| Background Endometrial carcinoma(EC)is one of the most important malignant tumors in the world and accounts for about 20-30% of the female reproductive system tumors.Endometrial cancer occurs in postmenopausal women,but in recent years,clinical statistics show that the incidence of the disease was gradually younger tendency.Although the 5-year survival rate of patients with advanced endometrial cancer has been greatly improved after treatment,the prognosis of patients with advanced,poorly differentiated endometrial cancer or with specific types of endometrial cancer is often poor,so is one of the important factors that cause the patient to die.In recent years,the incidence of endometrial cancer in China showed an upward trend year by year.Endometrial cancer is one of the clinical manifestations of perimenopausal irregular vaginal bleeding,it is often mistaken for menstrual disorders,when patients take the hospital when they are already endometrial cancer,thus delayed the best treatment opportunity.The occurrence of endometrial cancer has a more rigorous evolution process,if the precancerous lesions take effective interventions,it may reduce the incidence of the disease.So looking for molecular markers that can be effectively diagnosed and treated is of great significance for the treatment of endometrial cancer.Ovarian cancer(Ovarian cancer,OC)refers to the growth of ovarian cancer.Due to the early stage of ovarian cancer without typical symptoms and the limited role of screening,even if the symptoms are not specific,so early diagnosis is more difficult.About 60% to 70% of patients have been treated for advanced cases,even though the current progress has been made in cancer treatment and new treatments continue to emerge,but for the treatment of advanced ovarian cancer cases the effect is still not ideal,so the ovarian cancer mortality ranks first in gynecologic malignancies.Currently there is still a lack of effective identification of ovarian cancer tissue typesand benign and effective method.As ovarian cancer patients in the early onset of clinical symptoms is not significant,and ovarian cancer surgery found only 30% of the tumor confined to the ovary,most have spread to the uterine bilateral attachment,omentum and pelvic organs,so the ovarian cancer in terms of diagnosis and treatment is indeed a big problem.Mi RNAs belong to the endogenous single-stranded non-coding single-stranded small molecule RNA.Studies have shown that many miRNAs are involved in the development and progression of malignant tumors including endometrial and ovarian cancers.Mi R-548 is a member of the miRNA family.Up to now,studies have shown that miR-548 c is low-expressed in breast cancer,liver cancer and osteosarcoma and therefore has a similar anti-oncogene effect.However,no study has been done on the role of miR-548 c in ovarian cancer and endometrial cancer and related mechanisms.Metastasis is the leading cause of death in endometrial and ovarian cancer patients.Epithelial-mesenchymal transition(EMT)is thought to be a key step in the metastasis of endometrial and ovarian cancers,which enhances the migration and invasion of tumor cells and thus promotes their malignant progression.Therefore,understanding the molecular mechanisms that mediate the migration,invasion and EMT of endometrial and ovarian cancer cells can identify new molecular targets for the future treatment of these cancers.Twist belongs to the basic helix-loop-helix(b HLH)family of transcription factors.As a key gene regulating EMT,Twist protein can promote embryonic development by promoting the transformation of mesodermal epithelial cells into stromal cells,So mesoderm cells have the ability to migrate,eventually on the growth and development of various organs of biological organisms have a promoting effect.Up to now,Twist has been shown to be abnormally highly expressed in both endometrial and ovarian cancers,suggesting that Twist has a role of oncogene in both malignant tumors.Objective This study examined the expression of miR-548 c and Twist in endometrial and ovarian cancer,and analyzed the relationship between them and the clinicopathological parameters of the above two tumors.Meanwhile,the effect ofmiR-548 c on the biological behavior of endometrial and ovarian cancer cells was studied in vitro and the invasion and migration of endometrial and ovarian cancer cells were influenced by whether miR-548 c regulated the expression of Twist gene.To clarify the specific mechanism of miR-548 c in the process of malignant transformation of endometrial and ovarian cancer cells,and lay a theoretical and experimental foundation for finding new and effective molecular targets for the diagnosis and treatment of endometrial cancer and ovarian cancer.Method1.In this study,50 pairs of primary endometrial cancer tissues and adjacent normal tissues were collected,and 60 cases of epithelial ovarian cancer tissues and 20 cases of normal epithelial ovarian tissues were collected.Fluorescent quantitative PCR was used to detect the above tissue samples miR-548 c and Twist m RNA in ovarian cancer patients,and to analyze the relationship between miR-548 c and clinicopathological parameters in patients with ovarian cancer.Meanwhile,the relationship between miR-548 c and the prognosis of patients with endometrial cancer and ovarian cancer was analyzed.2.Human endometrial carcinoma cell lines(RL95-2 and HEC-1A)and human ovarian cancer cell lines(SKOV-3 and OVCAR3)were randomly divided into two groups: Anti-Ctr group: Transfected cells;Anti-548 c group: transfected cells with miR-548 c inhibitor.The proliferation activity,apoptosis rate,migration ability and invasion ability of each group were detected.3.The target gene of miR-548 c was predicted by biological software and verified by luciferase reporter system to detect the expression of Twist protein in RL95-2,HEC-1A and SKOV-3 cells after up-regulation and down-regulation of miR-548 c The impact of expression.To detect the effect of miR-548 c up-regulation and down-regulation on the expression of EMT related gene E-cadherin?N-cadherin,CD133 and MMP-9 m RNA in RL95-2,HEC-1A and SKOV-3 cells.RL95-2,HEC-1A