| Ovarian cancer(OC)is a gynecological cancer with a high mortality rate.Up to 80%of ovarian cancer patients are diagnosed with symptoms in the late stage of the disease.At this time,the tumor cells have spread to the pelvis(stage Ⅱ)and abdomen(Stage Ⅲ)or beyond the peritoneal cavity to other organs(stage Ⅳ).GLOBOCAN estimates that there will be 313,959 new cases of ovarian cancer in 2020,including 55,342 new cases of ovarian cancer in China.Despite the continuous development of surgical techniques and chemotherapy,most patients with advanced ovarian cancer will still relapse,and the median survival time is only 18 months.Therefore,the pathogenesis of ovarian cancer is still a poorly understood disease with a very poor prognosis.In order to provide insights that can develop new therapies,it is important to clarify the molecular mechanisms related to the migration and invasion characteristics of ovarian cancer cells.MicroRNA(miRNA)is a short(19-23 nucleotides)non-coding single-stranded RNA.Studies have found that the mechanism of miRNA is to bind to the 3’-untranslated region(3’-UTR)of the target gene mRNA to inhibit gene transcription or degrade the mRNA of the target gene to achieve the purpose of regulating the target gene.Since miRNAs has been discovered,it played an important role in many biological processes as post-transcriptional regulators,including cancer.Over the years,continuous research on miRNA has found that many miRNAs are directly related to human cancers,and these miRNAs can act as tumor suppressor genes or Oncogenes.For example,abnormally activated miRNAs can be silenced with antagomirs,and the re-expression of miRNAs lost in cancer can be achieved by inducing miRNA mimics,which provides new research directions for tumor data.Many studies have reported extensive changes in miRNA profiles during the initial and developmental stages of OC.More importantly,new studies have established the potential use of miRNAs as anti-cancer therapeutic molecules.It is reported that miR-181a is also up-regulated in various human cancers including OC,such as liver cancer,breast cancer and ovarian cancer.In the carcinogenic process of OC,miR-181a can promote tumor growth and metastasis,and is closely related to poor prognosis.Therefore,targeting miR-181a may control the development of OC and relieve the symptoms.More research focuses are needed to characterize the feasibility of targeting miR-181a in OC treatment and to develop simplified and cost-effective manipulation methods.Programmed cell death 4(PDCD4)is a tumor suppressor.It encodes a protein that can bind to eukaryotic translation initiation factor 4A1(EIF4A1)and inhibit its function by preventing RNA binding.At the same time,MAPK85 activation and consequent JUN-dependent transcription,they are of great significance to inhibit invasion by the function of modulating the activation of JUN kinase and down-regulating the expression of MAP4K1.Maybe it also plays a role in apoptosis.PDCD4 plays an important regulatory role in many tumors.However,the correlation between the expression of PDCD4 and ovarian cancer,as well as the mechanism of miRNA regulating PDCD4 in ovarian cancer remains unclear.This study is divided into three parts to illustrate the mechanism of miR-181a and PDCD4 regulating OC:Part one Exploration of the correlation of miR-181a/PDCD4 in ovarian cancerObjectivesBy detecting the expression of miR-181a and PDCD4 in ovarian cancer tissue specimens,we can explore the direct correlation between miR-181a and PDCD4 in ovarian cancer,and analyze the mechanism of miR-181a’s negative regulation of PDCD4 expression.Methods1.Using qPCR to detect the expression of miR-181a and PDCD4 in ovarian cancer and para-cancerous specimens,and analyzing the correlation between them;2.Bioinformatics analysis were used to predict the binding sites between miR-181a and PDCD4,and to analyze the expression and correlation of miR-181a and PDCD4 in ovarian cancer;3.Verifying the relationship between miR-181a and PDCD4 binding sites by constructing a dual luciferase reporter gene vector;4.Culturing normal human ovarian cells OV-1063 and ovarian cancer cell lines Caov-3,SKOV3,A2780,ES-2,and detecting the expression of miR-181a in these cells by qPCR.Furthermore,to detect the regulatory relationship between miR-181a and PDCD4 