| Background:Ovarian cancer is one of the most common malignant tumors in the female reproductive system,and its mortality rate ranks the first in gynecological malignant tumors.And epithelial ovarian cancer(EOC)is the most important pathological type of ovarian cancer.At present,"operation + chemotherapy" is still the main treatment for ovarian cancer,but the treatment is not ideal because of the high degree of malignancy and easy recurrence of ovarian cancer.Therefore,it is urgent to explore new therapeutic targets and improve the treatment strategy of ovarian cancer.In recent years,abnormal iron metabolism has been considered as one of the specific markers of tumor.Recent studies have shown that tumor can help itself to gain better growth advantage by changing its iron metabolism.Moreover,abnormal iron metabolism,especially iron overload,is closely related to the occurrence and development of cancer.At the same time,it has been reported that there is abnormal high expression of iron in patients with ovarian cancer.The transferrin receptor(TFRC),also known as CD71,is the most important membrane protein regulating the iron transport in cells.Although TFRC plays an essential role in maintaining the normal iron metabolism of human cells,the process of absorbing and ingesting iron through TFRC has also become one of the most important ways for tumor cells to help their survival and growth.When the cells become cancerous,the process of absorbing iron through TFRC has become one of the most important ways for tumor cells to help their own survival and growth.At present,the expression pattern of TFRC in EOC and its specific molecular mechanism are not clear.Based on this,this study aims to explore the role of TFRC in EOC pathophysiological process and its specific molecular mechanism by detecting and analyzing the expression ofTFRC in EOC clinical samples and its correlation with the expression of cell function related markers,and then combining with in vivo and in vitro experiments to inhibit the expression of TFRC in order to further clarify the role of TFRC in the development of human EOC.Through the research of this subject,it not only provides new target molecules for EOC treatment strategy,but also brings new hope to patients.Methods:1.Flow cytometry and immunofluorescence assays were used to detect the expression of TFRC in EOC clinical samples,and survival analysis and molecular expression correlation analysis were performed with online database.2.SKOV3 and A2780 cell lines with stable knockdown TFRC expression were constructed by lentivirus vector transfection technology,and the expression of TFRC at m RNA and protein levels was verified by qRT-PCR,flow cytometry and Western blot assays.3.Flow cytometry was used to detect the correlation between TFRC and Ki-67 expression in EOC clinical samples.4.The role of TFRC on the proliferation of SKOV3 and A2780 cells were studied by CCK-8,Ed U,cell cycle assays and nude mice subcutaneous tumor bearing experiment.5.The role of TFRC in the invasion and metastasis of SKOV3 and A2780 cells was studied by Transwell assay and nude mice peritoneal metastasis model experiment.6.AXIN2,the target molecule of TFRC,was found by RNA-seq.The expression of TFRC and AXIN2 and their functional correlation in both of cells and clinical tissues,were verified by qRT-PCR,flow cytometry and immunohistochemistry assays.7.Online database was used to analyze the survival and molecular expression correlation of AXIN2.8.The expression of AXIN2 in SKOV3 and A2780 cells was knocked down by transfecting si RNA,and the roles of AXIN2 in cell proliferation and migration were studied by CCK-8,Ed U and Transwell assays.9.The expression of AXIN2 in SKOV3 and A2780 cells was up-regulated by transfecting overexpression plasmid,and the functional relevance between TFRC and AXIN2 in the proliferation,invasion and metastasis of epithelial ovarian cancer cells was further confirmed by CCK-8 and Transwell experiments.Results:1.The results of flow cytometry and immunofluorescence showed that the expression of TFRC in EOC patients was higher than that in normal cancer tissues,and it was higher in metastasis tissues than that in primary tumor tissues.The analysis results of KM-plotter and progenev2 database showed that the higher expression of TFRC means the shorter total survival time and recurrence free survival time of patients;the analysis results of UCSC Xena database showed that the higher the expression of TFRC means that the patients were more likely to have cancer metastasis of blood vessels or lymph nodes;the analysis results of GEO database showed that TFRC was positively correlated with the expression of cell proliferation and invasion and metastasis markers.2.TFRC-downregulated SKOV3 and A2780 cell lines were stably cultured and subcultured.3.Flow cytometry result showed that TFRC was positively correlated with Ki-67 expression in EOC clinical samples.4.TFRC knockdown inhibited the proliferation of epithelial ovarian cancer cell and the growth of tumor: the results of CCK-8 and Ed U assays showed that the cell absorbance and EdU positive rate in TFRC knockdown group were significantly lower than those in the control group;the results of nude mice subcutaneous tumor bearing experiment showed that the tumor growth in TFRC knockdown group was significantly slower than that in the control group.5.TFRC knockdown inhibited the invasion and migration of epithelial ovarian cancer cell and the metastasis of tumor: the results of Transwell assay showed that the invasion and migration cells in TFRC knockdown group were significantly less than those in the control group;the results of nude mice peritoneal metastasis model experiment showed that tumor metastasis nodes in TFRC knockdown group were significantly less than that in the control group.6.TFRC may promote the proliferation,invasion and metastasis of epithelial ovarian cancer cells by positively regulating the expression of AXIN2: the bioinformatics analysis results of RNA-seq showed that AXIN2 might be a potential target molecule of TFRC in the proliferation,invasion and metastasis of epithelial ovarian cancer cells;the results ofq RT-PCR,flow cytometry and immunohistochemistry assays showed that the expression between TFRC and AXIN2 was positively correlated.7.AXIN2 is related to the poor prognosis of patients with ovarian cancer,and positively related to the expression of cell proliferation and invasion and metastasis markers8.AXIN2 knockdown inhibited the proliferation and migration of epithelial ovarian cancer cells: the results of CCK-8,EdU and Transwell assays showed that the cell absorbance,Ed U positive rate and the number of migration cells in AXIN2 knockdown group were significantly lower than those in the control group.9.Up-regulation of AXIN2 promoted the proliferation and migration of epithelial ovarian cancer cells,and reversed the inhibition caused by TFRC knockdown: the results of CCK-8 and Transwell assays showed that the cell absorbance and the number of migration cells in AXIN2 overexpression group were significantly higher than those in the control group,and the cell absorbance and the number of migration cells in the cotransfection group were significantly higher than those in TFRC knockdown group.Conclusions:1.TFRC is overexpressed in EOC and correlates with poor prognosis.2.TFRC knockdown inhibited the proliferation,invasion and metastasis of epithelial ovarian cancer cells.3.TFRC and AXIN2 are positively correlated in epithelial ovarian cancer cells and clinical samples.4.AXIN2 knockdown inhibited the proliferation and migration of epithelial ovarian cancer cells,however,up-regulation of AXIN2 promoted the proliferation and migration of epithelial ovarian cancer cells,and reverse the inhibition caused by TFRC knockdown.5.TFRC promotes epithelial ovarian cancer cell proliferation and metastasis by positively regulating the expression of AXIN2,and it plays an oncogenic role in the development of EOC. |
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