| Thyroid cancer(TC) is the most prevalent endocrine neoplasm, and has had a steadily increasing incidence over the last several decades. Papillary thyroid carcinoma(PTC), referred to as a differentiated neoplasia, is the most prevalent type of tumor among thyroid malignancies, accounting for approximately 80%-90% of all TC cases. Most patients with PTC have a favorable prognosis, but the overall recurrence rates may be as high as 35%, since clinical and biological behaviors cannot be properly predicted. Therefore, it is an urgent need to understand the molecular mechanisms of PTC progression to find novel diagnostic, prognostic, and therapeutic strategies for this disease.Micro RNAs(mi RNAs) are small non-coding RNAs(19–25 nucleotides) that regulate gene expression at the transcriptional or posttranscriptional level by binding to the complementary 3’-untranslated region(3’-UTR) of messenger RNAs(m RNAs).Mi RNAs may regulate thousands of human genes and accumulating evidence showed that mi RNAs regulate a variety of basic physiological processes such as cell differentiation, proliferation and survival, apoptosis, migration and invasion.mi RNAs has been reported to be associated with cancer development and progression through regulation of cellular proliferation, differentiation, and apoptosis. Therefore, analysis of the mi RNAs and the related target genes may provide unique insight into tumorigenesis and new strategies to improve the diagnosis or treatment of PTC.mi R-29 a is a member of mi R-29 s family, which is a conserved family of mi RNA.Decreased expression of mi R-29 s has been described in multiple cancers, such as gastric cancer, pancreatic cancer and prostate cancer. In contrast, upregulation of mi R-29 s was reported in breast cancer, nasopharyngeal carcinoma, glioma and acute myeloid leukemia. These studies suggested that mi R-29 a functions as an oncogenic or a tumor-suppressive mi RNA in different cancers. However, the clinical significance,and its role and underlying molecular mechanism in PTC remain unclear. Therefore,in the present study, we analyzed the association of mi R-29 a expression with clinicopathologic features in patients suffering PTC, and investigate the functional role of mi R-29 a and potential mechanism of PTC by several in vitro experiments and tumor growth of xenograft in vivo.Objective:Clarify the role and mechanism of mi R-29 a in Papillary Thyroid Carcinoma.Methods:1. The expression of mi R-29 a in PTC and the mechanism of proliferation and migration.(1) We assessed mi R-29 a expression by q RT-PCR in the 30 PTC tissues and matched adjacent normal tissues. To further investigate the clinicopathological significance of mi R-29 a level in PTC patients, the median value(0.59) of all 30 PTC samples were chosen as the cut-off point for separating tumors with low relative level of mi R-29a(<0.59, 14 cases) and high relative level of mi R-29 a group(>0.59,16 cases). Then correlations between mi R-29 a expression and clinicopathologic parameters were analyzed by the chi-square test.(2) To study the biological role of mi R-29 a in PTC, mi R-29 a stably expressing cell lines of K1 was established we by transfection of mi R-29 a mimic. Successful increase of mi R-29 a expression in K1 cells was confirmed by q RT-PCR. Then cell proliferation, cycle and apoptosis were determined in K1 cells transfected with mi R-29 a mimic or mi R-Ctrl.(3) We next investigated the role of mi R-29 a in regulating PTC cell migration and invasion by wound healing and invasion chamber assay.2. The mechanism of mi R-29 a target AKT3 inhibit the growth of PTC.(1) Potential targets of mi R-29 a were predicted using bioinformatic databases such as Target Scan, mi Randa and Pic Tar, AKT3 has been chosen as study object since it plays central role in the PI3K/ AKT signaling pathway, and involves in multiple cellular functions. To verify whether AKT3 is a direct target of mi R-29 a in PTC, a human AKT3 3’ UTR fragment containing the binding sites of mi R-29 a or the mutant sites were cloned into the p GL3 vector, then along with mi R-29 a mimic or mi R-Ctrl were co-transfected into K1 cells and cultured for 48 h, then luciferase activities were determined. Then, q RT-PCR and western blotting analysis confirmed that overexpression of mi R-29 a drastically inhibited AKT3 expression on m RNA level and protein level in K1 cells.(2) Knowing AKT3 was the target of mi R-29 a, we investigated expression of AKT3 in PTC specimens and their matched adjacent normal tissues from 30 PTC patients by q RT-PCR.(3) To investigate the biological role of AKT3 in PTC, K1 cells were transfected with si-AKT3 or the si-Ctrl. The knockdown efficiency of AKT3 was verified by q RT-PCR and western blot. After si-AKT3 treatment, MTT, apoptosis, would healing and invasion assays were performed.(4) To investigate the functional relevance of AKT3 targeting by miR-29 a, we assessed whether over-expression of AKT3 could rescue the effects of mi R-29 a on PTC cells proliferation, apoptosis, migration and invasion. To this end,overexpression AKT3 plasmid was transfected into K1 cells along with mi R-29 a mimics or mi R-Ctrl.3. miR-29 a suppresses PTC growth in vivo.Having shown in vitro that mi R-29 a possessed tumor suppressive activities, we undertook to evaluate it role in vivo. To this aim, the human K1 cells stably expression mi R-29 a or mi R-Ctrl was implanted subcutaneously into nude mice to allow tumor formation. At 30 th days post-injection, the mice was killed, tumor tissue was stripped. Tumor size and weight was observed in mice injected with K1/mi R-29 a compared to the group injected with K1/ mi R-Ctrl. Furthermore, we also determined mi R-29 a and AKT3 expression in tumor tissue.Results:1. The expression of miR-29 a in PTC and the mechanism of proliferation and migration.