by transfecting SKOV3 and A2780 cells into miR-181a mimics and inhibitor.Results1.In ovarian cancer specimens,the expression level of miR-181a in the ovarian cancer tissues is higher than the para-cancerous tissues,and the expression levels were 3.782±2.631 and 1.066±0.809 respectively.While the expression of PDCD4 is the opposite,which is lower in ovarian cancer tissues.The expression levels were 0.749±0.581 and 2.745±1.540 respectively,and the differences were statistically significant,p=0.000.The expression levels of miR-181a and PDCD4 in ovarian cancer tissues are negatively correlated,the correlation coefficient r=-0.689,p=0.027.2.The analysis of bioinformatics shows that there is a binding site of miR-181a at the 3’UTR end of PDCD4,and they have in a negative regulatory relationship.3.The results of the dual luciferase reporter gene verified that the fluorescence of the group with the miR-181a mimics and PDCD4-WT was significantly lower than the group of the miR-181a mimics and PDCD4-MUT,and the differences were statistically significant,p=0.048.4.Culturing normal human ovarian cells OV-1063 and ovarian cancer cell lines Caov-3,SKOV3,A2780,ES-2,and detecting the expression of miR-181a.The expression levels of miR-181a in the five groups of cells were 1.002±0.073,1.247±0.129,1.916±0.108,3.834±0.261 and 0.734±0.028,respectively.The expression level of miR-181a in ovarian cancer cell lines SKOV3 and A2780 was significantly higher than that of normal human ovarian cell lines,and the differences were statistically significant(p<0.01).5.Transfecting the SKOV3 and A2780 cell lines respectively to detect the expression of miR-181a and PDCD4.The results showed that the expression of miR-181a in the cells transfected with miR-181a mimics was significantly increased,and the expression of PDCD4 was significantly decreased;the expression of miR-181a in the cells transfected with miR-181a inhibitor was significantly decreased,and the expression of PDCD4 was significantly increased.The differences were statistically significant(p<0.01).Conclusions1.The expression of miR-181a is increased and the expression of PDCD4 is decreased in ovarian cancer tissue specimens,and the expression of the two is negatively correlated.2.MiR-181a negatively targets and regulates the expression level of PDCD4 in ovarian cancer cells.Part Two The effect of miR-181a/PDCD4 on the biological behavior of ovarian cancer cells and its mechanismObjectivesThis study explored the effect of miR-181a/PDCD4 on the biological behavior of epithelial ovarian cancer cell lines through in vitro experiments,further confirmed that the mutual regulation of miR-181a and PDCD4 and provided experimental and theoretical basis for the clinical treatment of ovarian cancer.Methods1.The miR-181a inhibitor transfected A2780 and SKOV3 ovarian cancer cell lines,verified the transfection effect,and tested the proliferation,migration,invasion ability,apoptosis of transfected cell lines and its influence on the epithelialmesenchymal transition ability;2.The PDCD4 overexpression plasmid was synthesized and transfected into A2780 and SKOV3 cell lines to verify the transfection effect and detect the proliferation,migration,invasion ability,apoptosis of the transfected cell line and the effect on the epithelial-mesenchymal transition ability;3.The miR-181a inhibitor and siRNA-PDCD4 were simultaneously transfected into A2780 and SKOV3 ovarian cancer cell lines to verify the transfection effect and detect the proliferation,migration,invasion ability,apoptosis of the transfected cell line and the effect on the epithelial-mesenchymal transition ability.Results1.After transfection of miR-181a inhibitor,the expression level of miR-181a in A2780 and SKOV3 ovarian cancer cells was significantly decreased,and the expression level of its target gene PDCD4 increased significantly;cell proliferation,migration and invasion capabilities were significantly decreased;apoptosis and apoptosis related proteins(Cleaved-Caspase-3 and Bax)increased significantly,and the degree of epithelial-mesenchymal transition decreased.Compared with the control group,there were significant differences(p<0.01).2.The expression level of PDCD4 in A2780 and SKOV3 ovarian cancer cells overexpressing PDCD4 increased significantly;cell proliferation,migration and