Effects of MicroRNA-106 on Proliferation of Gastric Cancer Cell through Regulating p 21 and E 2 F 5

In recent years, with the in-depth research of Nucleic Acids, the study of genome’s “dark matter” non-coding RNA has become hot spots to explore life sciences, such as miRNAs are widely used in the research of development and diseasea (Martin et al., 2012). In the tumor related miRNAs positioning study, we have found that 98 (52.5%) of the 186 miRNA-encoding genes located in the tumorassociated region of chromosome fragile sites, prompted miRNA may play an important role in the process of tumor formation (Calin et al., 2001). Currently, only a small part of the biological function of miRNAs are articulated, these miRNAs regulate cell growth and tissue differentiation, involved in several physiological processes (Moss., 2002; Anglicheau et al., 2010; Herranz et al., 2010). MiR-106b is located on chromosome 7q21 amplified region of gastric cancer. In a previous study, found its target genes are p21 and E2F5 (Li et al., 2011; Trompeter et al., 2011). It is significantly overexpressed in gastric cancer, but its main role is still unclear. This article is to explore the effect of miR-106b on malignant features of gastric cancer cells, by analyzing itself and its target genes expression in different gastric cancer cell lines. With the elucidation of miRNAs mechanism, we found


Introduction
In recent years, with the in-depth research of Nucleic Acids, the study of genome's "dark matter" -non-coding RNA has become hot spots to explore life sciences, such as miRNAs are widely used in the research of development and diseasea (Martin et al., 2012).In the tumor related miRNAs positioning study, we have found that 98 (52.5%) of the 186 miRNA-encoding genes located in the tumorassociated region of chromosome fragile sites, prompted miRNA may play an important role in the process of tumor formation (Calin et al., 2001).Currently, only a small part of the biological function of miRNAs are articulated, these miRNAs regulate cell growth and tissue differentiation, involved in several physiological processes (Moss., 2002; RESEARCH ARTICLE

Effects of MicroRNA-106 on Proliferation of Gastric Cancer Cell through Regulating p21 and E2F5
Yong-Liang Yao, Xiao-Yang Wu, Jian-Hong Wu, Tao Gu, Ling Chen, Jin-Hua Gu, Yun Liu, Qing-Hui Zhang* miRNAs also closely related to the development of a variety of human tumors, including gastric cancer (Gong et al., 2005).Numerous studies have shown that the use of miRNAs microarray\, Northern Blotting, quantitative stem-loop PCR technology, each stage of the tumor tissue-specific expression of miRNAs can be efficiently identified, thus further to diagnoses and foresee its prognosis (Manikandan et al., 2008).MiRNAs has become a new biological marker in the diagnosis of diseases, may also be to explore molecular drug targets or to simulate drug research, to provide new ideas and means to the treatment of a variety of human diseases in the future.

Cell culture
Human gastric cancer cell lines MKN-28, SGC-7901 and MKN-45, and normal gastric mucosa cell line GES were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).Cells were cultured in 25-cm 2 cell culture flask at 37℃ in a humidified atmosphere of 5% CO2 with RPMI-1640 Medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal calf serum with 50 U/ml penicillin and 50 μg/ml streptomycin.

Cell transfection
MiR-106b mimics were transfected into MKN-45 cell line.Cells with mutant mimics and without anything were used as the randomized and blank control groups, respectively.3 mmol mimics were diluted in 200 μl serum-free RPMI-1640 medium, and then 10 μl the liposome Lipofectamine TM2000 were diluted in 200 μl RPMI-1640 medium by mixing gently.After incubation at room temperature for 5 min, the two agents were mixed gently and incubated at room temperature for 20 min.The resulting complexes were added into cells and shaken gently, and were incubated overnight at a temperature of 37℃, and humidity of 5% CO 2 .In the follow-up experiments, the medium was replaced with containing 10% fetal calf serum and cultured for 48h.

