| Background and ObjectiveBreast cancer is a leading cause of cancer-related deaths in women worldwide. With the deepening of the medical research, We got to know that we can improve cancer treatment through changing the biological characteristics of breast cancer cells. The present study showed that the development of breast cancer involved in a series of changes at the genetic level.Micro RNAs(mi RNAs) are a class of small conserved non-coding RNAs that play an important role in kinds of physiological and pathological processes, including cell proliferation, differentiation, apoptosis, and epigenetic changes. mi RNAs can bind the 3′-UTRs of target m RNAs and thereby post-transcriptionally regulate gene expression via translational inhibition or target m RNA degradation. Studies revealed that mi RNAs participated in the initiation and development of breast cancer. In 2005, mi RNAs deregulation was first described in breast cancer. Subsequently, a growing number of studies reported altered mi RNA expression was connected with breast cancer. mi RNAs. Based on mi RNA manipulations, the development of new therapies is urgently needed.We aimed to evaluate mi R-365 and ADAMTS-1 m RNA expression in breast cancer tissues, and analyzed the relationship between their espression levels and clinicopathological characteristics of breast cancer patients. We also investigated the effect of mi R-365 on cell growth, cell cycle and cell invasion in MDA-MB-231 and MCF-7 cells, and explored the target relationship between mi R-365 and ADAMTS-1. Methods1、Breast cancer resection specimens(n=64) were collected. We evaluated mi R-365 and ADAMTS-1 m RNA expression levels in breast cancer using q RT-PCR, then we analyzed the relationship between their espression levels and clinicopathological characteristics of breast cancer patients.2、The mi R-365 mimics, mi R-365 inhibitor, and negative controls(NC) were transfected into MDA-MB-231 and MCF-7 cells using LipofectamineTM 2000 according to the manufacturer’s instructions, then the cells were maintained at 37°C in a humidified chamber with 5% CO2.3、CCK-8 assays were used to explore the effect of mi R-365 on cell growth in MDA-MB-231 and MCF-7 cells.4、Cell cycle assays were used to explore the effect of mi R-365 on cell cycle in MDA-MB-231 and MCF-7 cells.5、Transwell assays were used to explore the effect of mi R-365 on cell invasion in MDA-MB-231 cells.6、We investigated potential targets of mi R-365 using prediction algorithms(Target Scan, Pic Tar, and mi Randa), and conducted two reporter plasmids(pmir GLOADAMTS-1-wt and pmir GLO-ADAMTS-1-mut). Luciferase reporter and western blotting assays were used to test whether ADAMTS-1 is a direct target of mi R-365.7 、 We constructed an expression vector(pc DNA3.1-ADAMTS-1) that exogenously expressed ADAMTS-1 lacking the 3′-UTR. Restore assays were used to explored the mechanism that mi R-365 regulated the expression of ADAMTS-1. Results1. q RT-PCR was used to evaluate mi R-365 and ADAMTS-1 m RNA expression levels in breast cancer. We found that compared with adjacent normal tissues(the relative expression level was 1), mi R-365 expression level(2.711±0.893) was significantly higher in breast cancer tissues(P<0.05), and the mean ADAMTS-1 expression level(0.528±0.371) was significantly lower in breast cancer tissues(P<0.05). We also found that mi R-365 and ADAMTS-1 expression levels in breast cancer were associated with TNM stage(P <0.05), and there were no differences between mi R-365 expression and age, tumor size, lymph node metastasis, or estrogen receptor and progesterone receptor positivity(P>0.05).2. Compared with the Blank group or NC group, mi R-365 expression in cells transfected with mi R-365 mimics was significantly higher(MCF-7:6.274±0.591; MDA-MB-231:7.841±0.643), and mi R-365 expression levels in MDA-MB-231 and MCF-7 cells transfected with the mi R-365 inhibitor were significantly decreased(MCF-7:0.216±0.148; MDA-MB-231:0.126±0.104)(P<0.05).3. In CCK-8 assays we found that, compared to the Blank and NC groups, the OD450 values of cells transfected with mi R-365 mimics on days 2, 3 and 4 were increased, and the OD450 values of cells transfected with mi R-365 inhibitor on days 2, 3 and 4 were decreased, especially the difference on day 4 is statistically significant(P<0.05).4. Cell cycle assays showed that, compared to the Blank and NC groups, cells transfected with mi R-365 inhibitor had a significant increase in the percentage of G0/G1 phase cells, and had a significant decrease in the percentage of S phase cells(P<0.05).5. Trans-well assays showed that significantly fewer cells invaded the matrigel in the mi R-365 inhibitor group compared to the Blank and NC groups in MDA-MB-231 cells(P<0.05).6. Dual-luciferase showed that the luciferase activity was significantly reduced when the mi R-365 mimic was co-transfected with the wt-ADAMTS-1 reporter plasmid(P<0.05). In contrast, the mi R-365 inhibitor significantly promoted wt-ADAMTS-1 reporter plasmid luciferase activity(P<0.05).7. Restore assays showed that transfection with pc DNA3.1-ADAMTS-1 and co-transfection with mi R-365 mimics and pc DNA3.1-ADAMTS-1 markedly increased ADAMTS-1 expression and the population of cells in the G0/G1 phase, and decreased invasive cell number(P<0.05). While cells that transfected with mi R-365 mimics markedly decreased ADAMTS-1 expression and the population of cells in the G0/G1 phase, and increased invasive cell number(P<0.05). Conclusions1. Compared to adjacent normal tissues, the relative expression of mi R-365 was significantly higher in breast cancer tissues, the relative expression of ADAMTS-1 m RNA was significantly lower in breast cancer tissues, and their expression were associated with TNM stage.2. mi R-365 down-regulation reduced proliferation of MDA-MB-231 and MCF-7 celld, and restrained the invasion of MDA-MB-231 cells.3. mi R-365 may function as a novel oncogene in breast cancer by targeting the 3′-UTR of ADAMTS-1. |
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