| Micro RNAs(mi RNAs) are a kind of short non-coding RNA, from 19 to 25 nucleotides in size, which can post-translational down-regulate the protein expression of the target gene in organisms. Recent studies have shown that mi RNAs are playing a important role in wide array of critical cell processes,including drug resistance and prognosis, even involve in the initiation and progression of cancer which acts as oncogene or tumor suppressor gene. How to find the mi RNA relative to cancer and explore the function are the focuses of molecular medicine research.Gastric cancer(GC)is one of the most frequent malignant carcinomas in China and almost 400000 patients newly diagnosd every year. However, even the patients accepted surgery which be proven to the only method to cure GC, most of them would lead to recurrence or metastasis. Therefore, chemotherapy is necessary to control the recurrence or metastasis of GC and for advanced GC, but there is a big bottleneck that drug resistance response often appeared. So, it is the most crucial task to look for a effective and sensitive molecular markers, and illuminate the mechanism of multidrug resistance and find out the new target for treatment in GC.micro RNA-21(mi R-21)has been shown to be implicated in GC related processes including cell proliferation, apoptosis, invasion, metastasis and multidrug resistance. This mi RNA is frequently over-expressed in a wide variety of cancers and overexpressd mi R-21 may promote cell proliferation and inhibit apoptosis. Previous studies have also shown that mi R-21 was related with multidrug resistance in malignant tumors, including pancreatic cancer, bile duct cell cancer and breast cancer.However, the relationship between mi R-21 with drug resistance in GC was still not clear. In order to further explore the role for mi R-21 in GC drug resistance, we tested the expression of mi R-21, and verify the function and the mechanism of mi R-21 in GC. The general information of this article is as follows:Objective:To explore the expression level of the mi R21 in GC different types tissue, and analyze the correlation between mi R-21 expression and clinic-characteristics and prognosis of GC, while investigate the function and relationship with mi R-21 and doxorubicin resistance in GC cells.Methods:(1)The mi R-21 expression level was tested by q RT-PCR in 46 cases of primary GC tissues and their corresponding adjacent normal tissues. The correlation between mi R-21 expression level and clinic-characteristics of gastric cancer were analyzed by Mann-Whitney U and one-way ANOVA.(2) In this study, we successfully established the GC resistance cell line SGC7901/DOX3 by concentration gradient steadily increasing method and one-step drug selection. And then, Real-time PCR was used to detect expression of mi R-21 in SGC7901/DOX3 and SGC7901. The SGC7901 and SGC7901/DOX3 cells were transfected with the mimics or inhibitors of mi R-21 or negative control RNA(NC) by lipofectamine 3000. CCK-8 was used to analyze drug sensitivity. Apoptosis analysis and cell cycle were measured by fluorescence activated cell sorter and Western Blot.The cell cycle was measured by PI staining to detect.(3) We forecasted targets related to cancer by bioinformatics, then use dual luciferase reporter gene assay system and Western Blot were used to verify the real targets, and the targets gene m RNA expression were also determined by real-time q RT-PCR using GAPDH as an internal control in GC patient.(4) The expression of PTEN and TIMP3 m RNA and protein were measured by QRT-PCR and Western blot respectively, and the proliferation and apoptosis of GC after transfected by si-PTEN and si-TIMP3 were analyze by cell CCK-8 and fluorescence activated cell sorter and Western Blot.Results:(1) mi R-21 was significantly higher in tumor tissues than that in the corresponding normal tissues(P<0.05), what’s more, the mi R-21 expression was higher in recurrence GC than that in primary samples(P<0.05).The statistical analysis showed that the expression of mi R-21 were associated with degree of differentiation(P=0.017), TNM stage(P=0.047) and lymph node metastasis(P=0.013).(2) The expression of mi R-21 was an average of(8.5±0.887)-fold higher in SGC7901/DOX3 cells than in SGC7901 cells and there is a significant difference between the two groups(P<0.01). And mi R-21 expression gradually rised with the drug concentration, suggested that mi R-21 play an important role in drug resistance in GC cell. SGC7901/DOX3 cells transfection with inhibitor of mi R-21 showed that the IC50(4.476±0.014μg/m L) was less than the NC(23.085±0.0639μg/m L), and apoptosis rate was higher than the NC(P<0.05).(3) We forecast PTEN and TIMP3 were the targets of mi R-21 in GC, QRT-PCR and Western blot showed that anti-mi R-21 could promote the expression of ATM protein, while mi R-21 mimics could prevent that. All these results indicated that PTEN and TIMP3 is a downstream target gene of mi R-21 in different GC cell lines.Further, PTEN and TIMP3 were expressed at low levels in GC samples compared with their corresponding adjacent normal tissues, which negative correlate with the expression of mi R-21.(4)The expression of PTEN and TIMP3 were significantly lower in SGC7901/DOX3 cells than in SGC7901 cells(P<0.01).The drug resistance may could be partly reversed by transfected by si-PTEN or si-TIMP3 in SGC7901 cells,with a IC50 20.22±0.039μg/m L, 18.51±0.056μg/m L and 4.49±0.032μg/m L in si-PTEN or si-TIMP3 or si-NC respectively.Conclusion:In conclusion, our results demonstrated that dysregulated mi R-21, PTEN and TIMP3 in GC, and we identified PTEN and TIMP3 both as bona fide target of mi R-21 and Illustrated the high expression of mi R-21 was correlation with clinical characteristics and prognosis of GC, and reveals the mi R-21 involved in GC drug resistance mechanisms. It proved that the high expression of mi R-21 and change the cell cycle phase, promote cell apoptosis, cell resistance to change by targeting PTEN and TIMP3. Our data highlight an important role of mi R-21 in drug resistance of GC and implicate the potential application of mi R-21 in clinical cancer therapy. |
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