Expression And Correlation Of MiR-181a And CA125 In Human Type â…  And â…¡ Endometrial Carcinoma

Endometrial carcinoma (EC) is a kind of malignant tumor originated from endometrial epithelium. which often occurs in perimenopausal and postmenopausal women, The epidemiologic surveys showed EC is significantly related with obesity, high blood pressure, diabetes, smoking, history of cancer in family, infertility and so on. Vaginal bleeding is the most common symptom of endometrial carcinoma after menopause and menopause transition. EC, cervical carcinoma and ovarian cancer were named as the top three common malignant tumors of female genital tracts, It accounts for 20%～30% of female genital tract malignant tumors, and the incidence of EC has been second only to cervical cancer in our country, while ranked the first in western country, which rises year by year in the worldwide scale in recent years. and the patients become more younger. The treatment has made no breakthrough till now, and the postoperative survival rate is still low. What’s more, the causes and pathogeneses of this disease have not been illuminated clearly so far.According to the clinical and pathologic characteristics of endometrial carcinoma, Bohkman divided endometrial carcinoma into the estrogen dependent type (type Ⅰ) and non-estrogen dependent type(type Ⅱ), The two different types of EC have different risk factors. The former is related with endogenous or exogenous estrogen stimulation without progesterone as an antagonist for a long time, which tends to occur in patients with endometrial hyperplasia (simple hyperplasia, complex hyperplasia, atypical hyperplasia), on the basis of endometrial hyperplasia, it finally turned into endometrial carcinoma. The main pathological type is endometrial adenocarcinoma, usually with ER(estrogen receptor) and PR(progesterone receptor) positive expressed, The type Ⅰ EC accounts for most of the EC, with tumor highly differentiated、muscular shallowly infiltrated and a fine prognosis, PTEN gene inactivation and microsatellite instability are the common molecular events in Type Ⅰ EC; The Type Ⅱ EC have no clear relationship with estrogen, which is poorly differentiated, deeply muscularis infiltrated, and has a high degree of malignancy and a poor prognosis, tending to occur on the basis of atrophic endometrium. The top two pathological types are serous papillary adenocarcinoma and clear cell carcinoma, with the ER and PR lowly or negatively expressed. It mostly happened in the postmenopausal women, The P53 gene mutation and excessive expression of HER2 gene are common molecular events. Early detection, diagnosis and intervention can significantly improve the quality of life and the survival of patients with endometrial carcinoma. But EC at present is still lack of specific biomarkers in the early diagnosis phase.With the rapid development of the technique of molecular biology and information biology, small RNA (mirna, mir-RNA) has been a hot spot in recent years, it is a kind of highly conservated, time sequence specific, tissue specific and short (usually around 20-23 nt) non-coding single-stranded RNA, The miRNA combines after transcription to the target genes mRNA 3’-UTR (3’-untranslated region), inducing the degradation of mRNA, inhibiting translation of mRNA, or other forms of regulating mechanism in the inhibition of the expression of target genes, regulates the gene expression after the transcription phase, thus affects the biological character. MiRNAs act as oncogenes or anti-oncogenes. and the expressions of miRNAs are closely related with the diagnosis, treatment and prognosis. Recent research reported that the specific expression profiles of miRNAs in endometrial carcinoma were obvious correlated with the clinical features such as tumor stage, histological grade, myometrium invasion, recurrence and lymph node metastasis. Yoneyama’ study found that the expression of miR-200a, miR-200b and miR-429 were up-regulated obviously in the endometrial adenocarcinoma, probably by reducing the expression of tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10). Then as a result in the occurrence of endometrial carcinoma. Lee and other researchers found that anti-miR-200 family significantly inhibited the growth of HEC-1A cells and Ishikawa cells. What’s more, when HEC-1A cells were transfected with anti-miR-429, the cytotoxic effect of cisplatin was enhanced. Karaayvaz etc, found that the expression of miR-200c and miR-205 