| Objective: With the rapid growth of China’s economic level and the change of people’s diet and living habits,the incidence rate of endometrial cancer(EC),one of the three major malignant tumors in the female reproductive system,has gradually increased,and there is a younger trend.The existing treatment methods for patients with advanced or recurrent endometrial cancer are not satisfactory,and exploring more effective treatment methods is still the focus of current endometrial cancer research.A large number of studies believe that there are functional cell subsets with stem cell characteristics in malignant tumors,which are called tumor stem cells.Tumor stem cells have strong proliferation,self-renewal,migration and anti-chemotherapy drug capabilities,which are the basis of tumor occurrence,development and maintenance.Therefore,in-depth study of tumor stem cells is of great significance for refractory malignant tumors.Long non-coding RNA(LncRNA)is often used as a tumor promoter or tumor suppressor to regulate the occurrence and progression of cancer,and is also considered to be a key regulatory factor for the stemness characteristics of human tumor stem cells.The abnormal amplification of human plasmacytoma variant transfer gene 1(PVT1)often indicates a high risk of tumor development.Some studies have shown that PVT1 is up-regulated in endometrial cancer,but its mechanism of action in endometrial cancer stem cells is still unclear.Bioinformatics predicts that there is a binding site between PVT1 and miR-136,and that the expression of miR-136 is abnormal in endometrial cancer.Therefore,this study aims to explore the mechanism of the interaction between PVT1 and miR-136 on the malignant behavior and cell stemness of endometrial cancer parent cells and stem cells.PVT1 promotes the expression of transcription factor Sox2 by inhibiting the expression of miR-136,and improves the transcription level of UPF1,it affects the malignant behavior and cell stemness of endometrial carcinoma parental cells and stem cells.Methods: First,the expression of PVT1 and miR-136 in endometrial cancer tissue and endometrial cancer stem cells was detected by q RT-PCR,and the relationship between the expression of PVT1 and miR-136 and the prognosis of patients was analyzed.CCK-8and Ed U experiments were used to detect the effect of PVT1 on the proliferation of endometrial carcinoma parental cells and stem cells;Transwell test was used to detect the effect of PVT1 on the migration and invasion of the two cells;The changes of apoptosis rate and cell cycle were detected by flow cytometry;The change of self-renewal ability of endometrial cancer cells was detected by spheroid formation test;CCK-8 assay was used to detect the effect of PVT1 on carboplatin resistance of two kinds of cells;Western blot was used to detect the effect of PVT1 on the expression of cell stemness markers in the two cells.Then q RT-PCR was used to detect the expression change of miR-136 after regulating PVT1.The binding site of PVT1 and miR-136 was verified by using the double luciferase reporter gene system.FISH experiment was used to observe the co-location of PVT1 and miR-136 in endometrial cancer parental cells and stem cells.CCK-8,Ed U,Transwell,flow cytometry,spheroidization test,carboplatin resistance test and Western blot were used to detect the effect of miR-136 on the malignant biological behavior and cell stemness of two kinds of cells,and rescue experiments were designed to verify the rescue effect of miR-136 on PVT1.QRT-PCR and Western blot were used to detect the expression level of Sox2 and UPF1 in normal endometrial tissue and endometrial cancer tissue,as well as in endometrial cancer parental cells and stem cells.The binding sites of miR-136 and Sox2 were verified by the dual luciferase reporter gene system.The expression of Sox2 after regulating PVT1 and miR-136 was detected by q RT-PCR and Western blot.The effects of Sox2 on the malignant biological behavior and cell stemness of two kinds of cells were tested,and rescue experiments were designed to verify the recovery effect of Sox2 on miR-136.The binding action and binding site of transcription factor Sox2 and UPF1 promoter region were verified by Ch IP experiment and double luciferase reporter gene system.The expression of UPF1 after regulating PVT1,miR-136 and Sox2 was detected by q RT-PCR and Western blot.Finally,a nude mouse subcutaneous tumor model was constructed to verify the effect of PVT1 and