The Role Of Mir-26a During Development Of Leiomyoma Uteri And Its Regulation Mechanism Of Estrogen Receptors α, Progesterone Receptors A

Backgroud:Leiomyoma uteri is the most common benign tumors in women and nearly half of allwomen aged35to49years have leiomyoma uteri. They are originating from smooth musclecells of the leiomyoma uteri is often accompanied by abundant and long menstruations similarto hemorrhage, often causing secondary anemia. Furthermore, some cases report moderate tosevere pain abdomen. Pelvic discomfort and bowel and bladder dysfunction from pressure arealso common symptoms. Fibroids have also been associated with infertility and recurrentabortion These tumors tend to grow rapidly during pregnancy and can cause obstructed labournecessitating Caesarean section, fetal malpresentation and fetal anomalies, as well aspost-partum haemorrhage secondary to uterinene atony. These clinical complicationsseriously impact women's health.As we all know, leiomyoma uteri are estrogen and progesterone-dependent tumors.Although the clinical and biochemical observations have traditionally supported an importantrole for estrogen in the promotion of leiomyoma uteri growth, there is also increasingevidence to suggest the involvement of progesterone in the pathogenesis of leiomyomauteri.Estrogen receptor (ER) and Progesterone receptor(PR) play important roles in theestrogen-mediated carcinogenic process and the regulation of leiomyoma cell growth.MicroRNAs(miRNAs) are21-23nt long noncoding RNA sequences that are involved invarious biological processes,including cell proliferation, cell death, stress resistance, andtumorigenesis．Recent studies indicate that miRNAs may play a role in several cancers.Altered miRNA levels can result in aberrant expression of gene products that may contributeto cancer biology, which suggests that expression profiling of miRNAs may significantlycontribute to cancer development. Furthermore, it has also been shown that miRNAexpression patterns have relevance to the biological and clinical behavior of humancancers．These and other data demonstrate that miRNAs play a substantial role in thepathogenesis of cancers, and miRNA is a novel targeting approach for cancer therapy.The role of miR-26a in carcinogenesis appears to be a complicated one, in the sense thatboth oncogenic and tumor suppressive effects were reported in cancers such as glioblastomaand hepatocellular carcinoma, respectively. Surprisingly,despite the pivotal role of ERα,PRain the development and progression of leiomyoma uteri, there is no evidence of a linkbetween ERα, PRa expression and their potential regulation by miRNAs. Usingcomputational and rational miRNA:mRNA base pairing analyses, we identified a potentialtarget site for miR-26a in the3'UTR of ERα,PRa mRNA. Base aboved results, we wereinterested in miR-26a with potential effects on leiomyoma uteri development and show theexperimental validation of the predicted interaction between miR-26a and ERα,PRa3'UTR.Our data show that miR-26a represses ERα,PRa expression in leiomyoma uteri smoothmuscle cells, leading to inhibition of cellular growth. Results:1.Realtime PCR,hybridization in situ detected the expression of mir-26a in leiomyomauteri tissues and the paired myometrium tissues:15pairs leiomyoma uteri tissues and the paired myometrium tissues of leiomyoma uteripatients were collected and the expression of mir-26a in leiomyoma uteri tissues and thepaired myometrium tissues were detected respectively by realtime PCR.13pairs of them,themir-26a level of leiomyoma uteri tissues was obviously down-regulated.Morphologically,detected the expression of mir-26a in leiomyoma uteri tissues and thepaired myometrium tissues were detected by hybridization in situ,we found that theexpression of mir-26a in leiomyoma uteri tissues was significantly lower than in the pairedmyometrium tissues;2.The expression of ERα,PRa,Ki67,PCNA,CyclinD2and CyclinE2in leiomyoma uteritissues and the paired myometrium tissues were detected by immunol histochemistry,observedthe difference by morphology:15cases of leiomyoma uteri patients were diagnosed by clinical and pathology diagnosis,aged of33to54, These cases had not have been accepted hormone therapy one week beforeadmission.We used immunol histochemistry to detected the expression ofERα,PRa,Ki67,PCNA,CyclinD2and CyclinE2in the15pairs.The result revealed thatERα,PRa,Ki67,PCNA,CyclinD2and CyclinE2were expressed all in leiomyoma uteri tissuesand the paired myometrium tissues,The expression of four genes in leiomyoma uteri tissuesand the paired myometrium tissues were significant difference,the expression of the fourgenes in leiomyoma uteri tissues are all higher than in the paired myometrium tissues.3.Leiomyoma uteri cells were primary cultured, the regulation of ERα,PRa by mir-26awere confirmed in leiomyoma uteri cells:Fresh samples of the leiomyoma uteri from department of gynaecology and obstetrics ofchanghai hospital were obtained and primary cells cultured ERα,PRa as the potential targetgenes of mir-26a were predicted, the fragment of ERα,PRa 's3'UTR which contained thebinding site of mir-26a was inserted the plasmid named PmirGLO respectively,and a newreportor gene vector was constructed. Dual-Luciferase reporter gene detection system wasused to confirm that ERα,PRa are the target genes of mir-26a.After the leiomyoma uteri cells were transfected the mimics of mir-26a, the expressionof ERα,PRa was downregulated dectected by western blot, compared to the NC.4. The regulation of the leiomyoma uteri cell cycle and proliferation by mir-26a:To confirm the biological role of mir-26a on the leiomyoma uteri cells, leiomyoma uteriprimary cells were transfected with mir-26a's mimics, the change of cell cycle was detectedafter24hours,48hours by flow cytometry,we found that cell proliferation of leiomyomauteri cells was inhibited in48hours, the result demonstrate that high expression of mir-26acan inhibit the proliferation of leiomyoma uteri cells.Conclusions:1.The down regulation of mir-26a in leiomyoma uteri tissus has the potential relationshipwith leiomyoma uteri. 2.The expression of ERα,PRa,Ki67,PCNA,CyclinD2and CyclinE2in leiomyoma uteritissues are all higher than in myometrium tissues.There was a significant negative relationshipbetween the mir-26a and ERα,PRa,Ki67,PCNA CyclinD2and CyclinE2,it indicated theimportance of high regulation of ERα,PRa,Ki67,PCNA, CyclinD2and CyclinE2in thedevelopment and progression of leiomyoma uteri.3.The primary culture method of human leiomyoma uteri was established successfully.Two reportor gene vectors were constructed,and that mir-26a post transcriptionally regulatingERα,PRa were confirmed.4. It was shown that ERα,PRa protein level of leiomyoma uteri cells transfected by themimic of mir-26a was lower than that of leiomyoma uteri cells transfected by negativecontrol.These data suggest that mir-26a may become a significant clinical biomaker.5.The expression of mir-26a is related to the proliferation and cycle of leiomyoma utericells,high expression of mir-26a in leiomyoma uteri cells blocked the cell cycle and suppressproliferationof leiomyoma uteri cells.