increased significantly in endometrial carcinoma, the miR-205 is related with the prognosis obviously, The Kaplan-Meier survival analysis showed that the expression of miR-205 was negatively correlated with patients’ overall survival rate. The results implied that miR-205 can lead to the development of endometrial carcinoma and poor prognosis by reducing the expression of PTENMiR-181a, a newly discovered miRNA in recent years, widely exists in human cells, plays an important role in the regulation of cell growth, cell proliferation, differentiation and apoptosis. The abnormal expression often leads to cell dysfunction, for example immune regulation, leukemia, breast cancer, digestive system tumors, gliocytoma, lung cancer, oral squamous cell carcinoma and cervical carcinoma, et al. At present, A foreign research found that PR may be a target gene of miR-181a in fresh frozen endometrial carcinoma tissues, and the expression of PR is negatively correlated with the expression of miR-181a. And we have not yet seen a few research about the relationship between endometrial carcinoma and miR-181a in China except our team’s early research.Our team’s early research found that Hsa-miR-181a was expressed in formaldehyde-fixed paraffin-embedding endometrial carcinoma tissues, Hsa-miR-181a was gradually rised from the normal endometrium tissues and the endometrial hyperplasia tissues, to endometrial carcinoma tissues, and the expression of hsa-miR-181a is obviously up-regulated in type II endometrial carcinoma tissues compared with the type I endometrial carcinoma tissues. A large number of researchers have found that miRNA expression profiles were different in the type I and type II endometrial carcinoma with different target genes and molecular signaling pathways, they may participated in different pathogenesis of the type Ⅰ/Ⅱ endometrial carcinoma. Zhou jing and other researchers chosen endometrial carcinma cell lines Ishikawa (from type Ⅰ endometrial carcinoma)/KLE (from type Ⅱ endometrial carcinoma) in vitro and in vivo and 10 cases of clinical specimens in endometrial carcinoma tissues to evaluate miR-100 by qRT-PCR and its assumed target gene ERa, found that the expression of hsa-miR-100 in type Ⅱ EC was obviously higher than that of type Ⅰ EC, its target gene may be ERa. Zhang, etc. found that miR-143 and miR-145 were obviously down-regulated in type Ⅰ endometrial carcinoma, and was related to the excessive expression of DNMT3B (DNA methyltransferase 3b), while in type II endometrial carcinoma, there was no significant correlation between miR-143, miR-145 and DNMT3B expression. Therefor, what is the possible target gene in the different pathogenesis of endometrial carcinoma or signaling pathway of miR-181a in type Ⅰ and type Ⅱ endometrial carcinoma?CA125, a macromolecule glycoprotein, widely exists in the various of body epithelial tissues and epithelium origining tumor tissues, such as, normal endometriums, fallopian tubes and peritoneum still have extremely traces of CA125 expressed. And the expression of CA125 in ovarian cancer, fallopian tube cancer, endometrial carcinoma and mesothelioma also up-regluated. CA125, currently the world’s most widely used tumor markers of ovarian epithelial carcinoma, has been widely applied to the reproductive system, urinary system, digestive system and many other kinds in tumor diagnosis and tracking. and it was also used for identifying diagnosis of different pelvic masses, judging the clinical effects and prognosis.Currently, the detection of CA125 in endometrial carcinoma mostly confined to the serum detection, however, the relationship between endometrial carcinoma and serum CA125 and the research value have been controversial so far, some scholars thought that preoperative serum CA125 positive rate was significantly related to the histology, surgical-pathologic stage, pathological classification, muscular infiltration and lymph node metastasis. But most scholars considered that serum CA125 level cannot accurately reflect the degree of tumor progression, has nothing to do with muscular infiltration depth, and has no help in predicting prognosis. The quantity of CA125 antigen was insufficient,the causes may be that antigen that entered into the peripheral blood was quickly removed; or falling off the intrauterine without entering blood circulation, which resulted in reduced early endometrial carcinoma serum CA125 positive rate. Researchers have found that the sensitivity and specificity