The Study Of MiRNA-mRNA Regulatory Network Of Type â…  Endometrial Adenocarcinoma And The Effect Of CPEB1 On Its Biological Behavior

Introduction:Endometrial carcinoma, one of the three malignant tumors, formed by the endometrial epithelial cells, is a common malignant tumor of the female genital system. Its incidence rate only follows cervical cancer, which accounts for about 30% of gynecologic cancer. Although the survival rate of patients with endometrial carcinoma is not lower than other tumors of the female reproductive system, and its prognosis is good.But in China increased year by year. Serious harm women’s health. As a disease etiology is not clear, the endometrial carcinoma has a 85% to 90% for type I endometrial carcinoma; the pathogenesis of the tumor research on the disease is still not very good interpretation of endometrial cancer. Endometrial carcinoma in high-risk population is mainly in postmenopausal women, the age is between 50 and 60 years of age, accounting for about 75% of the total incidence of [2]. In recent years, related research reports at home and abroad, and endometrial cancer incidence and mortality in the world were significantly younger and rapidly rising trend [3],. The American Cancer Society The evaluation report shows that in 2005, the new discovery of endometrial cancer 40880 cases, where 7310 patients died. In 2010, new cases 43470 cases,795 cases [death.6], and the latest epidemiological survey data show, in 2015, the U.S. women all new of cancer, endometrial cancer incidence increased obviously, ranked only in breast cancer, lung cancer and colon cancer among the four, and is expected due to endometrial cancer death toll reached 10170 much. In our country, there is no large-scale fully the endometrial cancer statistics report However, according to incomplete survey statistics, the incidence of endometrial cancer in our country has been increasing year by year.According to the different pathological morphological characteristics of tumor and uterine endometrial carcinoma is divided into two types:the type Ⅰ estrogen dependent and type Ⅱ non estrogen dependent [8], the molecular mechanism of the development of both, morphological characteristics and prognosis of exist obvious difference. Ⅰ type and estrogen hyperstimulation, mainly for endometrial excessive growth based on the formation of endometrioid adenocarcinoma development, accounting for 80-90% of endometrial cancer; type Ⅱ major pathological type was clear cell carcinoma, papillary serous carcinoma, mucinous adenocarcinoma and adenosquamous carcinoma., clinical to estrogen dependent type Ⅰ[9], so this study mainly for type Ⅰ of the uterus Membrane adenoid carcinoma were studied and mentioned in front of endometrial cancer refers to generation of type Ⅰ endometrial cancer. Endometrial cancer early clinical performance for irregular vaginal bleeding symptoms can help to early diagnosis and 80% of patients with endometrial cancer lesions will be confined to the uterus in vivo, because of this, with respect to the other female genital system malignant tumor patients, patients with endometrial cancer most five years survival rate is not low, prognosis is good [10], studies have reported that patients with early endometrial cancer five years free disease survival rate can reach 98% and 5 year overall survival rate of up to 93%[11]. However, there are still some endometrial cancer patients Low degree of pathological differentiation, invasion and metastasis rate and poor prognosis, usually because of tumor metastasis and invasion, leading to the depth of the disease progression and death. At present, the pathogenesis of endometrial cancer is not entirely clear, for high pathological stage, patients with metastatic recurrence occurred there is no effective treatment measures. Therefore, in-depth study of endometrial cancer biological characteristics, search endometrial carcinoma associated markers and analysis its differentiation and endometrial cancer, difference and molecular mechanism of metastasis and invasion of and to improve the prediction of the judgement of endometrial cancer progression and clinical treatment guidance and the prognosis of patients with modified Good is of great significance.MicroRNAs (micro RNAs and miRNAs) is a composed of 22 to 25 nucleotide noncoding single stranded RNAs. Now it is considered that in mammals, miRNA and target genes, the mRNA of target gene in translation process after transcription is inhibited or degradation, resulted in expression level was lower in the target gene of egg white. In this way, miRNA involved in including growth, hematopoiesis, organ formation, cell proliferation, apoptosis and tumor occurrence of a variety of biological processes [12]. According to the study, each miRNA can control 200 target genes, also a target gene can be miRNA regulation. Bioinformatics is the use of miRNA and target gene mRNA can completely or partially complementary principle, through their respective nucleotide sequences predict may and specific miRNA and mRNA in all mRNA (miRNA), to narrow the scope of the study, further experiments verify their exact relationship.MiRNA-mRNA regulatory networks defined in similar biological processes in a group of miRNA S and a set of mRNAs. A control module contains miRNAs and their target mRNAs are highly correlated, they are thought to have similar biological function. At present, is widely used MRMs[13]. This method is the use of a variety of heterogeneous data source miRNA mRNA base pairing information, miRNAs and mRNAs expression information to build a network of miRNA mRNA regulation, they found their miRNA mRNA with high confidence degree and the expression pattern of miRNAs and mRNAs are highly correlated in the. Because of This, by mining the miRNA mRNA regulatory modules to understand disease of biological processes at the molecular level. Secondly, it can understand biological disease mechanism of cancer or tumor, including. In addition, it can provide a reference for gene diagnosis and gene therapy.Cytoplasmic poly adenylation original binding protein (cytoplasmic polyadenylation element binding protein, CPEBs) is a group of highly conserved RNA binding protein family. In the study of its relation with tumor and to CPEB 4 research and CPEB1 widely. But with the preparatory work for the foundation, indicating CPEBs in the family of of CPEB 1 endometrial carcinoma and endometrial carcinoma high-throughput sequencing more closely. Literature research, CPEBs human papillary tumor virus (H Uman papilloma virus (HPV) staining[14]. HPV persistent infection and protein E5, E6 and E7 expression is cervical cancer prerequisites, HPV E5, E6, E7 mRNA containing 3’do not translate region (3’UTR) specific sequences (U rich cytoplasmic polyadenylation original, CPE), they are the specific targets of CPEB1 and the polyadenylation translation by CPEB1 regulation.CPEB 1 in HPV positive tumor development may have Important regulatory function, mRNA CPEB as a marker of reproductive organ cancer should be very useful, so to explore the role of CPEB1 in the occurrence of endometrial cancer is of great significance. there are still some cases of endometrial cancer patients with low pathological differentiation, high invasion and metastasis rate, low prognosis ablitity, commonly duing to the tumor metastasis and growth, leading to the deterioration of the condition and death. At present, it is not clear about the pathogenesis of endometrial cancer, and there are not effective treatment for the severe patients. Therefore, it is important to study the biological characteristics of endometrial carcinoma and to analyze the differences and molecular mechanisms of endometrial carcinoma.Objective:This study was aimed to use biological network information theory, traits associated miRNA from endometrial cancer related miRNA literature mining, combined with the known function of the gene and miRNA target prediction database, build miRNA and target gene regulation network, and previous research work to combine and initially validated by the selected new and endometrial carcinoma is highly correlated with the target gene CPEB1 and target gene CPEB1 center regulation network; based on again to get clear and lack of function test selected target gene in endometrial carcinoma The molecular mechanism of CPEB1 in the development and progression of endometrial carcinoma.Methods:Using the free word retrieval, to facta tool and PubMed database literature mining endometrial cancer related miRNAs; four different prediction methods (targetscan, Miranda, was and Starbase) prediction excavated out of each miRNA target gene miRNA mRNA of; from the miRNA mRNA of selected at least five or more miRNA common targeting mRNA for endometrial cancer related gene to construct endometrial carcinoma of miRNA mRNA network. The QRT-PC Method R verification regulation network in comprehensive ranking near the front of the mRNA expression of selected specific differentially expressed genes CPEB1 as the target gene for further study. Expression in endometrial carcinoma tissues and cells by QRT PCR method for the detection of CPEB1; construction CPEB1 over expression vector, by in vitro transfection CPEB1 overexpression vector overexpression of CPEB1. By MTT, colony formation, flow cytometry, effects of scratch assay and Transwell experiments in vitro detection CPEB1 on the biological behavior of endometrial carcinoma cell line; construct stable over