Expression of miR-711 and mechanism of proliferation and apoptosis in human gastric carcinoma.

MicroRNAs (miRs) are involved in many aspects of cell biology, including cell proliferation and apoptosis, two critical aspects of tumor biology. We investigated the effect of miR-711 on Bcl-2 expression in human MGC803 gastric cancer cells and the mechanism of cell proliferation, apoptosis, and invasion. Expression of miR-711 and Bcl-2 was significantly increased in gastric adenocarcinoma compared to adjacent normal tissue. Inhibition of miR-711 in MGC803 gastric cancer cells decreased the expression of Bcl-2, decreased cell proliferation, decreased the invasion ability, and increased apoptosis. The expression of Bcl-2 protein correlated with clinical staging, lymph node metastasis, and tumor differentiation in patients with gastric cancer. The expression of miR-711 positively correlated with the expression of Bcl-2, suggesting that miR-711 and Bcl-2 are co-regulated and involved in the development of gastric cancer.

Introduction

The incidence of gastric cancer is 13.9% worldwide, representing the most common digestive tract cancer (1). With the changes of diet structure, the incidence of gastric cancer continues to rise annually (2). Many scholars suggested that gastric cancer is a multi-stage process with a variety of oncogenes and tumor suppressor genes involved. The development of gastric cancer may be due to oncogene activation and tumor suppressor gene inactivation that result in the excessive proliferation of tumor cells (3). In addition, gastric cancer may be the result of inhibiting apoptosis, which promotes the survival of malignant tumor cells. Overall, genetic changes that alter cell proliferation and/or apoptosis may lead to the occurrence of malignant tumors (4).

Bcl-2 is an inhibitor of apoptosis expressed mostly in human stem cells of specific tissues, such as basal cell collagen and intestinal crypt bottom cells (5). Bcl-2 inhibits apoptosis to ensure these cells have enough time to complete the transformation from stem cells into differentiated cells (6). On the other hand, Bcl-2 activity is closely related to the occurrence and prognosis of lymphoma, colorectal, breast, cervical and thyroid cancer, as well as other malignant tumors (7). In addition, the invasion and metastasis of malignant tumors are closely related to high expression of Bcl-2. Anticancer drug research found that suppressing Bcl-2 in tumor cells caused apoptosis and improved the sensitivity of cancer cells to chemotherapeutic drugs. In addition, inhibiting Bcl-2 induced apoptosis in a variety of cells and primary tumor cells (8).

MicroRNA (miR) is a type of non-coding RNA that regulates gene expression. Many studies have linked miRs with the occurrence of malignant tumors (9). In malignant tumors, some miRs demonstrate abnormal expression and seem to play the role of oncogenes or tumor suppressor genes. Therefore, miRs have a relevant impact on the incidence and progression of malignant tumors (10). In the present study, we investigated the role of miR-711 in gastric cancer cell proliferation, apoptosis, invasion, and metastasis, and used theoretical tools to determine the early diagnostic value of miR-711. We found the abnormal expression of miR-711 and correlation with Bcl-2 expression in human stomach adenocarcinoma tissues, making these molecules targets for surveillance, diagnosis, and treatment (11–13).

Materials and methods

Quantitative polymerase chain reaction (qPCR)

We stored gastric carcinoma and normal adjacent tissue samples at −80°C. We placed the samples in a porcelain mortar, added liquid nitrogen to grind them into powder, and extracted total RNA by TRIzol (Invitrogen, Carlsbad, CA, USA). In detail, we prepared 10 ng of total RNA, 1X miRNA-specific reverse transcription primers (Thermo Fisher Scientific, Waltham, MA, USA), 100 µM nucleoside triphosphates, 3.33 U/µl MultiScribe Reverse Transcriptase, 1X Reverse Transcription Buffer and 1.33 U/µl RNase inhibitor (all from Thermo Fisher Scientific) in a final volume of 15 µl. The reaction was conducted at 16°C for 30 min, followed by 30 min at 42°C and 5 min at 85°C. The qPCR reaction was performed using a 20 µl volume containing 1.33 µl reverse transcription products, 1X TaqMan Small RNA Assay solution (including specific primers and probes; Applied Biosystems Life Technologies, Foster City, CA, USA; Thermo Fisher Scientific) and 1X Universal PCR Master Mix II (no UNG; Thermo Fisher Scientific). The RT-qPCR was performed in triplicate for each sample using an Applied Biosystems PRISM 7900HT System (Thermo Fisher Scientific) with the following conditions: 50°C for 2 min; 95°C for 10 min; and 45 cycles of 95°C for 15 sec and 60°C for 60 sec. The primer sequences for qPCR were: Bcl-2, forward, 5′-GACTTCGCCGAGATGTCCAG-3′ and reverse, 5′-CATCCCAGCCTCCGTTATCC-3′; β-actin, forward, 5′-CTCCATCCTGGCCTCGCTG-3′ and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′.

