The Preliminary Study On The Expression Of Hsa-miR-181a In Endometrial Carcinoma And Its Significance

Endometrial carcinoma is a group of epithelium occurred in endometrial malignant tumor, the endometrial gland adenocarcinoma is the most common. It is one of the three most frequently malignancies of the female genital tract, accounted for7%of systemic cancer in women. There are differences in the incidence in different regions all over the world, the highest incidence in the North America and Nordic, but lower in Asia, Japan, India and Latin America. Our country is still lack of a more detailed epidemiology survey data on endometrial cancer, maybe the estimated incidence is similar in Japan. In recent years, the incidence of endometrial cancer is increasing.Up to now, the etiology of endometrial cancer is not very clear. As is known to all, the growth and function of endometrium is regulated by estrogen and progesterone, it relates with high levels of estrogen. Progesterone is usually exert effects on the basis of the role of estrogen. The long-term stimulation of endogenous or exogenous estrogen is one of the most important and direct reason of endometrial carcinoma. Excessive endometrial proliferation may be driven by endogenous or exogenous overexposure to oestrogens, then develop to atypical hyperplasia and finally develop to endometrial carcinoma.Thirty years ago, Bohkman classified the endometrial carcinoma into two types (type I and type II) based on the clinical and pathological features. Type I was estrogen dependent, which may be involved in the occurrence of endometrial hyperplasia and cancerous, without progesterone antagonist. This type accounts for the majority of endometrial cancer. They are endometrial adenocarcinoma, with well differentiation and high estrogen receptor (ER)-positive rate. Type II was non-estrogen-dependent. The occurrence of endometrial carcinoma has no definite relationship with estrogen. There can be observed many atrophy endometrial tissue around the primary tumor, with poor differentiation and low ER-positive rate.Mature small RNA (microRNA, miRNA) are a class of endogenous small non-coding and single-stranded RNA of20-23nucleotides. MicroRNAs thought to control gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs by binding with the3’region of target gene. miRNAs play important roles in various biologic processes in worm, fly, fish, mouse, and human cells, where they regulate apoptosis, proliferation, differentiation, development, and metabolism. In addition, miRNAs are dynamically regulated in response to physiological and developmental cues. It was suggested that over30%of protein-coding genes in humans are regulated by miRNAs. Since Lee et al found that in the first miRNA lin-4in1993, miRNAs is becoming a hot topic in the field of cancer research. miRNA dysregulation could drive tumorigenesis through the roles miRNAs can adopt as tumor suppressors or oncogenes.The estrogen receptor and progesterone receptor, which received the stimulation of estrogen and progesterone, can be found extensively in tissue and cell of female genital organs. There is many researches showed that estrogen can inhibit or induce the expression of miRNA, while the miRNA would affect the activity of estrogen, consequently influence the metabolism of estradiol. Therefore, miRNA may be involved in the in the occurrence and progression of materials used in detecting, such as breast cancer and endometrial carcinoma.Has-miR-181(miR-181) was a member in the miRNAs family, reports showed that the hemopoietic progenitor cell has an over-expression of miR-181, after some stimulation, the cell number of B cell was increased with T cell not affected, which in total suggested the important role of miR-181in the cell differentiation. The miR-181family include miR-181a, miR-181b, miR-181c and miR-181d. miR-181a was confirmed to be a key factor in the regulation of cell proliferation, development, and differentiation, the over-expression of which often resulted in abnormity of cell function. Previous data revealed the abnormal expression of miR-181a in many cancer, including leukemia, breast cancer, liver cancer, colorectal cancer and glial cell tumors. What’s more, miR-181a can regulate the expression Bc1-2、TGF-β、PTEN、 ATM, all of which was associated with the development of cancer. In a word, we can conclude that miR-181a plays an important role in the occurrence and progression of tumors.Currently, the research focusing on the relationship between endometrial carcinoma and miR-181a was rare in our country. Also there are only some reports in the worldwide. Using fresh frozen tissues, they suggested that the expression level of miR-181a was higher in endometrial carcinoma than normal endometrial tissues, with PR being target gene, then they concluded that miR-181a involved in the progression of endometrial carcinoma through suppressing the expression of PR. Until now, the materials used in detecting miRNAs were fresh frozen tissues, cultured cells, and blood serum. However, it is difficult and costly to keep the frozen tissues in storage, which limited its use in miRNAs. The formalin-fixed paraffin-embedded (FFPE) endometrial carcinoma tissue has been used in detecting the biomarker and molecular mechanism in some diseases. To date, there are few researches investigating the miRNAs with FFPE tissues. Therefore, first, we would examine whether it is feasible to detect miRNAs using FFPE tissues. Then, we carry out experiments to clarify1) whether miRNA can be detected in FFPE?2) Whether the expression level of miR-181a was similar as that in different races and regions?3) Whether it is involved in the morbidity of type I and type II endometrial carcinoma?4) Whether miR-181a can be used as biomarker for the diagnosis, treatment, and prognosis in clinical practice? The above questions are worth to be researched.Based on the above issues, the study was aimed to investigate the miR-181a expression in endometrial tissues and its related significance by real-time quantitative PCR. In the first section, the expression of miR-181a was determined in normal, hyperplastic, and cancerous endometrial tissue. Then