Gastric cancer is a digestive tract malignant tumor originating from gastric mucosal epithelial cells. It has the fifth highest global incidence rate and the third highest mortality rate. It has been at the forefront of the world’s malignant tumors [1,2]. Statistics showed that less than 25% of patients with early diagnosis and treatment of gastric cancer had a low survival rate within 5 years [3,4]. Gastric cancer has the characteristics of occult occultation, easy metastasis and recurrence, and metastasis and recurrence are the main reasons for poor prognosis and mortality. In recent years, great progress has been made in the mechanism research, early diagnosis and treatment of gastric cancer, but the effective diagnosis and treatment of gastric cancer is still a huge problem faced by human beings. With the development of molecular biology, microRNA (miRNA) provides a new direction for the early diagnosis and treatment of gastric cancer .
miRNAs are a class of non-coding RNAs with 19–24 nt in length . It regulates cell proliferation, cell cycle, differentiation, development, apoptosis, migration, invasion and metastasis [7,8]. A large number of studies have shown that the occurrence and development of tumors are accompanied by changes in the level of miRNAs and affect the radiotherapy or chemotherapy effects of tumors [9,10]. Abnormally expressed miRNAs affect the development and progression of cancer by regulating oncogenic or tumor suppressor pathways. Therefore, studying the function of miRNAs, especially regarding the occurrence and development of gastric cancer, will provide new diagnostic and therapeutic targets.
It has been detected that miR-216b is down-regulated in many cancer types, indicating that miR-216b can be used as a prognostic marker for these particular types of cancer. It was reported that miR-216b inhibited osteosarcoma cell and hepatocellular carcinoma cell proliferation, migration, and invasion by targeting Forkhead Box M1 [11,12]. MiR-216b also suppressed cell proliferation and migration by down-regulating HDAC8 or SDCBP expression in human breast cancer [13,14], by targeting FOXM1 in cervical cancer cells and associated with better prognosis , by decreasing MALAT1 expression to induce G2/M arrest and apoptosis in pancreatic cancer cells , by targeting HMGB1-mediated JAK2/STAT3 signaling way in colorectal cancer , by targeting FOXM1 in vitro and in vivo in human melanoma . It was also demonstrated that miR-216b played as a sensitizer in cancer treatment, including increasing cisplatin sensitivity in ovarian cancer cells by targeting PARP1 , enhancing cisplatin-induced apoptosis by targeting c-Jun in non-small cell lung cancer (NSCLC) cells , and regulating cisplatin sensitivity of NSCLC cells via modulating autophagy and apoptosis . However, the effect of miR-216b on gastric cancer remains unclear.
Therefore, this study collects tissue samples, observes the expression of miR-216b in gastric cancer tissues, and explores its role and mechanism in the proliferation and invasion of gastric cancer cells in vitro, and looks for target genes to provide a certain basis of diagnosis and treatment for gastric cancer.
Materials and methods
The levels of miR-216b in gastric cancer tissues and paracancerous tissues (at least 5 cm from the edge of the tumor) of 10 gastric cancer patients diagnosed and treated by the Department of Gastroenterology, Affiliated Hospital of Nantong University were measured. All patients who participated in the study received informed consent before surgery and did not receive any radiotherapy or chemotherapy. The collected specimens were placed in a liquid nitrogen tank and stored in a refrigerator at −80°C for later use.
Cell culture and transfection
Human gastric cancer cell lines (BGC-823, BGC-803, SGC-7901 and AGS), human normal gastric mucosal cells GES-1 and HEK-293 cells were purchased from the cell bank of Chinese Academy of Sciences; cells were cultured in RPMI-1640 medium containing 10% FBS and 1% streptomycin in a constant temperature incubator with 5% CO2 saturated humidity at 37°C.
MiR-216b mimics, miR-negative control (NC), CCNT overexpression vector (pcDNA3.1-CCNT) and empty vector (pcDNA3.1) were obtained from Shanghai Genechem (Shanghai, China). Cells were plated into six-well plates at a density of 5 × 105 cells per well and incubated at 37°C with 5% CO2 until 50% confluence was reached. Cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen Carlsbad, California, USA) following the manufacturer’s instructions.
RNA isolation and RT-qPCR
TRIzol Reagent (Invitrogen) was added to the pulverized tissue samples and the collected cells to extract total RNA. To examine miR-216b expression, reverse transcription was conducted using a TaqMan miRNA reverse transcription kit, followed by real-time PCR with a TaqMan miRNA PCR kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s protocol, and normalized to U6 snRNA. Expression of CCNT2 was determined using an SYBR Mix (Promega, Madison, Wisconsin, USA) and normalized to GAPDH. Data were calculated with 2−ΔΔCt.