Western blotting

Tissue (200 mg) was sheared and broken, 1 ml pyrolysis liquid was added, homogenized, centrifuged at 8,000 × g for 10 min, and the supernatant was transferred to a new tube. Then, centrifugation was performed at 12,000 × g for 60 min, and the supernatant was transferred to a new tube. The protein content was determined according to the BCA Protein kit operation manual. Protein expression was analyzed by electrophoresis (PAGE), transferred to membrane, and detected by immunoreaction following standard procedures.

Flow cytometry

Human gastric cancer MGC803 cells were transfected with miR-711 mimics and apoptosis was detected after 48 h by Annexin V-FITC/propidium iodide (PI) double staining. MGC803 cells were serum starved for synchronization, cells were transfected with miR-711 mimic, cultured for 48 h, the cells were digested with 0.25% trypsin and suspended, and counted. Equal number of cells were inoculated into the cell culture bottle and 2% Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) culture medium was added. When cell confluence reached 50%, DMEM containing 10% FBS was added. At confluence of 80–90%, medium, was discarded and washed 2–3 times with PBS, then suspend in culture with 0.25% trypsin. Cells were collected into a 1.5 ml tube, centrifuged at 1,500 × g for 5 min, and the supernatant was discarded. The tube was added with 100 µl binding buffer, gently mix, and 5 µl Annexin V-FITC was added at room temperature in the dark and incubate for 10 min, then 1 µl PI (100 µg/µl) was added, and incubated for 5 min, adding 400 µl binding buffer. Flow cytometry was carried out 30 min later. A total of 1×104 cells from each sample was detected on the scatter plot of double variable flow cytometry. The left lower quadrant showed living cells (FITC−/PI−) and the upper right quadrant showed necrotic cells (FITC+/PI+). The lower right quadrant showed apoptotic cells (FITC+/PI−).

MTT cell proliferation detection

DMEM cell culture medium containing 10% FBS was used to re-suspend human gastric cancer MGC803 cells after transfection, adding 1×103-1×104 cells/ml inoculum density into 96-well cell culture plates in a volume of 200 µl each. The 96-well culture plates were incubated at 37°C and 5% CO2 in saturated humidity for 3–5 days. MTT solution (20 µl) was added into each well, then the cells were incubated for 4 h. Culture medium was discarded, 150 µl DMSO/well was added, rocking the reaction for 10 min to lyse the crystal within the cells. The absorbance was measured at 92 nm by enzyme-linked immunosorbent assay, and then the cell growth curve was drawn with the time as the horizontal coordinate and absorbance value as the vertical coordinate.

Cell transfection

MGC803 cells were cultured in DMEM supplemented with 10% FBS (Thermo Fisher Scientific) at 37°C with an atmosphere of 5% CO2. The miR-711 mimic, inhibitor and miRNA negative control (NC) were designed and synthesized by GenePharma (Shanghai, China). When the cells reached 60–70% confluence, Invitrogen Lipofectamine® 2000 RNAiMAX reagent was used to perform the transfection of cells with 100 nM miR-711 mimic or inhibitor, or NC, according to the manufacturer's protocol.

Detection of invasion of cells by Transwell assay

Human gastric cancer MGC803 cells were cultured for 24 h in serum-free DMEM, pipette was used to remove supernatant as chemotaxis fluid, and 0.05–0.2% BSA was added. MGC803 cells were washed at the logarithmic phase of each group after transfection with PBS 2–3 times, and cultured for 24 h. The upper compartment was add with 300 µl pre-warmed free serum DMEM medium. A total of 200 µl chemotaxis solution was added to the lower compartment to match the artificial matrix. Diluted 400 µl single cell suspension MGC803 was added with 95% ethanol solution to fix. Following standard H&E staining, five fields were select under ×200 magnification in an inverted microscope, and then counted the relative number of invaded cells as the invasion ability of the tumor cells.

