A Preliminary Study On Expression Of MicroRNA-21and Its Regulation On PTEN Expression In Endometrioid Endometrial Cancer

Objective1. To investigate the expression of microRNA-21(miR-21) in endometrioid endometrial cancer tissue and their corresponding adjacent nontumor tissues.2. To evaluate the correlation of clinicopathologic factors with miR-21level in endometrioid endometrial cancer.3. To detect the PTEN protein expression, and to analyse its correlation with miR-21level in endometrioid endometrial cancer.Methods1. Paired fresh endometrioid endometrial cancer tissue and their adjacent nontumor tissues were collected from untreated patients subjected to radical hysterectomy in Department of Gynecology, Provincial Hospital affiliated to Shandong University.2. Real-time RT-PCR was used to detect the expression of miR-21expression in endometrioid and endometrial cancer and their adjacent nontumor tissues.3. Western blot assay was used to detect the expession of PTEN protein level in tumor and nontumor tissues of endometrioid endometrial cancer.Results1. The expression of miR-21in EEC tumor tissues was about2.879-fold change (1.021-7.402folds, with an SD of1.625, P<0.05) than that of adjacent nontumor tissues in all38EEC tumor tissues.2. The relative expression of miR-21in each clinical stage was:2.178±1.158in stage Ⅰ,3.510±1.749in stage Ⅱ+Ⅲ. The difference between stage I versus stage Ⅱ+Ⅲ was significant (P=0.01); The relative expression of miR-21in patients with>1/2myometrial invasion (3.972±1.797) was higher than that with<1/2myometrial invasion (2.084±0.881)(P=0.001). The relative expression of miR-21in each pathologic grade was:1.376±0.229in grade1,2.452±0.447in grade2and5.002±1.299in grade3. Both differences between grade1versus grade2and grade2versus grade3were significant (P<0.001).3. Using western blot analysis, we proved that tumor tissues expressed significantly lower PTEN protein compared with their adjacent nontumor tissues (0.64vs0.91, P<0.05).4. Pearson correlation analysis showed a significantly inverse correlation between miR-21and protein expression of PTEN (Pearson correlation=—0.762, P<0.001).ConclusionMiR-21was over-expressed in tumor tissues than the adjacent nontumor tissue of patients with endometrioid endometrial cancer. MiR-21expression correlated with advanced clinical stage, pathologic grade and deep myometrial invasion and also inversly correlated with PTEN protein expression, which may play an important role in the pathogenesis and progression of endometrioid endometrial cancer. Objective1. To investigate whether PTEN could be modulated by miR-21in EEC.2. To evaluate the potential oncogenic activity of miR-21on endometrial tumor cell proliferation. Methods1. In EEC cell line KLE, gain-and loss-function of miR-21was achieved by transfection with chemical synthetic miR-21mimic and miR-21inhibitor oligonucleotides with Lipofectamine2000Reagent. The efficiency of transfection was evaluated by inverted fluorescence microscope.2. The influence of miR-21on mRNA and protein level of PTEN was evaluated by qRT-PCR and western blot assay, respectively.3. The functional effect of miR-21on cell proliferation, migration and invasion was evaluated using Cell Couting Kit8(CCK-8), scratch wound test and matrigel-coated transwell invasion assay, respectively.Results1. MiR-21mimic, inhibitor and their respective scrambled control were respectively and successfully transfected into KLE cell line, and the efficiency of transfection was up to90%.2. Compared with control group, transfection of miR-21mimic resulted in a6.898±0.312fold up-regulation (P=0.003) of miR-21expression, a53.8±2.7%decrease of PTEN protein expression (P=0.007) and a significant promotion to the tumor cell proliferation, migration and invasion (P<0.05). In contrast, administration of miR-21inhibitor showed a33.4±5.4%decrease (P=0.007) of miR-21expression, a1.888±0.147-fold increase (P=0.002) of PTEN protein expression and a significant restraint of tumor cell proliferation, migration and invasion (P<0.05). While the corresponding mRNA level of PTEN showed no significant changes (P>0.05) compaired with negative control.ConclusionThe over-and down-expression of miR-21could significantly promote or inhibit the proliferation, migration and invasion of KLE cell line, suggesting that miR-21could play an important role in the tumorigenesis and development of endometrioid endometrial cancer. These findings also demonstrated that miR-21might be a gene therapeutic target in endometrioid endometrial cancer. PTEN is the endometrioid endometrial cancer-associated targeted gene of miR-21. MiR-21 might regulate the biological behavior by targeting PTEN mRNA.