| Objective: Endometrial cancer is one of the three major malignant tumors of the female reproductive system,and its incidence has been increasing year by year.Long non-coding RNA has been shown to play an important role in tumorigenesis and development.CASC2 is the earliest tumor suppressor gene found in endometrial cancer.In recent years,CASC2 has also been found in other tumors.Although CASC2 was first discovered in endometrial cancer,no research has been reported on its mechanism in endometrial cancer.miR-103a-3p,mir-107 is a member of the mir-15 / 107 family.The literature reports increased expression of miR-103 and mir-107 in patients with endometrial cancer.In recent years,abnormal expressions of mir-103a-3p and mir-107 have also been found in other cancers.Bioinformatics software predicts that CASC2 has binding sites to miR-103a-3p,mir-107.Targetscan predicts that PLAG1 is a target gene of miR-103a-3p,mir-107,and the literature reports that PLAG1 binds to the promoter region of IGF2.The purpose of this study was to investigate the effects of CASC2 on miR-103a-3p and mir-107 on the biological behavior of endometrial cancer and the expression of PLAG1,IGF2 and their possible mechanisms through clinical histological samples and in vitro and in vivo experiments.The study of endometrial cancer provides a new perspective and provides a new idea for clinical diagnosis and treatment.Methods: Bioinformatics software predicts the expression of miR-103a-3p,mir-107,PLAG1,and IGF2 in endometrial cancer tissues and normal endometrial tissues,their relationship with staging.QRT-PCR was used to detect the RNA expression levels of CASC2,miR-103a-3p,mir-107,PLAG1,and IGF2 in endometrial cancer tissues and normal endometrial tissues,and to analyze the expression of CASC2 and clinical characteristics Relationship of pathological parameters.Western blot was used to detect the protein expression levels of PLAG1 and IGF2 in endometrial cancer tissues and normal endometrial tissues.The correlation between CASC2 and miR-103a-3p,mir-107 was analyzed.Bioinformatics software predicts the binding sites of CASC2 to miR-103a-3p,mir-107,and constructs wild-type and mutant vectors of CASC2,co-transfects mimics of miR-103a-3p,mir-107 with the vector,Double luciferase experiments verified the binding sites of the two.Endometrial cancer Ishikawa and HEC-1A cells were transfected with over-expressed and silenced CASC2 lentivirus,and stable transfected cell lines were selected.The transfection efficiency was verified by q RT-PCR.Endometrial cancer Ishikawa and HEC-1A cells were transfected with miR-103a-3p,mimic and inhibitor of mir-107,and the transfection efficiency was verified by q RT-PCR.QRT-PCR was used to detect the expression of miR-103a-3p and mir-107 after CASC2 was over-expressed or silenced.Changes in biological behavior of endometrial cancer Ishikawa and HEC-1A cells after overexpression or silencing of CASC2 were detected.CCK-8 and Edu experiments detect changes in cell proliferation ability after transfection;scratch tests detect changes in cell migration ability after transfection;Transwell experiments detect changes in cell invasion ability after transfection.Flow cytometry was used to detect changes in cell cycle and apoptosis after transfection.The same method was used to detect changes in the biological behavior of endometrial cancer Ishikawa and HEC-1A cells after transfection with miR-103a-3p,mimic and inhibitor of mir-107.Target Scan predicts that miR-103a-3p,mir-107 has a binding site with the transcription factor PLAG1,and the literature reports that PLAG1 binds to the promoter region of IGF2.Construct wild-type and mutant vectors of PLAG1,co-transfect the vector with mimics of miR-103a-3p,mir-107,and double luciferase experiments to verify the binding sites of PLAG1 and miR-103a-3p,mir-107;QRT-PCR and Western blot were used to detect the expression of PLAG1 and IGF2 in endometrial carcinoma Ishikawa and HEC-1A cells after CASC2 overexpression or silence;q RT-PCR and Western blot were used to detect miR-103a-3p,mir-107 Expression of PLAG1 and IGF2 in endometrial carcinoma Ishikawa and HEC-1A cells after mimic and inhibitor.Endometrial cancer Ishikawa and HEC-1A cells were transfected with PLAG1 overexpression vector and si RNA,and the effects of transfection on the proliferation,migration,invasion,cycle,and apoptosis