Results

Expression of miR-711 mRNA in gastric cancer and adjacent tissues

We used fluorescence qPCR to determine miR-711 and Bcl-2 mRNA expression in 50 pairs of gastric cancer samples and the corresponding normal tissue. We found that the level of miR-711 in gastric adenocarcinoma was significantly higher than in adjacent normal tissues (Table I). Also, Bcl-2 mRNA levels in gastric adenocarcinoma were significantly higher than in adjacent normal tissues (Table II). Thus, gastric cancer cells showed increased levels of miR-711 and Bcl-2, suggesting a role for these candidate genes in the development and growth of the cancer cells.

Table I.

Expression of miR-711 mRNA.

Group	ΔCq	ΔΔCq	2-ΔΔCq
Normal tissue adjacent to cancer (n=50)	14.78±0.15	6.69±0.32	1.06±0.13
Gastric adenocarcinoma (n=50)	8.63±0.26	0.54±0.12	8.12±0.21[a]

P<0.01, compared with adjacent normal tissue.

Table II.

Expression of Bcl-2 mRNA.

Group	ΔCq	ΔΔCq	2-ΔΔCq
Normal tissue adjacent to cancer (n=50)	12.18±0.15	7.81±0.19	1.13±0.55
Gastric adenocarcinoma (n=50)	5.63±0.26	0.43±0.27	9.26±0.37[a]

P<0.01, compared with adjacent normal tissue.

Expression of Bcl-2 protein in gastric carcinoma

To further study the expression of Bcl-2 protein in gastric cancer tissue, we used western blotting. Bcl-2 was significantly higher in cancer tissue than in adjacent normal tissues (Fig. 1). This result is consistent with the elevated levels of Bcl-2 mRNA, further supporting a role for Bcl-2 in the survival of the cancer cells.

Figure 1.

Bcl-2 expression in two different tissues. (A) Western blotting showed Bcl-2 expression in normal tissue and gastric adenocarcinoma (GA). (B) The level of Bcl-2 was significantly increased in cancer tissues. **P<0.01, compared with the normal group.

Bcl-2 expression in gastric carcinoma and clinical pathological factors

To understand the relevance of Bcl-2 protein expression in gastric adenocarcinoma, we analyzed its correlation with clinical and pathological factors affecting gastric cancer patients (Table III). The Bcl-2 protein level was not associated with patient age or tumor location. However, we found association with clinical stage, lymph node metastasis, and tumor differentiation degree. We used Spearman's rank correlation to analyze the correlation between miR-711 and Bcl-2 mRNA expression in gastric cancer tissue. The Spearman rank correlation coefficient was rs=−1.131, P=0.0042, indicating a strong positive correlation between the two markers.

Table III.

Correlation between Bcl-2 protein expression and clinical pathology.

Group	Cases, no.	Bcl-2 high expression, no.	Bcl-2 low expression, no.	P-value
Age, years				0.72
≤65	14	8	6
>65	36	18	18
Clinical stages
T1	14	3	11	0.024
T2	20	8	12
T3	10	8	2
T4	6	5	1
Lymph node metastasis
Yes	9	8	1	0.025
No	41	13	28
Degree of tumor differentiation
High	21	18	3	0.041
Medium	16	10	6
Low	11	4	7

Proliferation of miR-711-transfected MGC803 cells

The proliferation of MGC803 human gastric cancer cells was analyzed 24, 48, and 72 h after transfection using an MTT assay. Compared with the control group, cell proliferation after miR-711 mimic transfection was significantly lower than that of the control group at each time point (Fig. 2). Inhibition of miR-711 had the opposite effect, increasing proliferation (Fig. 2). Thus, miR-711 had an obvious inhibitory effect on the proliferation of human gastric adenocarcinoma cells.

Figure 2.

Proliferation of MGC803 human gastric cancer cells detected 24, 48 and 72 h after transfection using an MTT assay. *P<0.05, compared with the control group.

Effects of miR-711 on proliferation, apoptosis and cell cycle of MGC803 cells

To better understand the activity of miR-711, we used flow cytometry to detect MGC803 cell apoptosis before and after transfection. The number of MGC803 cells undergoing apoptosis increased gradually in the miR-711 mimic group (Fig. 3A). MGC803 cells transfected miR-711 mimic, the G2 phase was significantly elevated (Fig. 3B). Detection of Bcl-2 expression levels determined that Bcl-2 was significantly higher in the miR-711 group.