its significance was analyzed in the transition from normal endometrial to cancerous states. In the second section, first, the endometrial carcinoma was classified into type Ⅰ and type Ⅱ based on the immunohistochemical results. Then the expression of miR-181a was detected in type Ⅰ and type Ⅱ endometrial carcinoma respectively, in order to examine whether miR-181a was involved in the origin of the two different type cancer.Chapter1Expression of miR-181a in endometrial carcinoma and its clinical significanceContents and MethodsThe present study was aimed at exploring the expression of miR-181a in the progression of endometrial carcinoma and its clinical significance. Seventy-two endometrial tissues were obtained from Xiao Lan People Hospital Affiliated to Southern Medical University, four type Ⅱ endometrial carcinoma tissues from Zhongshan Hospital and two from Southern Hospital during January2011to December2013. All the samples were formalin-fixed paraffin-embedded (FFPE) tissues. The age of patients ranged from24to69yr, with average48yr. Among the tissues,13were normal endometrium,18were endometrial hyperplasia, and47were endometrial carcinoma. No radiotherapy, chemotherapy and endocrine therapy were taken pre-operatively, and the diagnosis was confirmed by pathological test post-operatively. Ten sections were obtained from each case with thickness lOpm. The section was used only if the cells covered over50%of the area. Purification of total RNA from FFPE tissue was performed following the instructions, and then real-time PCR of SYBR green was carried out to detect the expression level of miR-181a in endometrial tissue in each group. Comparative Ct (threshold cycle) method was used to present the expression level of miRNA, which calculate△Ct=Ct (miR-181a)-CtU6to indicate the relative gene expression, calculated△△Ct=(Ct (miR-181a)-CtU6) experimental grou-(Ct (miR-181a)-CtU6) control group,2-△△Ct fold change represents the fold change of miRNA.Results1The expression of miR-181a can be detected in formalin-fixed paraffin-embedded tissue specimens.2Comparing with normal endometrium, the expression of miR-181a in endometrial carcinoma and endometrial hyperplasia tissues were increased to11.228and4.073folds, respectively. Expression of miR-181a in endometrial carcinoma was about2.757times higher than that in hyperplastic tissues, and each pairwise comparison were statistically significant differences (P<0.05).3The expression of miR-181a in endometrial carcinoma was associated with FIGO stages (P<0.05).Summary The increasing expression of miR-181a in endometrial carcinoma may play the role of oncogenes. Abnormal expression of miR-181a is probably associated with the occurrence and development of endometrial carcinoma. Our data thus indicated the potential of miR-181a as a novel diagnostic and prognostic biomarker for endometrial carcinoma. In addition, miR-181a can be detected in formalin-fixed paraffin-embedded tissue specimens, so formalin-fixed paraffin-embedded tissue specimens can be used for the study on the relevant aspects of miR-181a.Chapter2Expression of miR-181a in human type Ⅰ and Ⅱ endometrial carcinoma and its significanceContents and MethodsImmunohistochemical experiments were performed to classified the endometrial carcinoma into two types by examining the expression of estrogen receptor and progestogen receptor. Staining (-) was negative expression, and staining (+)-(+++) were positive. According to the results of immunohistochemisty, the tissue with both ER+and PR+are grouped into estrogen-dependent endometrial cancer, and non-estrogen-dependent endometrial cancer that type Ⅱ (ER-, PR-). The expression of miR-181a was examined in type Ⅰ and type Ⅰ endometrial cancer, respectively.Results1Compared with normal endometrium, the expression of miR-181a in type Ⅰ and type Ⅱ endometrial cancer tissues were up-regulated8.515and31.233times (P <0.05).2The expression level of miR-181a in type Ⅱ is significant higher than that in type Ⅰ, the difference was statistically significant (P<0.05). 3The expression level of miR-181a was also significantly increased in type Ⅱ endometrial carcinoma compared with endometrial hyperplasia (P<0.05).SummaryOur study demonstrates that there is different expression level of miR-181a between type Ⅰ and type Ⅱ endometrial carcinoma and it may be related to the pathogenesis of type Ⅱ endometrial carcinoma. Moreover, over-expression of miR-181a might be involved in development of endometrial carcinoma, and thus played the role of oncogenes. In addition, miR-181a maybe related to the pathogenesis of type Ⅱ endometrial cancer, can provide a basis for further study on the molecular mechanisms of endometrial cancer development.Conclusion1The expression of miR-181a can be detected in formalin-fixed paraffin-embedded tissue specimens, so formalin-fixed paraffin-embedded tissue specimens can be used for the study on the relevant aspects of miRNA. Paraffin-embedded tissue samples have improved enormously, storaged simply and conveniently, but long-term storage of fresh or frozen tissue samples is expensive and laborious, so we can expand the utilization of paraffin-embedded tissue.2The up-regulation of miR-181a expression in endometrial carcinoma, may play the role of oncogenes. And from normal endometrium to endometrial hyperplasia and endometrial cancer tissues, the expression level of miR-181a gradually increased, suggesting that miR-181a may be related to early events in endometrial cancer. It indicated the potential of miR-181a as a novel diagnostic and prognostic biomarker for endometrial carcinoma.3Compared with normal endometrium, the expression of miR-181a in type Ⅰ and type II endometrial cancer tissues were higher, and the expression level of miR-181a in type II is significant higher than that in type I. So we hypothesized that the different expression level of miR-181a between type I and type II endometrial carcinoma may be related to the development of different type endometrial carcinoma, and it maybe related to the pathogenesis of type II endometrial cancer, can provide a basis for further study on the molecular mechanisms of endometrial cancer development.