| Background:Endometrial carcinoma(EC)derives from the epithelium and accounts for 20%-30% of malignant genital tumors,a death threat to women worldwide with high occurrence and mortality.Recent studies show that EC occurrence is on the rise and is more and more common among young women.Though standard treatment is available and the 5-year survival rate increases,the treatment outcome for patients with advanced,poorly-differentiated or recurrent EC remains gloom.Therefore,it is urgent to figure out the cellular mechanism of EC progression,to identify specific biomarkers for EC diagnosis and prognosis,to work out new therapeutic strategy for EC.There is accumulating evidence that EC is closely correlated to obesity,diabetes,hypertension and long-term unavoidable exposure to estrogen;however,most of them were halted at the abnormal gene regulation such as epigenetic regulation.Recently,microRNAs(miRNA)have been highlighted in many studies for the impressive role in cellular process for cancers including EC.miRNAs,small non-coding RNA molecules,contains 19-25 nucleotide and can bind to the mRNA of a specific gene at 3'UTR through complementary base pairing and thereby regulate the gene at the post-transcriptional level by suppressing mRNA translation or degrading the mRNA.Since the first identification of miRNA(lin-4)in caenorhabditis elegans by Lee and his co-workers in 1993,miRNAs have been a hotspot in cancer research.It is proven that differentially-expressed miRNAs are oncogenes or anti-oncogenes in the progression of cancers,including EC.miR-200 family and miR-205 are known as tumor-promoting miRNAs while miR-200 c,miR-424,miR-223 and miRNA-124 as tumor-suppressing miRNAs.On this ground,a detailed understanding of EC-specific miRNAs and their target genes may facilitates determining progression,treatment and prognosis of EC.The miR-29 family,covering miR-29 a,miR-29 b and miR-29 c,is first discovered in Hela cell line and functions as an oncogene in many cancers.Previous studies show that miR-29 b,the most important member in the family,is involved in proliferation,differentiation,apoptosis and invasion of tumor cells and its abnormal expression results in cell dysfunction.Differentially-expressed miR-29 b is observed in patients with such cancers as breast cancer,liver cancer and prostatic cancer,suggesting that miR-29 b is a promising biomarker for diagnosis,treatment and prognosis of cancers.Regretfully,few studies have ever explored the miR-29 b expression in EC patients,its correlation to the EC clinicopathological features and its role in EC.Angiogenesis is a process whereby the endothelial cells of the pre-existing vessels grows into new blood vessels.It nourishes tumors and produces multiple angiogenic growth factors.Among such angiogenic growth factors,Vascular endothelial growth factor A(VEGFA)is a significant participant in proliferation,differentiation and migration of endothelial cells.Mitogenactivated protein kinase/extracellular regulated protein kinases(MAPK/ERK)pathway and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathway are two major signaling pathway for signal transmission into the cell nucleus,and thus regulate cell proliferation,angiogenesis and metabolism.It is reported that estrogen can activate various pathways in the uterus including MAPK/ERK and PI3K/Akt pathway.Nevertheless,the role of MAPK/ERK and PI3K/Akt pathway in angiogenesis is unclear.In this context,we determined the expression of miR-29 b in EC tissues and cell lines,investigated the role of miR-29 b in angiogenesis in EC and looked into the mechanism for such relationship.Methods:?.miR-29 b expression in EC tissues and cell lines and its clinical significanceEC tissues and paired adjacent normal tissues were resected from EC patients in surgeries between February,2013 and April,2015 hospitalized in the First Affiliated Hospital of Nanchang University.Those tissues were fixed in formalin and then embedded in paraffin.qRT-PCR was adopted to detect miR-29 b in the collected tissues and EC cell lines(type I endometrial cancer cell,RL-95-2;type I endometrial cancer cell,HEC-1-B).Statistical analysis was performed to define the relation of miR-29 b expression in the tissues to the clinicopatholoical features of EC.CD34 antibody was used to label vascular endothelial cells with a string of endothelial cells in brownish yellow to be defined a new vessel.Immunohistochemistry