Cell proliferation was assessed using MTT assays. The cells were plated in the 96-well plate at a density of 6000 cells/well. Transfection was carried out when the cell fusion degree in the well reached about 80%. MTT solution at the concentration of 5mg/mL was added to each well at 0, 12, 24, 48 h after transfection. After 4 h of incubation, the reaction was stopped by adding DMSO. The absorbance of each well at 490 nm was measured by microplate reader.
Cell migration and invasion assay
Millipore Transwell chamber assay was used to detect the migration and invasion of gastric cancer cells. Cells with different transfections were digested, resuspended in serum-free RPMI-1640 medium, and adjusted to a cell density of 5 × 105 cells/ml. A cell suspension of 100 μl/well was implanted in a Transwell chamber (8 µm pore-size; Corning Inc., Corning, New York, USA) with an polycarbonate membrane (pre-coated with Matrigel in invasion assay), and the chamber was placed in a 24-well plate in RPMI-1640 medium containing 10% FBS. After 24 h, the cells in the upper chamber were removed, and cells invading to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of migrated or invading cells was counted under a microscope in five randomly selected fields of view.
Cell cycle and apoptosis assay
Cell cycle was performed using a FACSVerse flow cytometer (Becton Dickinson, San Jose, California, USA). The transfected cells were seeded into six-well plates at 1 × 104 cells/well, collected 48 h later. The cells were washed with pre-cooled phosphate buffer, centrifuged, and fixed overnight with 70% ice-cold ethanol. The propidium iodide (PI) was added for 30 min-staining in the dark, and the cycle distribution of each group was detected by flow cytometry. The cell apoptosis was determined with the Annexin V/PI kit (Beyotime, Nanjing, China).
Luciferase reporter assay
The potential targets of miR-216b were predicted using the algorithms TargetScan (http://www.targetscan.org/vert_72/). In order to clarify the direct targeting relationship between miR-216b and CCNT2 mRNA, the wild-type and mutant CCNT2 mRNA 3′-untranslated region (UTR) sequences containing the miR-216b binding site were designed and cloned into the reporter gene. The plasmids were named CCNT2 3′-UTR-WT and CCNT2 3′-UTR-MUT, respectively. The reporter plasmid was co-transfected with miR-216b mimics and miR-NC, respectively. The transfected cells were planted in 96-well plates at a density of 5 × 105 per well, and cultured for 48 h. The Dual-Glo dual luciferase reporter assay system was used to analyze and calculate the ratio of luminescence intensity.
After transfection for 48 h, the cells of each group were collected, and the total protein was extracted. The same amount of protein samples was taken from each group. The protein was denatured by adding SDS loading buffer to the bath for 10 min, and then subjected to conventional SDS-PAGE gel electrophoresis. The protein on the gel was transferred to a polyvinylidene fluoride membrane, blocked with 5% skim milk powder for 1 h at room temperature, and a 1:1000 diluted primary antibody was added and incubated overnight at 4°C. The membrane was washed with Tris-HCl buffer solution and Tween (TBST) buffer (5 min × 3 times) the next day, and then incubated with 1:5000 diluted secondary antibody for 1 h at room temperature. The TBST buffer was washed thoroughly and then chemiluminescence was developed with electrochemiluminescence reagent. The results were expressed as the ratio of the optical density of the target band to the internal reference GAPDH.
Data were analyzed using GraphPad Prism 7.0. The data is expressed as ‘mean ± SD’. Student’s t-test was utilized for comparison between the two groups. One-way analysis of variance was applied to compare the difference between multiple groups. P < 0.05 was considered statistically significant.
MiRNA-216b was low-expressed in gastric cancer tissues and cell lines
To investigate the role of miR-216b in the development of gastric cancer, we first examined the expression of miR-216b in 10 pairs of gastric cancer tissues and adjacent normal tissues by qRT-PCR. The results of qRT-PCR showed that the relative expression of miR-216b in gastric cancer tissues was significantly lower than that in adjacent tissues (Fig. 1a); compared with normal gastric mucosal epithelial cell line GES-1, miR-216b levels in human gastric cancer cell lines BGC-823, BGC-803, SGC-7901 and AGS were all significantly lower, and the difference was statistically significant (P < 0.001) (Fig. 1b).
