| Background:Gastric cancer is one of the most common cancers in the world. Although its diagnosis and therapy have made some progress, the prognosis of gastric cancer patient is still unsatisfactory. microRNAs (miRNAs) is one of the hottest points about biological molecules in present tumor research. Many research groups found that miRNAs played a vital role in the invasion and metastasis of tumor, and they may participate in the extracellular matrix degrading, the epidermis mesenchymal transformation (EMT) and angiogenesis through suppressing the target gene expression to affect the metastasis and invasion of tumor.The studing on miRNAs related to the metastasis of gastric cancer is very few. Rsearchers in Japan found that high expression of miR-214 was associated with unfavourable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis and stage. Our group had examined miR-214 expression level of gastric cancer cell lines-BGC823, MKN45 and SGC7901, discovered that it was upregulated, compared with GES-1, the human normal gastric mucosal cell line.There have been some studies about the target gene and the mechanism of miR-214. Li et al found that miR-214 specifically binds to the PTEN mRNA 3'-untranslated region to inhibit PTEN expression and delayed apoptosis of THP-1 cells in the development of inflammatory responses. Jindra et al found that miR-214 was upregulated after T cell activation, and the upregulation of miR-214 inversely correlated with levels of PTEN. Another research also discovered that miR-214 may suppress the expression of PTEN to activate the PI3K/AKT signal pathway, suppress the tumor cells to apoptosis and induce the cisplatin resistance in the ovarian cancer.It still needs massive experimental studies to confirm that whether miR-214 is related with the metastasis of gastric cancer. The mechanism is also unclear. Objectives:To explore the effect of miR-214 on the invasive ability of gastric cancer cell line SGC7901 and its mechanism.Methods:1. Use the prediction website miRanda and the database TarBase to analyse whether PTEN is the target gene of miR-214, and the possible binding site between PTEN mRNA and miR-214.2. miR-214 mimics, inhibitors or the independent sequences (NC group) were transfected transiently into SGC7901 cell line respectively. The NC group is the negative control and the blank transfection group is the blank control. The transfection efficiency was observed under fluorescence microscope when cultured for 24h after transfection.3. The miR-214 expression level of SGC7901 cell line was investigated by Real-time PCR.4. The PTEN protein expression level of SGC7901 was investigated by Western blot.5. The invasive ability of SGC7901 cells transfected with miR-214 mimics and inhibitors was detected by transwell chamber assay and transwell migration assay and in vitro wound healing assay.Results:1. Using TarBase and miRanda discovered that PTEN is one of miR-214's target genes, the 5'-seed sequence region of miR-214 matches the conservative sequence of PTEN mRNA, and the miRNA:mRNA dimer has certain stability.2. The miR-214 mimics and inhibitors was successfully transfected into human gastric cancer cell line SGC7901. The transfection efficiency was over 80%.3. The miR-214 expression level of miR-214 mimics transfection group and miR-214 inhibitors transfection group detected by Real-time PCR is respectively 82902.31 times,0.16 times as much as the control group 24h after transfection.4. Tested by Western blot, the ratio of PTEN/GAPDH protein of miR-214 mimics transfection group (0.049±0.007) is lower than NC group (0.087±0.007), miR-214 inhibitors transfection group (0.093 ±0.010), and the control group (0.099±0.012), and the difference was statistical significance (P<0.05).5. Transwell chamber assay showed the SGC7901 cells through the martrigel coated transwell membrane of miR-214 mimics transfection group (43.60±4.39) was more than NC group (25.80±7.79), miR-214 inhibitors transfection group (16.60±5.32) and control group (24.60±6.54), the difference was statistical significance (P< 0.05). The SGC7901 cells through the martrigel coated transwell membrane have no significant difference between miR-214 inhibitors transfection group and the control group. (P>0.05)6. Transwell migration assay indicated the SGC7901 cells through the transwell membrane of miR-214 mimics transfection group (39.00±6.04) was more than NC group (26.20±2.39), miR-214 inhibitors transfection group (20.00±2.55) and control group (20.40±4.77), the difference was statistical significance (P<0.05). The SGC7901 cells through the transwell membrane have no statistically difference between miR-214 inhibitors transfection group and the control group. (P>0.05)7. In vitro wound healing assay demonstrated the scratch width of miR-214 mimics transfection group was narrower than other three groups at the same wound healing time, and there was statistical significance (P<0.05). The factor analysis exhibited the main effect of the transfection factor (F= 34.913, P< 0.01), the main effect of the time factor (F=417.910, P<0.01) and the interaction between the two factors (F= 8.520, P<0.01)Conclusions:1. miR-214 could promote the invasive ability of the gastric cancer cell line SGC7901.2. miR-214 could down-regulate PTEN expression to promote the invasive ability in gastric cancer cell line SGC7901. |
PDF Full Text Request