miR-136 on tumor growth.Western blot was used to detect the expression of Sox2,UPF1 and cell stem markers in the transplanted tumors of each group.Results: The expression of PVT1 in endometrial cancer tissue and endometrial cancer stem cells was significantly increased.The increased expression of PVT1 was associated with FIGO stage III-IV,LVSI positive,lymph node metastasis positive and poor prognosis of endometrial cancer.CCK-8 and Ed U experiments after silencing PVT1 showed that the proliferative ability of endometrial cancer parental cells and stem cells decreased.Transwell experiments showed that the migration and invasion ability of cells decreased,and flow cytometry showed that after silencing PVT1,cell apoptosis increased,G0 phase cells increased,and cell cycle arrest.Spherical formation test showed that silencing PVT1 inhibited cell self-renewal,carboplatin resistance test showed that silencing PVT1 reduced cell resistance,and Western blot showed that the expression of cell stem markers decreased after silencing PVT1(P<0.05 for all the above).The expression of miR-136 was decreased in endometrial cancer tissue and endometrial cancer stem cells.The decreased expression of miR-136 was associated with FIGO III-IV stage,pathological grade G2-3 and poor prognosis of endometrial cancer.It was confirmed by q RT-PCR that PVT1 could negatively regulate miR-136,and the double luciferase gene reporting system showed that there was a binding site between the two.CCK-8,Ed U,Transwell,flow cytometry,spheroid formation test,carboplatin resistance test and Western blot test showed that overexpression of miR-136 could inhibit the proliferation,migration and invasion of endometrial cancer parental cells and stem cells,increase the rate of cell apoptosis,block cell cycle,inhibit self-renewal,and reduce the expression of drug resistance and stem markers.The results of salvage experiment showed that miR-136 could reverse the effect of PVT1 on endometrial cancer parent cells and stem cells(P<0.05 for all the above).The expression of Sox2 is increased in endometrial cancer tissue and endometrial cancer stem cells.The double luciferase gene reporting system shows that there is a binding site in the 3 ’-UTR region of miR-136 and Sox2.QRT-PCR shows that Sox2 is negatively regulated by miR-136 and positively regulated by PVT1.CCK-8,Ed U,Transwell,flow cytometry,spheroidization test,carboplatin resistance test and Western blot test show that silencing Sox2 can inhibit the malignant behavior and cell stem of endometrial cancer parental cells and stem cells,and the rescue test proves that Sox2 can reverse the effect of miR-136 on endometrial cancer parental cells and stem cells(P<0.05 for all the above).The results of Chi IP test and double luciferase reporter gene system showed that Sox2 as a transcription factor combined with UPF1 promoter region,and the results of q RT-PCR and Western blot showed that Sox2 promoted UPF1 expression(P<0.05 for all the above).In vivo,silencing PVT1 combined with overexpression of miR-136 has the strongest inhibitory effect on the growth of transplanted tumor in nude mice,and reduces the expression of Sox2,UPF1 and stemness markers to the greatest extent(P<0.05 for all the above).Conclusion: 1.PVT1 is highly expressed in endometrial cancer,which is associated with high malignancy and poor prognosis.PVT1 promotes the malignant biological behavior and cell stemness of endometrial cancer stem cells;2.The expression of miR-136 is low in endometrial cancer,which is associated with high malignancy and poor prognosis of tumor.PVT1 can target and negatively regulate miR-136,and the cancer-promoting effect of PVT1 can be achieved by inhibiting miR-136;3.Sox2 and UPF1 are highly expressed in endometrial cancer,and Sox2 promotes the malignant biological behavior and cell stemness of endometrial cancer stem cells.The 3 ’-UTR of Sox2 can bind to miR-136,while Sox2 acts as a transcription factor to bind to the UPF1 promoter region and promote UPF1 expression.PVT1 promotes the expression of Sox2 by inhibiting miR-136,improves the transcription level of UPF1,and promotes the malignant behavior and stemness characteristics of endometrial cancer parental cells and stem cells.LncRNA PVT1/miR-136/Sox2/UPF1 regulatory axis plays a role in the development of endometrial cancer and may become a potential target for molecular therapy of endometrial cancer in clinical practice. |
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