Reduced miR-29a-3p expression is linked to the cell proliferation and cell migration in gastric cancer
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MicroRNAs (miRNAs) play an important role in a tumor-suppressive or oncogenic manner in carcinogenesis. Alteration expression patterns of miRNAs in gastric cancer (GC) are associated with cancer initiation and progression. In the present study, we evaluated miR-29a-3p expression pattern and its function in gastric carcinogenesis.
The expression of miR-29a-3p in GC tissue samples and cell lines was detected by quantitative real-time PCR (qRT-PCR). After transfected with miR-29a-3p mimics or inhibitor, the cell proliferation, cell migration, and invasion ability were assessed by CCK-8 assay, wound healing assay, and Trans-well assay, respectively. The level of CDK2, CDK4, CDK6, and CyclinD1 were determined by qRT-PCR and Western blot.
Compared with the corresponding non-tumor tissues, miR-29a-3p showed a significant down-regulated expression in tumor tissues. In vitro functional assays demonstrated that enforced miR-29a-3p expression inhibited cell proliferation by reducing the expression of CDK2, CDK4, and CDK6. Wound healing and Transwell assays revealed that miR-29a-3p suppressed tumor metastasis in GC.
Our preliminary results suggest that altered expression of miR-29a-3p is involved in gastric cancer process. The present study provides the first insight into the specific role of miR-29a-3p in gastric carcinogenesis.
KeywordsmiR-29a-3p Migration Cell proliferation Gastric cancer
quantitative reverse-transcription polymerase chain reaction
Gastric cancer (GC) is one of the malignant digestive tract tumors which seriously threatens human health , accounting for a major cause of cancer-related mortality in China . Although the tremendous improvements in diagnosis and treatment technologies, the difficulty for early diagnosis and the poor prognosis of advanced GC still results in a low survival rate . Multiple encoding or non-coding gene expression alteration is involved in the occurrence and progression of GC. MicroRNAs (miRNAs) are small endogenous, non-coding, single-stranded RNAs , which inhibit the translation and stability of messenger RNAs (mRNAs), controlling genes expression involved in cellular processes such as cell-cycle regulation, differentiation, apoptosis, and migration. Nowadays, it is shown that miRNA closely relates to the process of tumor and the development of tumor invasion and migration . miRNA also acts as a biomarker stably expressed in serum and provides new target for molecular target therapy of various cancers . Accumulating evidences have strongly suggested that altered miRNA expression profiles and patterns could play an important role in tumorigenesis as oncogenes and tumor-suppressor genes . Currently, multiple abnormal expressions of miRNAs in gastric cancer have been observed, which are involved in gastric cancer cell proliferation, invasion, metastasis, and apoptosis, as well as in radiotherapy and chemotherapy sensitivity via regulating different tumor-related target genes [8-11]. Hence, the extensive analysis of miRNA expression in GC could contribute to deeply understand the mechanisms of GC development and identify diagnostic biomarkers and therapeutic targets.
miR-29a-3p is a member of miR-29s family, which is a conserved family of miRNA. Decreased expression of miR-29s has been described in multiple cancers, including GC [12,13]. Despite plenty of evidences showing that miR-29s can function as a tumor-suppressor gene, miR-29a-3p expression pattern in GCs still remains partially unresolved. The present study revealed that miR-29a-3p was significantly decreased in GC tissues and was involved in cell growth, migration, and invasion. These studies also provided novel insight into the role of miR-29a-3p in gastric carcinogenesis which would be crucial for the development of new strategies for GC treatment.
Cell culture and oligonucleotides transfection
Human gastric adenoma cell lines, including GES-1, SGC-7901, AGS, MCG803, and BGC-823, were obtained from the Cell Bank of Chinese Academy of Science and maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 U/ml of penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator with 5% CO2 atmosphere at 37°C.
miR-29a-3p mimics/inhibitors and negative control molecules (scramble control mimic and inhibitor) were synthesized and purified by the GenePharma Company (Shanghai, China). The sequences were shown in Additional file 1: Table S1. They were transfected into cells at a final concentration of 50 nM using Lipofectamine-2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Tissue sample preparation
Fifty pairs of histopathologically confirmed GC tissues and their adjacent non-cancerous tissue specimens were collected between 2010 and 2013 from the Jiangning Hospital of Nanjing, China. The study was approved by the Committee for Ethical Review of Research at the Jiangning Hospital of Nanjing in China, and the patients signed informed consent forms. All tissue samples were obtained from patients undergoing GC and immediately snap frozen in liquid nitrogen until RNA and protein extraction.