Real-time polymerase chain reaction (Real-time PCR)
Total RNAs were extracted by using Trizol in accordance with the manufacturer's instructions.Reverse transcriptase reactions contained RNA samples, 50 nM stem-loop RT primer (Table 1), 1×RT buffer (Biosystems), 0.25 mM each of dNTPs, 3.33 U/ml MultiScibe reverse transcriptase (Biosystems), and 0.25 U/ml RNase inhibitor (Applied Biosystems).The 7.5 μl reactions were incubated in a ABI 7900 cycler for 30 min at 16 ℃, 30 min at 42 ℃, 5 min at 85 ℃ and then held at a constant temperature of 4 ℃.Quantitative PCR reaction conditions: 95 ° C for 10 min, 95 ° C for 15 s, 60 ° C for 1 min, 35 cycles (Chen et al., 2005;Xu et al., 2013), the specific primer sequences are shown in Table 1.Each sample was measured three times.

Western blot
For detecting the changes of miR-106b targets in protein levels, Western blot analysis were performed in MKN-45 cell lines transfected with miR-106b mimics and several control groups.For the detection of E2F5 and P21 proteins, antibodies for proteins (Santa Cruz Biotechnology, at 1:500 dilution) were diluted.The secondary HRP conjugate antibodies were diluted 1:1500.The immunocomplexes were detected using the ECL system (Beyotime Biotechnology, China).

Analysis of cell viability
Cell viability was measured by using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan), which assessed the number of cells based on the reduction of a water-soluble formazan by the dehydrogenases present in viable cells.Briefly, cells (1~2×10 3 cells/well) were seeded in 96-well plates (200 μl medium/well).Three wells of each group were used in repeated experiments.After 24 h post-attachment, cells were transfected.At the appropriate times , 10 μl of WST-8 solution was added to each well, and cells were incubated at 37℃ for 3 hours.Absorbance was read at 450 nm on a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria).

Flow cytometry cell cycle
collection suppress most efficient at 48 h after transfection of the interference group of MKN-45 cells were washed twice with PBS, fixed with 70% ethanol, 4 ℃ overnight adding 50 μg/mL propidium iodide piperidine (PI) 250 μl of 1mg/mL of RNase 10 μl of dark 30min, flow cytometry measurement of cell cycle.

Cell migration and invasion assay
Cell migration assays were performed using transwell chambers with 8 μm pores (Chemicon).The lower chambers of the transwell plates were filled with 500 μl medium containing 10% fetal bovine serum as a chemoattractant.The cell suspension (300 μl) was then added to the upper chamber, and plates were incubated at 37℃ for 24 h.Cells that migrated to the lower surface of the polycarbonate membrane were stained with Giemsa solution.The cells that migrated to the lower surface were quantified by counting 5 randomly selected microscope fields at ×400 magnification.Cell invasion assays were performed using same way, transwell chambers covered wiht Matrigel and calculated to cultivate through the number of cells after 36 hours.

Statistical analysis
All results are expressed as mean ± standard deviation (X ± s).Real time-PCR data were analyzed using paired t-test comparison analysis (Livak et al., 2001).Western blot, cell cycle, transwell migration and invasion assay were used ANOVA test for analysis.P <0.05 was statistically significant.
To characterize the function of miR-106b on GC cell proliferation, miR-106b mimics transfected MKN-45 cells were cultured and detected the cell proliferation rate in different time using cell counting kit 8 (CCK8).The assay manifested that miR-106b can play a catalytic role of gastric cancer cell proliferation (F = 18.204,P <0.001), as Figure 4 shows in vitro miR-06b can significantly enhanced cell proliferation effect, and presented in a certain range of time-dependent, and then the slope of the proliferation rate was decline after 48 hours, suggesting the efficiency begins to reduce in unit concentration of miR-106b, its effect gradually reached a plateau.

miR-106b has no effect on gastric cancer cell migration and invasion
Metastasis is an important character of gastric cancer that influences both clinical treatment and prognosis.To test the impact of miR-106b is necessary in GC cell metastasis, the function of miR-106b on GC cell migration and invasion is studied.The results of Transwell chamber assay showed that the relative percentage of cell migration and invasion were similar between the transfected cells group and negative or blank control groups.The migration or invasion ability of the cells of the experimental group is no significant changes, indicating that miR-106b can not affect the invasive ability of cancer cells (Figure 5A  and B).