miR-216b inhibits proliferation, migration and invasion of gastric cancer cells
To investigate the effect of miR-216b on the growth of gastric cancer cells, we first established a stable cell line of BGC-823 and SGC-7901 overexpressing miR-216b by transfection. The expression level of miR-216b in BGC-823 and SGC-7901 was significantly increased after transfection with miR-216b mimics (Fig. 2a). The proliferation of BGC-823 and SGC-7901 cells was detected by MTT assay at 0, 24, 48 and 72 h after transfection. Compared with miR-NC group, vitality of BGC-823 and SGC-7901 cells in miR-216b mimics group were significantly diminished after transfection for 24–72 h (Fig. 2b). In the cell migration and invasion experiments, the average number of migrated and invaded cells in the miR-216b mimics group were significantly decreased, suggesting that upregulation of miR-216b can reduce the migration and invasion of gastric cancer cells (Fig. 2c and d).
miR-216b arrested gastric cancer cell cycle and promoted apoptosis
After transfected with miR-216b mimics for 48 h, the cell cycle distribution in each group was detected by flow cytometry. The results shown in Fig. 3a revealed that compared with the NC group, the proportion of cells in the G1 phase was significant elevated due to upregulated miR-216b, while the proportion of cells in S phase decreased significantly, indicating that upregulation of miR-216b caused gastric cancer cell cycle arrest in G0/G1 phase. At the same time, overexpression of miR-216b in gastric cancer cells induced apoptosis, and the apoptotic rate was significantly higher than that in NC group (Fig. 3b), which was statistically significant.
CCNT2 is a direct target of miR-216b
To explore the molecular mechanism by which miR-216b inhibits proliferation and invasion of gastric cancer cells, we identified the target of miR-216b in gastric cancer. Bioinformation prediction results showed that there was a continuous CCNT2 binding site on the miR-216b gene sequence (Fig. 4a). At the same time, we further performed a luciferase reporter assay to determine whether the 3′-UTR of CCNT2 can be directly targeted by miR-216b, and the results showed that the reporter plasmid containing the CCNT2 wild type 3′-UTR sequence was co-linked with miR-126b mimics, the relative activity of luciferase was decreased, which was significantly lower than that of cells co-transfected with miR-NC (P < 0.05); whereas the reporter plasmid containing CCNT2 mutant 3′-UTR sequence co-transfected with miR-126b was identical to co-transfected with miR-NC, and there was no significant change in the relative activity of luciferase (Fig. 4b). These evidences suggest that miR-126b can directly bind to CCNT2 3′-UTR to inhibit CCNT2 expression in gastric cancer cells.
We used qRT-PCR and Western blot analysis to explore the regulation of CCNT2 expression by miR-126b at the mRNA and protein levels. The results showed that miR-126b upregulation significantly reduced the levels of CCNT2 mRNA (Fig. 4c) and protein (Fig. 4d) expression in BGC-823 and SGC-7901 cells compared with miR-NC group.
Overexpression of CCNT2 reverses the inhibitory effect of miR-126b on gastric cancer cell proliferation and cell cycle arrest
The effect of miR-126b overexpression on CCNT2 at the mRNA and protein levels was analyzed by qRT-PCR and Western blot. To further verify that the inhibitory effect of miR-216b on gastric cancer cell proliferation and invasion is mediated by CCNT2, we performed a series of reversal experiments. BGC-823 and SGC-7901 cells were co-transfected with the miR-216b mimics, the CCNT2 overexpression vector pcDNA3.1-CCNT2 or the empty vector pcDNA3.1. qRT-PCR analysis showed that the co-transfected portion of pcDNA3.1-CCNT2 restored the decrease in CCNT2 mRNA levels in BGC-823 and SGC-7901 cells induced by miR-216b mimics (P < 0.05; Fig. 5a). At the same time, the results of Western blotting also confirmed the above results of CCNT2 protein level (P < 0.05; Fig. 5b). The proliferation activity of cells was detected by MTT assay at 0, 24, 48 and 72 h after transfection. The results showed that the co-transfected of pcDNA3.1-CCNT2 restored the inhibition of cell proliferation caused by overexpression of miR-216b in BGC-823 and SGC-7901 cells (Fig. 5c).
Flow cytometry results showed that overexpression of CCNT2 significantly attenuated G0/G1 arrest induced by upregulation of miR-162b (P < 0.01) (Fig. 6a). At the same time, it can reduce the apoptosis induced by upregulation of miR-162b (Fig. 6b). Therefore, these results indicate that CCNT2 overexpression can reverse the anti-tumor effect of miR-126b on cell cycle arrest of gastric cancer cells, further confirming that CCNT2 is a downstream target gene of miR-126b in gastric cancer cells.