(1) mi R-29 a is down-regulated in human PTC tissues.It was found that the relative expression of mi R-29 a in PTC tissues was significantly lower than that those of their matched adjacent normal tissues. To further investigate the clinicopathological significance of mi R-29 a level in PTC patients, the median value(0.59) of all 30 PTC samples were chosen as the cut-off point for separating tumors with low relative level of mi R-29a(<0.59, 14 cases) and high relative level of mi R-29 a group(>0.59, 16 cases). Then correlations between mi R-29 a expression and clinicopathologic parameters were analyzed by the chi-square test. Correlation analysis showed that mi R-29 a expression was not significantly associated with age and gender, while the level of mi R-29 a negatively correlated with TNM stage(P <0.01), Tumor size(P <0.01) and lymph node metastasis(P <0.01).(2) miR-29 a inhibits PTC cell proliferation and induces apoptosis.Successful increase of miR-29 a expression in K1 cells was confirmed by q RT-PCR. Then cell proliferation, cycle and apoptosis were determined in K1 cells transfected with mi R-29 a mimic or mi R-Ctrl. MTT assay showed that restoration of mi R-29 a significantly inhibited cell proliferation in K1 cells compared to mi R-Ctrl group. Cell cycle assay showed that restoration of mi R-29 a in K1 cells significantly decreased the percentage of S phase, and increased percentage of G0/ G1 phase compared to mi R-Ctrl group. Cell apoptosis assay revealed that restoration of mi R-29 a in K1 cells significantly induced cells apoptosis compared to mi R-Ctrl group(P <0.01).(3) mi R-29 a inhibits PTC cells invasion and migration.It was found that restoration of mi R-29 a significantly inhibited cell migration and invasion capacities of K1 cells.2. The mechanism of mi R-29 a target AKT3 inhibit the growth of PTC.(1) AKT3 is a direct target of mi R-29 a.It was found that overexpression of miR-29 a expression obviously suppressed the luciferase activity of wide-type AKT3 site, but the activity of the mutant AKT3 site was not change, suggesting that AKT3 is a directly target of mi R-29 a. Then,q RT-PCR and western blotting analysis confirmed that overexpression of mi R-29 a drastically inhibited AKT3 expression on m RNA level and protein level in K1 cells.In addition, we also found that overexpression of mi R-29 a also inhibited phospho-Akt(p-AKT) expression, suggesting that mi R-29 a could inhibite PI3K/AKT signal pathway activation. These results indicated that mi R-29 a can bind directly to AKT3 and inhibits its expression.(2) mi R-29 a expression is inversely correlated with AKT3 expression in PTC tissues.It was found that AKT3 m RNA expression levels were increased in PTC tissues compared to matched adjacent normal tissues( P<0.01), and was negatively correlated with mi R-29 a expression in PTC tissues(P<0.01).(3) Downregulation of AKT3 had similar effects of mi R-29 a.As presented in, downregulation of AKT3 by si-AKT3 in K1 cells inhibited cell proliferation and induced apoptosis, as well as suppressed cell migration and invasion capabilities. In other words, reduction of AKT3 mimicked the effect of mi R-29 a over-expression.(4) Overexpression of AKT3 rescues the effects of mi R-29 a.It was found that overexpression AKT3 plasmid could increase AKT3 expression on m RNA level and protein level. In addition, our results also showed that overexpression of AKT3 in K1 cells could reverse the effect of mi R-29 a on cell proliferation, apoptosis, migration and invasion. These data indicated that mi R-29 a exerts inhibition effect on PTC growth and metastasis partially by targeting AKT3.3. miR-29 a suppresses PTC growth in vivo.A significant decrease in tumor size and weight was observed in mice injected with K1/mi R-29 a compared to the group injected with K1/ mi R-Ctrl( P<0.01).Furthermore, we also determined mi R-29 a and AKT3 expression in tumor tissue. We found that the mi R-29 a expression significantly increased(P<0.01), whereas AKT3 expression remarkably decreased both on m RNA level(P<0.01) and protein level in K1/mi R-29 a group compared to K1/mi R-Ctrl group.Conclusions:1. mi R-29 a was frequently down-regulated in PTC tissues, and its expression was significantly associated with tumor size, TNM stage and lymph node metastasis.2. Overexpression of mi R-29 a markedly suppressed PTC cell proliferation,migration and invasion, and promoted PTC apoptosis and cell cycle arrest at G0/G1 phase. In vivo, mi R-29 a overexpression decreased tumor growth by targeting AKT3 in a xenograft mouse model.3. mi R-29 a can directly bind to the 3’ untranslated region(UTR) of AKT3 in PTC cells. Overexpreesion of mi R-29 a obviously decreased AKT3 expression thereby suppressing phosphatidylinositol 3-kinase(PI3K)/AKT pathway activation.4. AKT3 expression was increased in PTC tissue, and was inversely correlated mi R-29 a expression in PTC tissue. Downregulation of AKT3 by si RNA mimicked the effects of mi R-29 a overexpression, and upregulation of AKT3 partially reversed the inhibitory effects of mi R-29 a. |