Discussion
MicroRNAs (miRNAs) are small, non-coding RNA controlling the activity of protein-coding genes by combining 3'-UTR of target mRNA and impacting its transcription or post-transcription level (Zeng et al., 2002).MiRNAs involved in various processes from early development of life processes to cell differentiation and apoptosis.With the depth of miRNAs research, in a variety of human tumors detected changes in miRNA expression levels, and the expression of different combinations of abnormal miRNA expression profiling in different types of tumors, suggesting that tumors have a tissue-specific miRNA expression (Ferdin et al., 2010;Kahlert et al., 2013).Multiple miRNA is associated with the formation of gastric cancer, which functions as a tumor suppressor, can also play the role of oncogenes.A lot of specific miRNAs differentially expressed in gastric cancer and normal gastric mucosa, such as miR-21, miR-34b/c, miR-221/222 and miR-106a is highly expressed in gastric cancer, compared with low expression of miR-124a, miR-128b, miR-148 and miR-129 (Konishi et al., 2012;Li et al., 2012).Murakami found that miR-222, miR-106a, miR-92, miR-17-5p, miR-20 and miR-18 are related to the degree of differentiation, indicating that specific miRNAs and disease processes (Chu et al., 2008).
In this study, we found significant differences in the differentiation of gastric cancer cells, and to investigate the effects on gastric cancer cell growth, proliferation and migration and invasion.The results showed, with expression of mkiR-106b, its target genes p21 and E2F5 transcriptional and translational levels are suppressed, further study found that miR-106b can effectively promote the growth and proliferation of tumor cells, gastric cancer cells were shorten in the G0/G1 phase and facilitate their entry into the subsequent mitosis proliferation, and speed up cell cycle progression, but had no effect on gastric cancer cell migration and invasion abilities.So we're guessing that the abnormal expression of miR-106b in gastric cancer upset the balance of cell cycle network, and promote gastric cancer progress which may is possible mechanisms of miR-106b for cancers.
Gastric cancer is one of the most common malignant tumor, its occurrence and development involved in multiple genes, multi-step and multi-stage molecular patterns of events, including activation of oncogenes, inactivation of tumor suppressors, abnormalities of mismatch repair genes, and mutations of cell cycle regulatory factors.MiRNAs were considered the switch of the regulation of tumor, because it can control the activity of oncogenes and tumor suppressors in the process of neoplastic transformation network, which impact on important characteristics of the tumor cells.Currently miRNAs are considered that can be formed microvesicles, similar to the endocrine cells of the vesicles, secreted out of the cell, to transfer information between cells through the fluid circulation, regulation of gene transcription and expression of a variety of cell (Carlsbecker et al., 2010;Zhang et al., 2010).Visible, miRNAs have hormone-like role, regulation and participate in a wide range of physiological and pathological reactions, their researches will contribute to the deepening of a variety of malignancies, including gastric cancer, future clinical diagnosis, treatment and prevention.

Figure 1 .
Figure 1.The Expression of miR-106b and its Target genes in Gastric Cancer Cell Lines and Normal Gastric Mucosa Cell.p < 0.05 (*), p < 0.01 (**) was considered a statistically significant difference

Figure 3 .
Figure 3. Cell Growth Curve of Gastric Cancer Cell Transfected with miR-106b Mimics.miR-106b effects on the cell cycle of gastric cancer cell

Figure 4 .
Figure 4. Effects on Tumor Cell Cycle of Gastric Cancer Cell after miR-106b Mimics Transfection.

Figure 5 .
Figure 5. Effects on Cell Migration and Invasion of Gastric Cancer Cell after miR-106b Mimics Transfection.A: effects on cell migration, B: effects on cell invasion