of endometrial cancer cells were detected by the same method.q RT-PCR and Western blot were used to detect the expression of IGF2 after PLAG1 was overexpressed or silenced.Transfection of CASC2,miR-103a-3p,and mir-107 by single or co-transfection was used to test the effects of transfection on the proliferation,migration,invasion,cycle and apoptosis of endometrial cancer cells by the same method.To verify whether CASC2 can rescue miR-103a-3p and mir-107 from inhibiting cell biological behavior.Western blot was used to detect the effect of salvage experiments on downstream PLAG1 and IGF2 protein expression.Western blot was used to detect the effects of CASC2,miR-103a-3p,and mir-107 on pathway-related proteins.Nude mouse tumor formation experiments were performed to examine the effects of CASC2,miR-103a-3p,and mir-107 on the size of transplanted tumors alone or in combination;q RT-PCR and Western blot were used to detect the changes of PLAG1 and IGF2 expression in tumor tissues of each group.Results: The TCGA database suggested that CASC2,PLAG1,and IGF2 were underexpressed in endometrial cancer tissues,and miR-103a-3p and mir-107 were overexpressed in endometrial cancer tissues,and that CASC2 was related to the stage classification of endometrial cancer.Patients with low expression of CASC2 have a poor prognosis.CASC2 is negatively correlated with miR-103a-3p and mir-107.Clinical histology samples showed the same expression difference results,and the expression of CASC2 was related to the degree of differentiation of endometrial cancer,and CASC2 was negatively correlated with miR-103a-3p and mir-107.Both CCK-8 and Ed U experiments suggest that cell proliferation ability decreases after over-expressing CASC2,and scratch healing experiments suggest that scratch healing ability decreases after over-expressing CASC2,and Transwell experiments suggest that cell invasion ability decreases after overexpressing CASC2,and cell cycle experiments suggest over-expression After CASC2,G0-G1 phase cells increased significantly.Apoptosis experiments showed that after overexpressing CASC2,the overall apoptosis rate(early apoptosis + late apoptosis)increased.Double luciferase experiments confirmed that CASC2 had binding sites to miR-103a-3p and mir-107.CASC2 negatively regulates miR-103a-3p,mir-107.Silencing miR-103a-3p,mir-107 can inhibit the malignant biological behavior of endometrial cancer cells.Double luciferase experiments confirmed that miR-103a-3p,mir-107 and PLAG1 3?UTR had binding sites.After over-expressing CASC2,PLAG1 and IGF2 expression increased at both the RNA and protein levels.After miR-103a-3p and mir-107 were over-expressed,PLAG1 and IGF2 expressions were reduced at both the RNA and protein levels.Overexpression of PLAG1 can inhibit the biological behavior of endometrial cancer cells.It is reported in the literature that there are binding sites in the promoter region of PLAG1 and IGF2.QRT-PCR and Western blot experiments have confirmed that the expression of PLAG1 can regulate the expression of IGF2,and the two are positively related.Low CASC2 expression can rescue miR-103a-3p and mir-107 low expression inhibited biological behavior,and the protein expression levels of PLAG1 and IGF2 also recovered.CASC2,miR-103a-3p,and mir-107 alone or in combination can affect related pathway proteins.With the increase of CASC2 and the decrease of miR-103a-3p and mir-107,the tumor formation volume of nude mice decreased,and the expression levels of PLAG1 and IGF2 increased(both results above p <0.05).Conclusion: In endometrial cancer tissues,CASC2,PLAG1,and IGF2 are underexpressed,and mir-103a-3p?mir-107 is overexpressed;CASC2 targeted binding to mir-103a-3p?mir-107 affects endometrial cancer cells.Malignant biological behavior;mir-103a-3p?mir-107 affects the malignant biological behavior of endometrial cancer cells through the3'UTR region of PLAG1,while PLAG1 acts as a transcription factor to regulate IGF2expression;CASC2 combined with miR-103a-3p Mir-107 can inhibit the formation of subcutaneous xenografts in nude mice and affect the expression of PLAG1 and IGF2.CASC2 combined with miR-103a-3p and mir-107 affect the expression of pathway proteins.The CASC2-mir-103a-3p ? mir-107-PLAG1-IGF2 regulatory axis plays an important role in the malignant process of endometrial cancer. |
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