of CA125 in endometrial carcinoma tissues were significantly higher than that of serum CA125, and the expression of CA125 in tissues was positively associated with the pathology classification, the pathological staging.Our Early experiments found that the expression level of CA125 in endometrium and endometrial lesions kept up with the pathological evolution process of endometrial tissues, the severer the disease was, the more CA125 the glandular cell secreted, but the expression of CA125 weakened in the low differentiated adenocarcinomas and malignant tumors. So, does the expression of CA125 in type II endometrial carcinoma have a reduced expression phenomenon? Is there any difference in the expression of CA125 between different types of endometrial carcinoma?Studies have found that the preoperative serum CA125 ovarian was correlated with the expression of miR-181a in the patients with ovarian cancers. Correlation analysis showed that:in the patients with ovarian cancer, the preoperative serum CA125 expression was closely relative to the miR-181a expression level, with the increasing of serum CA125, the expression of miR-181a increased. Therefor, we cann’t help thinking if there was also a correlation between miR-181a and the expression of tissues CA125 in Type I and type II endometrial carcinoma? If there did exist a correlation, what’s the possible pathological mechanism or signaling pathway? Is there any significance for clinical diagnosis or prognosis judgement?Therefore, this study aims to detect the expression of miR-181a and CA125 in the formalin-fixed paraffin-embedded endometrium tissues in human type I and II endometrial carcinomas, then to analyze their correlation. And to explore the significance and the possible mechanism in the development of endometrial carcinoma.Chapter 1 Expression of miR-181a in type Ⅰ and type Ⅱ endometrial carcinoma respectively and its clinical significanceContents and Methods:1 A total of 78 formalin-fixed paraffin-embedded endometrium tissue specimens (72 from Xiao Lan People Hospital Affiliated to Southern Medical University, the other 6 from Zhongshan Hospital and Southern Hospital) were used in this study during January 2011 to December 2013. Of all the specimens,47 cases were endometrial carcinoma,13 cases were normal endometrium, and the rest cases (18 cases) were endometrial hyperplasia. The patients’age ranged from 24 to 69 years old, with the average 48 year. All the treatments included chemotherapy, radiotherapy, and or endocrine therapy were pre-operatively not taken, and the post-operatively diagnosis was affirmed by pathology.2 Examined the expression of the ER and PR, The endometrium tissues were measured by immunohistochemical experiments to classify the endometrial carcinoma into two types. Taking tan particles appeared in the nucleus as a positive signal. Staining (-) was negative expression, Staining(+)～(+++) are all positive expression. According to the Staining result, the tissues were grouped into the estrogen-dependent endometrial carcinoma (type Ⅱ) and non-estrogen-dependent endometrial carcinoma (type Ⅱ), of which, type Ⅰ endometrial carcinoma (ER+, PR +) and type Ⅱ endometrial carcinoma (ER-, PR-).3 All endometrial tissues were formalin-fixed paraffin-embedded endometrium tissue specimens, Each specimens serially were sliced 10 sections with 10 microns’ thickness, then take the sections that cells covered more than 50% of the area of the section about 2～5(4 sections on average) to dewax and extract the total RNA according to the Instructions of the miReasy FFPE Kit.4 The Uv spectrophotometer was used to detected the absorbanceto of the total RNA (OD value),1% agarose denatured gel electrophoresis were used to detect the integrity of the RNA.5 The reverse transcription was conducted through miRNA Stem loop primers, then rt-PCR was conducted to detect the expression level of miR-181a and U6 at the same time in each endometrial tissue. The reaction parameters included:95 ℃ pre-modification 6 min,95 ℃ degeneration 10 s,55 ℃ annealing 10s,72 ℃ extending 30 s, a total of 50 PCR cycles. Record the A Ct value.The miRNA expression level uses the Relative Quantification method, namely the calculation Δ Ct=Ct (miR-181a)-CtU6 represented the relative expression, calculated ΔΔ Ct= (Ct (miR-181a)-CtU6) experimental group-(Ct (miR-181a)-CtU6) control group,2-ΔΔCt represented the fold changes of the experimental group comparing with the control group.Results:1 The expression