expression Endometrial cancer cell lines CPEB1. The mice into tumor in vivo biological function analysis of CPEB1; by transfection CPEB1 overexpression vector and to observe the morphological changes of cells, using Western blot analysis of apoptosis related proteins and key molecular protein marker of EMT expression and preliminary study CPEB1 molecular mechanisms involved in endometrial cancer cell biological behavior regulation.Results:1.208 EEC-relatived miRNAs were found based on the existing 41 relevant literature and multi-step analysis, and finally 35 endometrial cancer related miRNA were got after 4 different database comparison analysis.2. The targeted genes of 35 miRNAs were predicted by 4 kinds of prediction methods, and 110995 miRNA-mRNA pairs were obtained, about 7919 miRNA-mRNA pairs were exist in more than 3 prediction methods, and they contained 27 miRNAs and 3082 genes, among them, about 5 miRNA targeted and more than 457 genes.3. Proteins interaction of the 457 candidate genes was analysis, there were 61 genes each had more than 5 interacion proteins,14 genes each had more than 10 interacion proteins. Signaling pathway analysis of these 61 candidate genes were enrich in cancer related pathways.4. The expression of 10 of the 61 candidate genes in EEC were verified by QRT-PCR experiments,among them,3 genes, IGF1R and CDC25A were up-regulated in endometrial. carcinoma,which had been reported to be associated with endometrial cancer, but the expression of CPEB1 was down regulated, so it was selected for further studied.5. The expression of CPEB1 in 20 pairs of endometrial carcinoma tissues and its normal endometrial epithelial cells,3 endometrial carcinoma cell lines and 1 normal endometrial epithelial were detected by QRT-PCR. The results showed that the expression of CPEB1 in endometrial carcinoma tissues and cells was significantly down regulated, suggesting that CPEB1 may be a molecular marker of endometrial carcinoma.6. The in vitro functional studies showed that CPEB1 could inhibited the proliferation of endometrial cancer cells in vitro by MTT and clone formation expriments, CPEB1 can promote apoptosis of endometrial cancer cells by cell flow detection, CPEB1 could significantly inhibit the invasion of endometrial cancer cells by Transwell assay. CPEB1 could inhibit the formation of tumor in vivo.7.CPEB1 in ISK cell line is extremely low expression and/or non expression, CPEB1-siRNA experiment failed to build CPEB1 low expression vector.8.Through biological information science database analysis and prediction, suggesting that CPEB1 may participate in genesis of tumor diseases through cell apoptosis and EMT pathway; apoptosis related molecule detection results show that overexpression of CPEB1, isk of endometrial cancer cells apoptosis related p53, Bax, caspase-3 expression upregulation and downregulation of bcl-2 expression; prompt CPEB1 may participate in p53 mediated endometrial carcinoma cell apoptosis.9.EMT related molecule detection results showed that over expression of CPEB1 after cell epithelial morphological change. Endometrial cancer cell isk epithelial mesenchymal transformation characteristics of markers and associated transcription factor E-cadherin, beta catenin expression was significantly up-regulated, vimentin, SMA, snail reached significantly down regulated, twist does not change significantly, which confirmed, CPEB1 high expression promotes cell mesenchymal to epithelial change (MET).Conclusions:1. successfully constructed the miRNA-mRNA network of endometrial carcinoma, and screened CPEB1 for endometrial cancer closely related genes.2. The expression of CPEB1 was low in endometrial carcinoma, and low expression of CPEB1 may be a molecular marker for the occurrence and metastasis of endometrial carcinoma.3. CPEB1, as a cancer-supress factor, could inhibit the growth, proliferation, invasion and metastasis of endometrial cancer cells, and inhibit the biological function of tumor cells in vivo.4. CPEB1 could affected the expression of apoptosis relatived genes, such as p53, bax, bcl-2 and caspase3, suggested it may be involved in the development of endometrial carcinoma by p53 induced apoptosis.5. CPEB1 could affected the expression of EMT relatived genes, such as E-cadherin, Vimentin, SMA, β-catenin and Snail, suggested it maybe inhibit the invasion and metastasis of endometrial carcinoma by discreasing the expression of EMT-relatived factors.6. The expression of key molecules pAKT and pPTEN protein in PI3K/AKT signaling pathway were significantly effected by CPEB1, suggested CPEB1 could regulated the invasion and metastasis of endometrial carcinoma by down-regulated the phosphorylation level of PTEN and AKT, inhibit the PI3K/AKT signal pathway.