Real-time reverse transcriptase quantitative PCR
Total RNA was extracted from cell and tissue samples with Trizol reagent (Invitrogen, Carlsbad, CA). For the detection of miRNA expression, the primers used for stem-loop reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) were synthesized and purified by RiboBio (Guangzhou, China). The PCR conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The reactions were monitored using a preheated real-time instrument (ABI step one, Life Technologies). The relative expression ratio of miRNA in gastric cancer tissues and cells was quantified by the 2−ΔΔCT method. The primer sequences of gene ITGA6 and LAMA2 were shown in Additional file 2: Table S2.
Western blots were performed using Mouse anti-CDK2, anti-CDK4, anti-CDK6, and anti-cyclin D1 were purchased as part of the Cell Cycle Regulation Sampler Kit (Cell Signaling Technology, Danvers, MS, USA). Mouse anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein detection was performed with Super Signal chemiluminescence substrate (Pierce, Rockford, IL, USA).
Cell proliferation assay
The miRNA mimics/inhibitor was synthesized and purified by GenePharma Company (Shanghai, China) and transfected into the cells at a final concentration of 50 nM/100 nM using Lipofectamine-2000 transfection reagent (Invitrogen). One day before transfection, 5.0 × 103 BGC-823 cells and MCG803 in a 100-μl growth medium were plated in each well of a 96-well plate. The cells were then transfected with 50 nM of various synthetic miRNA mimics and corresponding control, and 100-nM inhibitor and corresponding control, using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s instruction. A cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was used to measure the cellular growth following the manufacturer’s instruction. Cell proliferation was assessed at different time points (0, 24, 48, and 72 h). Triplicate independent experiments were performed.
Migration and invasion assay
Cell mobility was subjected to the wound healing assay analysis as described previously . A scratch wound was generated using a 200-μl pipette tip on confluent cell monolayers in a six-well plate. Cells were then washed with a fresh medium to remove floating cells, and the spread of the wound closure was observed after 48 h and photographed under a microscope (Olympus, Tokyo, Japan). The potential for migration and invasion of transfected cells were evaluated by a Transwell assay (Life Technologies). Cells were grown to 70% confluence and transfected for 24 h with miR-29a-3p mimics or control mimics and miR-29a-3p inhibitor or control inhibitor, respectively. In the migration assay, cells were cultured in a 200-ml medium with 1% FBS in the upper chamber of a non-coated Transwell insert. In the lower chamber, a 600-ml medium with 10% FBS was used as a chemo-attractant to encourage cell migration. In the invasion assay, the upper chamber of the Transwell inserts were coated with 50 ml of 1.0 mg/ml Matrigel (Millipore, Billerica, MA, USA), and cells were plated in the upper chamber of the Matrigel-coated Transwell insert (Millipore, Billerica, MA, USA). After incubated for 24 h, the non-migrating or non-invading cells were gently removed with a cotton swab. All cells were stained using 0.1% crystal violet staining and counted in five fields under an inverted microscope. The independent experiments were repeated three times.
The independent Student’s t-test was used to compare the results expressed as mean ± SD between any two pre-selected groups. A P value less than 0.05 was considered statistically significant. To determine correlations between variables, Pearson’s correlation coefficient was calculated.