and SKOV-3 cells were transfected with miR-548 c inhibitor /miR-548 cmimic and / or Twist si RNA / Twist respectively to detect the proliferation,invasion and migration of these cells.Result1.The expression of miR-548 c in endometrial carcinoma was significantly lower than that in corresponding normal tissues(P <0.01).Compared with normal ovarian tissue,miR-548 c expression was significantly decreased in ovarian cancer(P <0.01).Mi R-548 c expression was significantly lower in serous carcinomas,stage III-IV and stage 3 tumor tissue samples than in non-serous,stage I-II and grade 1-2 tumor tissue samples.The overall survival rate of patients with endometrial cancer with low expression of miR-548 c is poor compared with patients with endometrial cancer with high miR-548 c expression.Although the use of TCGA data for meta-analysis in this study failed to show the relationship between miR-548 c expression and the risk of ovarian cancer(P = 0.425),the survival of ovarian cancer patients with miR-548 c downregulation was shorter.The expression of Twist m RNA in endometrial carcinoma was significantly higher than that in corresponding normal tissues(P <0.01).Compared with normal ovarian tissue,Twist m RNA expression in ovarian cancer was significantly increased(P <0.01).Twist m RNA expression was significantly higher in serous carcinomas,stage III-IV and stage 3 tumor tissue samples than in non-serous,stage I-II and grade 1-2 tumor tissue samples.2.Compared with Anti-Ctr group,the proliferation,invasion and migration of Anti-548 c group were significantly increased(P <0.01)and the apoptosis rate was significantly decreased(P <0.01)Compared with Anti-Ctr group,the proliferation,invasion and migration of Anti-548 c group were significantly increased(P <0.01)and the apoptosis rate was significantly decreased(P <0.01)(P <0.01),and the apoptosis rate of Pre-548 c group was significantly increased(P <0.01).In SKOV-3 cells,compared with Pre-Ctr group Compared with the control group,the proliferation,invasion and migration of Pre-548 c group were significantly decreased(P <0.01),and the apoptosis rate was significantly increased(P <0.01).3.Twist was identified as a direct target of miR-548 c.In RL95-2 cells,luciferase expression was significantly increased(P<0.01)in cells transfected with Twist-WT plasmid when miR-548 c expression was inhibited,whereas cellstransfected with Twist-Mut plasmid The luciferase expression level had no significant change(P> 0.05).In HEC-1A and SKOV-3 cells,luciferase expression was significantly decreased in cells transfected with Twist-WT plasmid(P <0.01)after miR-548 c expression was up-regulated,while transfection of Twist-Mut plasmid of cells in the luciferase expression levels did not change significantly(P>0.05).Compared with Anti-Ctr group,the expression of Twist protein in Anti-548 c group was significantly increased(P<0.01)and the expression of E-cadherin m RNA was significantly decreased in RL95-2 cells(P<0.01)The expression of N-cadherin,CD133 and MMP-9 m RNA were significantly increased(P<0.01).Compared with Pre-Ctr group,the expression of Twist protein in Pre-548 c group was significantly decreased(P<0.01)and the expression of E-cadherin m RNA was significantly increased in HEC-1A and SKOV-3 cells<0.01).The expression of N-cadherin,CD133 and MMP-9 m RNA were significantly decreased(P<0.01).Compared with Anti-548 c group,the proliferation activity of Anti-548 c + Twist si RNA group was significantly lower than that of Anti-548 c group(P<0.01).Compared with Pre-Ctr group,the proliferation activity of Pre-548 c group was significantly decreased in RL95-2cells(P<0.01).Compared with Pre-548 c group,the proliferation activity of Pre-548 c + Twist c DNA group was significantly higher(P<0.01).Compared with Pre-Ctr group,the proliferation activity of Pre-548 c group was significantly decreased in SKOV-3 cells(P<0.01).Compared with Pre-548 c group,the proliferation activity of Pre-548 c + Twist c DNA group was no significant change(P>0.05).Compared with Anti-Ctr group,the invasion and migration ability of Anti-548 c group was significantly increased(P <0.01).Compared with Anti-548 c group,the invasion and migration ability of Anti-548 c + Twist si RNA group was significantly decreased(P<0.01).In RL95-2 cells,the invasion and migration ability of Pre-548 c cells was significantly lower than that of Pre-548 c cells(P<0.01).Compared with Pre-548 c cells,Pre-548 c + Twist c DNA cells invasion and migration ability was significantly increased(P<0.01).Compared with Pre-Ctr group,the invasion and migration ability of Pre-548 c group was significantlydecreased in HEC-1A and SKOV-3 cells(P<0.01).Compared with Pre-548 c group,Pre-548c+Twist c DNA group invasion and migration ability was significantly increased(P>0.05).Compared with Anti-Ctr group,the expression level of Twist protein in Anti-548 c group was significantly increased(P<0.01).Compared with Anti-548 c group,Anti-548c+Twist si RNA group Twist protein expression levels was significantly lower(P<0.01).Compared with Pre-548 c group,the expression of Twist protein in Pre-548 c group was significantly lower than that in Pre-548 c group(P<0.01).The expression level was significantly increased(P<0.01).Compared with Pre-548 c group,the expression of Twist protein in Pre-548 c group was significantly lower than that in Pre-548 c group(P<0.01).The expression level was significantly higher(P>0.05).Conclusion1.The expression of miR-548 c in endometrial cancer and ovarian cancer tissues is abnormally downregulated,and the down-regulation of miR-548 c expression is associated with poor prognosis in patients with endometrial cancer.2.Twist expression in the above-mentioned tumor tissue abnormally increased.Both miR-548 c and Twist are related to the histological type,case classification and clinical stage of ovarian cancer.3.Mi R-548 c inhibits the development and progression of endometrial and ovarian cancers by inhibiting the expression of Twist and thus inhibiting the proliferation,invasion,migration and apoptosis of endometrial and ovarian cancer cells. |
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