invasion capabilities decreased significantly,and apoptosis and apoptosis-related proteins(Cleaved-Caspase-3 and Bax)increased significantly;the degree of epithelial-mesenchymal transition was reduced.There were significant differences compared with the control group(p<0.01).3.After transfection of miR-181a inhibitor,the expression of miR-181a in A2780 and SKOV3 cells was significantly inhibited,and the expression of PDCD4 was significantly increased;after transfection of miR-181a inhibitor and siRNA-PDCD4 at the same time,the expression of miR-181a in A2780 and SKOV3 compared with the inhibitor group,there was no significant change,and the expression of PDCD4 was significantly lower than that in the inhibitor group.The difference was statistically significant(p<0.01).The proliferation ability of A2780 and SKOV3 ovarian cancer cells transfected with miR-181a inhibitor significantly decreased,while the proliferation ability of cells transfected with miR-181a inhibitor and siRNA-PDCD4 was significantly restored;further experiments proved that the cell migration,invasion ability,apoptosis and the transition level of epithelial-mesenchymal were recovered.The difference was statistically significant(p<0.01).Conclusions1.In ovarian cancer cell lines,inhibiting the expression of miR-181a or overexpression of PDCD4 can significantly inhibit the proliferation,migration and invasion of ovarian cancer cells,reduce the EMT of ovarian cancer cells and promote the apoptosis of cancer cells.2.The regulation of miR-181a on the proliferation,apoptosis,migration and invasion and EMT of ovarian cancer cells is achieved through negative regulation of PDCD4.Part Three In vivo experiments to explore the effect of miR-181a/PDCD4 on EMT of ovarian cancerObjectivesIn vivo transplantation tumor experiments were carried out by constructing a nude mouse tumorigenesis model to verify the regulatory relationship between miR-181a and PDCD4,and its effect on EMT of ovarian cancer.Further improve the new experimental basis and theoretical basis for the clinical treatment of ovarian cancer.Methods1.Culture ovarian cancer SKOV3 cells for tumor formation experiments in nude mice,and inject miR-181a antagomir into the tumor to detect the effect of miR-181a antagomir on the tumor formation ability of ovarian cancer cells;2.The transfection efficiency of antagomir in tumors in vivo was verified by qPCR experiment,and the expression of the target gene PDCD4 was detected.Western blot and immunohistochemical staining experiments was used to detect the expression of PDCD4 and EMT-related proteins E-Cadherin and N-Cadherin.Results1.In the continuous injection of miR-181a antagomir group,the growth of nude mice tumors was significantly inhibited,and the weight of tumors was also decreased significantly.The mass of transplanted tumors in the miR-181 a antagomir group,NC group and blank group were 0.381±0.06 g、0.904±0.145 g and 0.932±0.165 g,respectively.Compared with the NC group,the mass of transplanted tumors in the miR-181a antagomir group was significantly different(p<0.01).It shows that in the continuous injection of miR-181a antagomir group,the growth of the tumor is significantly suppressed,and the weight of the tumor is also significantly reduced.2.The relative expression levels of miR-181a and PDCD4 in the transplanted tumor tissues of the NC group were 1.051 ±0.111 and 0.946±0.010,and the relative expression levels of the two in the transplanted tumor tissues of the miR-181a antagomir group were 0.494±0.041 and 2.997±0.083.All are statistically significant(p<0.01).It shows that in tumors injected with miR-181a antagomir,the expression of miR-181a is inhibited,and the expression of PDCD4 increases significantly.3.Western blot detected that in the group injected with miR-181a antagomir,the expression of PDCD4 increased significantly,the expression of EMT-related protein E-Cadherin increased,while the expression of N-Cadherin decreased,and EMT was suppressed.The immunohistochemical structure obtained the same experimental conclusion.Conclusions1.In in vivo experiments,miR-181a regulates the tumorigenesis ability of ovarian cancer cells.2.MiR-181a regulates the expression of PDCD4,affects EMT and participates in the development of ovarian cancer. |
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