or miR-143 inhibitor+si-DNMT3A. All data are mean ± SD of three independent experiments, *P < 0.05.

[Figure omitted. See PDF]

Discussion

MicroRNAs (miRNA) have been frequently indicated to be dysregulated in different human cancers and play important roles in tumourigenesis.^11,12 Studies have revealed that miR-143 functioned to act as anti-oncogenes and was frequently reduced in some cancers.¹³ For example, miR-143 expression is downregulated in human prostate cancer, and acts as a tumor suppressor by targeting hexokinase 2 and eventually decreased glucose metabolism.¹⁴ MiR-143 is significantly downregulated in nasopharyngeal carcinoma and suppressed NPC cell viability, colony formation, and anchorage-independent growth by targeting KRAS.¹⁵ Wei et al.¹⁶ reported that miR-143 inhibits cell proliferation by targeting autophagy-related 2B in non-small cell lung cancer H1299 cells. In breast cancer, MiR-143 synergistically regulated ERBB3 to suppress cell proliferation and cell invasion.¹⁷ MiR-143-3p functioned as a tumor suppressor by regulating cell proliferation, invasion and epithelial-mesenchymal transition by targeting QKI-5 in esophageal squamous cell carcinoma.¹⁸

In GC development and progression, Naito et al.¹⁹ demonstrated that microRNA-143 regulates collagen type III expression in stromal fibroblasts of scirrhous type gastric cancer. Iio et al.²⁰ found that DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in GC cells. Du et al.²¹ showed that MicroRNA-143 enhances chemosensitivity of Quercetin through autophagy inhibition via target GABARAPL1 in gastric cancer cells. Overexpressed miR-143 sensitized SGC7901/DDP cells to cisplatin via targeting IGF1R and BCL2.²² In our study, we further explored the role of miR-143 in GC, we found that miR-143 was decreased in GC cells and upregulation of miR-143 inhibited the cell proliferation and invasion, whereas, down-regulation of miR-143 promoted the GC cell proliferation and invasion. Furthermore, by flow cytometry analysis and western-blot analysis, we found that upregulation of miR-143 inhibited S phase cell number and cell cycle protein expression of Cyclin D1, CDK4, CDK6 expression. Based on these findings, we demonstrated that miR-143 functioned as tumor suppressor in GC.

We further investigated the underlying mechanism of miR-143 in GC, by online target predicted software, we found that DNMT3A was a potential target of miR-143. Cancer initiation and progression has been found to be association with aberrant expression of the DNA methyltransferases (DNMT), over-expression of DNMT3A predicts the risk of recurrent vulvar squamous cell carcinomas.²³ Downregulation of DNMT3a expression increased miR-182-induced apoptosis of ovarian cancer through caspase-3 and caspase-9-mediated apoptosis and DNA damage response.²⁴ In GC development and progression, DNMTs are over-expressed in gastric cancer and play a significant role in the development of aberrant promoter methylation during tumorigenesis.²⁵ DNA methyltransferase 3A promoted cell proliferation by silencing CDK inhibitor p18INK4C in gastric carcinogenesis.²⁶ Deregulation between miR-29b/c and DNMT3A was associated with epigenetic silencing of the CDH1 gene, affecting cell migration and invasion in gastric cancer.²⁷ Moreover, Zhang et al.²⁸ reported that a regulatory circuit involving miR-143 and DNMT3a mediates vascular smooth muscle cell proliferation induced by homocysteine. Based on these previous finding, we speculated that miR-143 may affect DNMT3A expression in GC. Furthermore, we demonstrated that DNMT3A was a direct target of miR-143 by luciferase reporter assays. Upregulation of miR-143 inhibited the DNMT3A mRNA and protein expression in GC cells. Moreover, we demonstrated that cell proliferation was promoted when miR-143 inhibitor was transfected into GC cells, but was partially reversed by co-transfecting with miR-143 inhibitor and si-DNMT3A oligos. Thus, these findings demonstrated that miR-143 inhibit GC cell proliferation by regulating DNMT3A.

In conclusion, our results showed miR-143 was down-regulated in GC cells and miR-143 inhibited the GC progression by targeting DNMT3A. Thus, miR-143 may serve as a potential target for treatment of gastric cancer.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.