The Effect And Mechanism Of MiRNA-135a-5p/STMN1 On The Proliferation And Migration Of Ovarian Cancer Cells Through Regulating HIF1α

BackgroundOvarian Cancer is one of the most common female reproducti ve system malignancies.The incidence of ovarian cancer is lower than that of endometrial cancer and cervical cancer,but the mortality rate exceeds that of endometrial cancer and cervical cancer,ranking the first.At present,the treatment strategy for ovarian cancer includes surgery,chemotherapy,targeted drug maintenance therapy and so on.Among them,maintenance therapy,mainly the application of PARP inhibitors,is the most remarkable progress based on the research of molecular mechanism in recent years.Individualized treatment based on biomarkers is the trend of tumor treatment in the future.Although molecular targeted therapy for ovarian cancer has made some progress,the prognosis of advanced ovarian cancer is still poor.Meanwhile,platinum-based chemotherapy is the cornerstone of ovarian cancer treatment.Once platinum resistance relapse occurs,the survival time is significantly shortened.We hope to explore the pathogenesis of ovarian cancer,find valuable molecular markers,improve the sensitivity of platinumbased chemotherapy,and provide new targets and targeted treatment strategies for ovanan cancer.Stathmin 1(STMN 1),a member of the stathmin protein family,which is associated with the regulation of the depolymerization and dynamics of microtubules,affects the stability and function of the spindle,and further affecting cell mitosis.STMN 1 is associated with the development and progression of a variety of malignant tumors,including gastric cancer,cervical cancer,esophageal squamous cell carcinoma,etc.The overexpression of STMN1 promotes tumor metastasis and is associated with poor prognosis.STMN 1 has been previously identified as an oncogenic gene in ovarian cancer.However,the correlation between the expression of STMN1 and clinical characteristics is not clear.The effect of STMN1 on ovarian cancer cells in vitro has not been confirmed,and the molecular mechanism of STMN 1 in ovarian cancer is not clear.Glycolysis is activated in cancer cells and glycolysis metabolism is closely related to the development of cancer.However,whether the expression of STMN1 affects glycolysis remains unknown.If the expression of STMN1 affects glycolysis,it needs to be further explored through what mechanism and whether it will further affect the biological behavior of ovarian cancer cells.microRNA(miRNA)is a non-coding single-stranded RNA molecule with 18 to 25 nucleotides,which degrades or silences mRNA of target gene to regulate its further transcription and translation.MiRNAs play a variety of roles in the process of cell growth and development.MiRNAs also play an important role in the formation of tumors.miRNAs are divided into tumor suppressor miRNAs and tumor promoting miRNAs.The weakened function of tumor suppressor miRNAs or hyperfunction of tumor promoting miRNAs can lead to the occurrence and development of tumor.STMN1 was predicted to be the target gene of miRNA-135a-5p by Target Scan Human and Star Base prediction software.MiRNA-135a-5p has been proved to be a tumor suppressor miRNA with low expression in a variety of tumors.It is closely associated with high tumor invasiveness and poor prognosis.Up to now,the expression of miRNA135A-5p in ovarian cancer has not been studied.We proposed the following hypothesis:STMN1 promoted the proliferation and migration of ovarian cancer cells by affecting glycolysis.MiRNA-135a-5p can target the expression of STMN1.If this hypothesis is true,then regulation of STMN1 or miRNA-135a-5p may change the biological characteristics of ovarian cancer,providing a new target for the treatment of ovarian cancer.Part Ⅰ The Expression and clinical significance of STMN1 in ovarian cancerObjective:To explore the expression of STMN1 in ovarian cancer,its correlation with clinicopathologic factors and its influence on prognosis.Methods:1.The expression of STMN1 in ovarian cancer was analyzed by database.Immunohistochemical methods were used to detect the expression of STMN1 in ovarian cancer,and the expression of STMN1 in ovarian cancer tissues and adjacent tissues was compared.2.Patients were included according to the inclusion