Gastric cancer is one of the most common malignant tumors of the digestive tract in the world with high morbidity and mortality. Due to the lack of specific clinical symptoms in early gastric cancer, most patients are in advanced stage at the time of diagnosis. In recent years, molecular targeted therapies targeting endogenous molecules have received increasing attention. Studies have shown that miRNAs play an important role in maintaining gene regulatory network balance and regulating cell proliferation, apoptosis, differentiation, migration and invasion [22,23]. Studies have found that miRNAs can influence tumorigenesis and development by negatively regulating target gene mRNA levels, which mediate their degradation, binding to functional proteins, activating TLR receptor proteins, and negatively regulating other non-coding RNA precursor RNAs [24,25].
miR-216b is a member of the miR-216 family and located on chromosome 2p16.1, which is reported to be associated with many types of human cancers . miR-216b is significantly down-regulated in lung cancer cells, it can regulate the proliferation and invasion of non-small cell lung cancer by targeting SRY-Box 9 gene in lung cancer cells . In addition, miR-216b has been shown to be low-expressed in breast cancer. miR-216 targets the purinergic receptor P2X7 and reduces the growth of breast cancer cells . These studies indicate that miR-216b can be used as a prognostic marker for these specific types of cancer. However, the effect of miR-216b expression levels on gastric cancer remains unclear.
In order to explore the relationship between miR-216b and gastric cancer, 10 cases of clinical patient tissues, four gastric cancer cells and one gastric mucosal epithelial cell were studied in this study. miR-216b was found significantly down-regulated in gastric cancer tissues and cell lines compared to adjacent non-tumor lung tissue and normal gastric mucosal cells, respectively. It is speculated that miR-216b may be a tumor suppressor, and downregulation of miR-216b may promote the development of gastric cancer. To further understand its related functions, this study use BGC-823 and SGC-7901 cells transfected with miR-216b mimics to assess the proliferative, migration and invasive effect. It was revealed that upregulation of miR-216b inhibited the proliferation of gastric cancer cells and regulates the cell cycle, resulting in a decrease in the number of cells in the S phase and an increase in the percentage of cells in the G0/G1 phase, and induction of G0/G1 arrest. At the same time, the migration and invasion abilities of BGC-823 and SGC-7901 cells were weakened after transfected with miR-216b mimics.
In addition, bioinformatics analysis demonstrated that CCNT2 is a target gene of miR-216b. Luciferase reporter assays showed that miR-216b binds to CCNT2 3′-UTR and reduces the level of CCNT2 mRNA and protein expression in gastric cancer cells. These results suggest that the miR-216b and CCNT2 interactions can be evaluated.
Deregulation of the cell cycle may result in an imbalance between cell proliferation and cell differentiation [28,29], leading to tumorigenesis. CCNT2 is a cyclin that plays an important role in gene transcription , and CCNT2 was identified as a direct target of miR-216b, which is regulated by miR-126b. CCNT2 is a component of the p-TEFb complex that is required for transcription initiation and elongation mediated by RNA polymerase II. The p-TEFb complex plays an important role in embryonic development and various cellular processes . It has been reported that CCNT2 can increase the proliferation of THP-1 cells and inhibit monocyte differentiatio .
We further verified that the inhibitory effect of miR-216b on proliferation and invasion of gastric cancer cells is mediated by CCNT2, and we conducted a series of reversal experiments. BGC-823 and SGC-7901 cells were co-transfected with the miR-216b mimics and the CCNT2 overexpression vector pcDNA3.1-CCNT2 or the empty vector pcDNA3.1. The results showed that overexpression of CCNT2 restored the inhibition of cell proliferation induced by overexpression of miR-216b in BGC-823 and SGC-7901 cells, and attenuated G0/G1 arrest induced by upregulation of miR-216b. These results indicate that miR-216b can regulate the proliferation and cell cycle arrest of gastric cancer cells via interaction with CCNT2.
In summary, miR-216b is low-expressed in gastric cancer tissues and cell lines. miR-216b may inhibit the growth, migration and invasion of gastric cancer cells by down-regulating CCNT2. The results of this study further enriched the mechanism of miR-216b in the development and progression of gastric cancer. Thus, miR-216b may be a potential novel target for the treatment of gastric cancer.
X.C. and Z.C. designed and managed all the experiments. X.C. and L.Z. did most of the experiments and wrote the draft. Q.S. analyzed all the data. Z.C. revised the manuscript. All of them approved this manuscript.
Conflicts of interest
There are no conflicts of interest.
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