miR-29a-3p was decreased in GC tissue samples and cell lines
Correlation of the expression of miR-29a-3p with clinicopathologic feature
T ≥ N
T < N
Mucous layer outside
miR-29a-3p has cell proliferation suppression effects in vitro
miR-29a-3p suppressed GC cell migration and invasion
miRNA is an important regulator of gene expression at the post-transcriptional level and participates in cell development, differentiation, proliferation, cell-cycle control, apoptosis, and metabolism . Increased evidences have implied that miRNAs function as oncogene and tumor-suppressor genes, regulating the expression and function of their related target genes during the biological process of cancer cells. Our previous study showed that there was distinct miRNA profiling between GC tissues and matched non-tumor tissues based on the miRNA microarray analysis (Additional file 4: Table S4). In the present study, miR-29a-3p was selected as a candidate miRNA due to its down-regulation in tumor tissues. miR-29a-3p is one of the members of miR-29 family, which also includes miR-29b and miR-29c. Accumulating evidences have shown that the aberrant expression of miR-29s are prevalent in multiple cancer types and are involved in complex regulatory process by targeting multiple factors associated with several common pathways, indicating its critical role in carcinogenesis and cancer progression [14-18]. miR-29a has two different transcripts, which are miR-29a-3p and miR-29a-5p, respectively. Neither of them has been reported specific aberrant expression in tumors. Recent studies have found that miR-29a is up-regulated in indolent human B cell chronic lymphocytic leukemia (B-CLL) and acute myeloid leukemia (AML) [19,20]. However, some researches reveal that miR-29a is down-regulated in neuroblastoma, sarcomas, and brain tumors [12,21]. In fact, miR-29a could suppress cell proliferation and induce cell-cycle arrest via the down-regulation of p42.3 expression . These inconsistent findings indicate that dysregulation of miR-29a in various cancers may be dependent on the cellular microenvironment, especially with regard to detecting different transcripts of miR-29a-3p. In the present study, we analyzed the expression levels of miR-29a-3p transcript in GC tissue samples and GC cells and its primary biological function on GC cells. We found that decreased expression of miR-29a-3p in GC tumor tissues was related to the degree of cell differentiation. Decreased miR-29a-3p expression promoted gastric cancer cell proliferation via reducing the expression of cell-cycle regulators including: CDK2, CDK4, and CDK6. In a way, our finding might be consistent with the reporting of Cui Y et al. .
Many miRNAs have been identified as key players in metastasis and invasion process of tumor. In prostate cancer, miR-29a is considered as a putative tumor-suppressive miRNA, contributing to cell migration and invasion . miR-29 family plays a dominant role in regulating extracellular matrix genes, such as collagens, LAMA2, integrin β, Mmp2, fibrillin, secreted protein, acidic, and Sparc [16,24], consequently contributing to the promotion of cancer cell migration and metastasis. The present findings showed that miR-29a-3p significantly inhibits the cell invasion and migration ability in GC cells. With the help of bioinformatics prediction (Target scan, miRanda, miRWalk, and miRDB), ITGA6, LAMA2, and DNMT3A were identified as direct targets of miR-29a-3p. Thus, the function of miR-29a-3p in metastasis depends on its target regulation. Although we evaluated its potential target gene ITGA6, LAMA2 expression in miR-29a-3p mimic, and inhibitor-transfected cells (Additional file 5: Figure S5), future studies should be required to validate the association between miR-29a-3p and targets. These results indicate that miR-29a-3p may serve as a potential predictor for prognosis of gastric cancer patients.
In conclusion, the present study reveals that miR-29a-3p is down-regulated in GC, and it may contribute to the occurrence of gastric cancer via influencing cell proliferation, migration, and invasion. These findings provide evidence for the clinical value of miR-29a-3p as targets for GC therapy, although the precise regulatory mechanism and specific targets should be explored in the future study.
This work was supported by The National Natural Science Foundation of China, No. 91229107. This work was also supported by the National 973 Basic Research Program of China 2013CB911302, the National Natural Science Foundation of China, No. 81171915 and No. 81472548, and the Fundamental Research Funds for the Central Universities (CXLX12_0074).
- 11.Song JH, Meltzer SJ. MicroRNAs in pathogenesis, diagnosis, and treatment of gastroesophageal cancers. Gastroenterol. 2012;143(1):35–47. e32.Google Scholar
- 15.Zhao JJ, Lin J, Lwin T, Yang H, Guo J, Kong W, et al. microRNA expression profile and identification of miR-29 as a prognostic marker and pathogenetic factor by targeting CDK6 in mantle cell lymphoma. Blood. 2010;115(13):2630–9.Google Scholar
- 17.Mott JL, Kobayashi S, Bronk SF, Gores GJ. mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene. 2007;26(42):6133–40.Google Scholar
- 20.Han YC, Park CY, Bhagat G, Zhang J, Wang Y, Fan JB, et al. microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloid development, and acute myeloid leukemia. J Exp Med. 2010;207(3):475–89.Google Scholar
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