of miR-181 a in endometrial tissues:miR-181a can be detected in the normal endometrium, and it in endometrial carcinoma group and endometrial hyperplasia group (ΔCt-3.356±2.40,-1.893±2.568 respectively) increased to 11.228 and 4.073 folds compared with the normal endometrium group respectively. Expression of miR-181a in endometrial carcinoma group was about 2.757 times higher than the endometrial hyperplasia group, and each experimental group was statistically significantly different from the control group(P< 0.05).2 The expression of miR-181a in two types of endometrial carcinoma tissues (type Ⅰ ΔCt-2.957±2.453, type Ⅱ ΔCt-4.832±1.524 respectively) significantly increased 8.515 and 31.233 times respectively (P<0.05) compared with normal endometrium. The expression level of miR-181a in type II EC group was 3.668 times higher than that in type I EC group (P< 0.05). The expression of miR-181a in type Ⅰand type Ⅱ endometrial carcinoma increased 2.091(P>0.05, no significant difference existed) and 7.669 times (P< 0.05) compared with endometrial hyperplasia.Summary:In the normal endometrium tissues, miR-181a was lowly expressed. The expression of miR-181a increased gradually from normal endometrium, endometrial hyperplasia, to endometrial carcinoma, It may have played an important role as an oncogene in the process of endometrial carcinoma. MiR-181a may participate in the early molecular events in the formation of endometrial carcinoma, the expression of miR-181a in endometrial hyperplasia tissues is not significant different from type Ⅰ endometrial carcinoma. While it was significantly reduced compared with type Ⅱ endometrial carcinoma tissues, and the expression of miR-181a in type II endometrial carcinoma tissues is significantly higher than type I endometrial carcinoma, Suggesting that miR-181a may have play an important role in different pathogenesis in different types of endometrial carcinomas, especially associated with the pathogenesis of type II endometrial carcinoma.Chapter 2 Expression of CA125 in type I and type II endometrial carcinoma respectively and its correlation with miR-181aContents and Methods:The endometrial tissue specimens were fixed by 4% formaldehyde.2-5(average 4) sections were obtained from each case with thickness 10μm. Immunohistochemical experiments (MaxVision TM immunohistochemical staining method) were performed to detect the expression level of CA125 according to the Maxvision TM detection KIT-5001 strictly. The tan particles that mainly located in cell membrane and cytoplasm represented positive expression of CA125. No staining represented negative expression (-), The weakly positive expression (+) meant staining was light brown and positive cells rate was less than 10% of the 10 times field of vision. The strong positive expression (+++) represented the positive cells rate was more than 50%, the tissues were dyed dark tan, and the structure is clear. The moderately positive expression (++) was between weakly and strongly positive expression.Results:1 The expression of CA125 was mainly weakly positive expressed in normal endometrial tissues, while it’s moderate positive and strong positive expression in endometrial hyperplasia tissues and endometrial carcinoma tissues. In addition, the CA125 was strongly positive expressed in type I endometrial carcinoma,while moderate positive expressed in type II endometrial carcinoma. And the expression of CA125 was significant different in different types of endometrial carcinoma tissues (Z= 4.717, P<0.05)2 The expression of miR-181a was negatively related with the expression of CA125 in the development of endometrial carcinoma (P=0.026, r=-0.25).Summary:In the carcinogenesis process of endometrium, CA125 is negatively associated with the expression of miR-181a, It may be related to the reduced expression of CA125 in the low differentiated adenocarcinomas and tumors with high degrees of malignance.Conclusion:miR-181a expressed differently in type Ⅰ and type Ⅱ endometrial carcinoma, which may be related to the development and progression of different types of endometrial carcinoma. Overexpression of miR-181a might be involved in the pathomechanism of endometrial carcinoma, which may act as an oncogene in the endometrial carcinoma, especially in type Ⅱ endometrial carcinoma. The mechanism may be that miR-181a regulated the expression of CA125 in the carcinogenesis of endometrium, but the specific mechanism still need to be validated in cell level experiments.