was conducted to measure the CD34 expression in the EC tissues and paired adjacent normal tissues for a decision of angiogenesis.?.The role of miR-29 b in angiogenesis of the EC tissuesmiR-29 b was predicted to target VEGFA by bioinformatics software,and such predication was afterward validated using qRT-PCR,Western Blot and dual-luciferase reporter gene system.VEGFA was measured using qRT-PCR for the analysis on the relation of VEGFA mRNA to clinicopathological features in EC patient.There were 8 cell groups: Normal group(normal human endometrial epithelial cells),Blank group(non-transfected RL-95-2 cells),pMIR-control group(RL-95-2 cells transfected with plasmid of empty vector),pMIR-miR-29 b group(RL-95-2 cells transfected with miR-29 b recombinant plasmid),LNA-control group(RL-95-2 cells transfected with oligonucleotides of non-specific effects),LNA-miR-29 b inhibitor group(RL-95-2 cells transfected with miRCURY LNA? miR-29 b inhibitor),LNA-miR-29 b inhibitor + PD98059(RL-95-2 cells transfected with miRCURY LNA? miR-29 b inhibitor and then cultured with MAPK/ERK signaling inhibitor PD98059)and LNA-miR-29 b inhibitor + wortmanin group(RL-95-2 cells transfected with miRCURY LNA? miR-29 b inhibitor and then cultured with PI3K/Akt signaling inhibitor wortmanin).ERK,Akt,mTOR and Bcl-2(downstream of MAPK/ERK and PI3K/Akt signaling pathway were)in the 8 groups were measured with qRT-PCR and Western Blot along with miR-29 b and VEGFA.A mouse model of EC was built with cells of the 8 groups transplanted in.The transplanted tumors were under close observation of the growth rate.The microvessel density in the tumors was measured with immunohistochemistry.Results:?.miR-29 b was downregualted in EC tissues and cells and close correlarted with clinicopathological features;1.miR-29 b was found significantly downregualted in EC tissues and cell lines in respective comparison with paired adjacent normal tissues and normal human endometrial epithelial cells(both P < 0.05).2.miR-29 b was found significantly higher in patients at ? + ?stage and patients with no lymphatic metastasis when compared with that in patients at ? + ? stage and patients with lymphatic metastasis,respectively(both P < 0.05).3.EC tissues had a significantly higher microvessel density than paired adjacent normal tissues(P < 0.05)and the MVD was found to be negatively correlated with expression level of miR-29b(Pearson r = 0.751,P < 0.01;Spearman rho = 0.772,P < 0.01).?.miR-29 b reduction and higher microvessel density1.miR-29 b directly targeting VEGFA2.VEGFA mRNA was found significantly lower in patients at ? + ?stage and patients with no lymphatic metastasis in parallel with that in patients at ? + ? stage and patients with lymphatic metastasis,respectively(both P < 0.01).3.When compared with normal group,the other 7 groups of RL-95-2 cells had significantly reduced miR-29 b mRNA and protein while remarkably raised mRNA and protein of VEGFA,ERK,Akt,mTOR and Bcl-2(all P < 0.05).When compared with blank group,miR-29 b group exhibited upregulation in miR-29 b while downregulation in mRNA and protein of VEGFA,ERK,Akt,mTOR and Bcl-2 and LNA-miR-29 b inhibitor group showed suppressed miR-29 b and while increased VEGFA,ERK,Akt,mTOR and Bcl-2 in both mRNA and protein expression(all P < 0.05).As revealed by LNA-miR-29 b inhibitor + PD98059 and LNA-miR-29 b inhibitor + wortmanin group,such tension between miR-29 b and VEGFA,ERK,Akt,mTOR and Bcl-2 was weakened by inhibition of MAPK/ERK and PI3K/Akt signaling pathway.4.In the mouse model,it was found that the mice treated with RL-95-2 cells presented increased tumor size,tumor weight and microvessel density by reference to the mice with treated with normal cells(all P < 0.05).In parallel with mice treated with cells of Blank group,mice treated with cells of miR-29 b group had tumors of smaller size and less weight and lower microvessel density,while mice treated with cells of LNA-miR-29 b inhibitor grew tumors of larger size and more weight(all P < 0.05);In mice treated with cells of LNA-miR-29 b inhibitor + PD98059 group and LNA-miR-29 b inhibitor + wortmanin,the trend of tumor growth was attenuated.Conclusion:In EC,miR-29 b may suppress MAPK/ERK and PI3K/Akt signaling pathway via directly targeting VEGFA and thereby inhibit angiogenesis in EC tumors.For this reason,miR-29 b may be a new target for EC therapy. |
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