Figure 3.

Effects of miR-711 on apoptosis and cell cycle of MGC803 cells. (A) Apoptosis of MGC803 cells in four groups were detected by Annexin V-FITC/PI double staining and flow cytometry. The results showed that the number of MGC803 cell apoptosis increased gradually in the miR-711 mimic group (P<0.05). (B) Effect of MGC803 cell cycle in each group after transfection. (C) The level of Bcl-2 expression transferred to the miR-711 group was the highest (*P<0.05).

Effect of miR-711 on the invasion ability of MGC803 cells

Increased cell invasion is the biological basis of malignant tumor metastasis. To study the metastasis of gastric cancer cells, we used the Transwell assay after miR-711 transfection. miR-711 mimic decreased significantly the invasive ability of cells, whereas the miR-711 inhibitor significantly enhanced invasion (Fig. 4).

Figure 4.

Invasion ability of miR-711 to MGC803 cells after transfection. (A) Transwell assay was performed to detect cell invasion ability. (B) miR-711 inhibitor significantly enhanced invasion (*P<0.05).

Discussion

The rapid development of molecular biology and modern cancer medicine has identified a strong correlation between miRs and the occurrence and development of malignant tumors. miRs can regulate the expression of one third of the total genes, modulating a number of physiological processes such as early development, cell proliferation, differentiation, apoptosis, and metabolism. Recent studies have found that miR expression in the majority of malignant tumor tissues was either increased or decreased, suggesting a robust connection between miR and tumor formation (14–17). Therefore, miRs may act as oncogenes or tumor suppressors in different tissues. Malignant tumor cell growth, proliferation, invasion, metastasis, apoptosis, and tumor angiogenesis strongly correlate with abnormal miR expression. Among the physiological and pathological activities of miRs are the activation or inactivation of relevant signaling pathways (18).

It was shown that miR-711 mediated the RASSF1A downregulation of CDK4 expression, which inhibited the proliferation of gastric cancer cell lines and increased apoptosis (19). The opposite result was observed in breast cancer, which showed that elevated expression of miR-711 significantly promoted the proliferation of breast cancer cells and indicated poor prognosis (20). Our study results suggest that Bcl-2 may be miR-711 downstream of other gastric cancer genes. Their abnormal expression can promote the occurrence of malignant phenotypes by inhibiting the expression of apoptosis-related genes. The inhibitory effect of Bcl-2 on cell apoptosis was mainly expressed as: i) The formation of channel proteins through the change of permeability of the cell membrane inhibits the release of mitochondrial apoptosis proteins, and ultimately inhibits cell apoptosis. ii) Improve the antioxidant function of the cells and scavenge oxygen-free radicals to inhibit cell apoptosis. iii) The blocking effect on the transmembrane flow of calcium ions to inhibit the cell apoptosis by regulating the intracellular calcium concentration (14,21–24).

The results of the present study showed that miR-711 and Bcl-2 mRNA, and Bcl-2 protein levels were higher in gastric adenocarcinoma cells. The relative expression of miR-711 and Bcl-2 mRNA in gastric cancer tissue showed a positive correlation. This result suggests that there may be some common regulatory relationship between the two. Since normal proliferation and apoptosis are genetically regulated, maintaining a balance between apoptotic and anti-apoptotic genes is critical for regulating cell proliferation and differentiation. The elevated expression of Bcl-2 is associated with the regulation of anti-apoptotic genes, but which signaling pathway regulates this process needs to be confirmed experimentally.

In addition, Bcl-2 protein was not associated with patient age and tumor location, but was related to patient's clinical stage, lymph node metastasis and tumor differentiation degree, and the difference had statistical significance.

In vitro experiments showed that the miR-711 mimic decreased the proliferation of MGC803 cells, suggesting that miR-711 inhibited the proliferation of human gastric adenocarcinoma cells. The miR-711 mimic also increased apoptosis in MGC803 cells, whereas more cells demonstrated cell cycle arrest at G2 phase. Based on this result, we hypothesize that miR-711 upregulates Bcl-2 to promote the apoptosis of MGC803 human gastric cancer cells and reduce the invasive ability, inhibit cell proliferation, and play a protective role. In conclusion, this study elucidated that miR-711 could provide a reliable theoretical support for the early diagnosis and targeted treatment of gastric cancer.