criteria and their clinical characteristics were analyzed.Immunohistochemistry were used to detect the expression of STMN1 in ovarian cancer,and the samples were divided into high expression group and low expression group.To explore the correlation between the expression of STMN1 and clinicopathologic factors,including age,FIGO stage,pathological classification,tumor differentiation grade,tumor size,lymph node metastasis,and CA125 level.3.Kaplan-Meier method was used to investigate the relationship between STMN1 expression level and prognosis of patients.Results:1.The expression of STMN1 in ovarian cancer tissues is higher than that in paracancer tissues.2.The high expression of STMN1 in ovarian cancer patients was correlated with advanced tumor stage and higher tumor differentiation grade(P<0.01),but had no statistical significance with age,pathological classification,tumor size,lymph node metastasis,and CA125 level.3.Patients with high expression of STMN1 had worse prognosis(P<0.05).Conclusions:The high expression of STMN1 in ovarian cancer was closely related to advanced tumor stage and higher tumor differentiation grade of the patients and was negatively correlated with the long-term survival.Part Ⅱ STMN1 can promote the proliferation and migration of ovarian cancer cells by regulating HIF1αObjective:To explore the effect and mechanism of STMN1 on the proliferation and migration of ovarian cancer cells.Methods:1.In ovarian cancer SKOV3 and A2780 cell lines,the expression of STMN1 was interfered by siRNA transfection or overexpression plasmid transfection.The effect of STMN1 on the biological function of ovarian cancer cells was verified by CCK-8 assays,colony formation assays,wound healing assays,and Transwell assays.2.The effect of STMN1 expression on tumor growth was also verified in vivo.3.Extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)of ovarian cancer cells were monitored by seahorse assay,and glucose uptake and lactic acid secretion of ovarian cancer cells were detected to verify the effect of STMN1 expression on glycolysis.4.After inhibiting glycolysis with 2-DG,CCK-8 and Transwell assays were used to verify the effects of STMN1 on the proliferation and migration of ovarian cancer cells.5.A series of glycolytic related molecules were detected by PCR,and the expression of STMN1 was closely related to HIF1 α.The effect of STMN1 on HIF1α promoter was verified by Dual luciferase assay.At the same time,WB was used to detect the change of HIF1α protein expression after the change of STMN 1 expression.6.After interfering the expression of HIF 1α,the effects of STMN1 on the proliferation and migration of ovarian cancer cells were verified by CCK-8 and Transwell assays.Results:1.After transfection with siRNA-STMN1 to interfere with the expression of STMN1,compared with siRNA-NC,the proliferation and migration of ovarian cancer cells were significantly decreased.2.After transfection with pcDNA3.1-STMN1 overexpression plasmid,compared with pcDNA3.1-control,the proliferation and migration of ovarian cancer cells were significantly increased.3.The volume of subcutaneous xenografts overexpressing STMN1 in nude mice was significantly larger than that of the control group.4.STMN1 enhanced the transformation of ovarian cancer cells to glycolysis metabolism.After inhibiting glycolysis,STMN1 decreased the ability to promote the proliferation and migration of ovarian cancer cells.5.STMN1 can affect the expression of HIF 1α.Dual luciferase assay confirmed that STMN1 enhanced the activity of HIF 1α promoter,and the expression of HIF1αincreased after overexpression of STMN 1.6.STMN1 can affect glycolysis by regulating HIF 1α.After interfering with HIF1α,the ability of STMN1 to promote glycolysis disappears,and the ability to promote proliferation and migration of ovarian cancer cells is weakened.Conclusions:1.The expression of STMN1 can affect the proliferation,migration and cisplatin sensitivity of ovarian cancer cells.2.STMN1 enhances the glycolysis level of ovarian cancer cells by promoting the expression of HIF1α,thus affecting the proliferation and migration of ovarian cancer cells.Part Ⅲ microRNA-135a-5p affects the proliferation and migration of ovarian cancer cells by targeting STMN1Objective:To investigate the regulatory effect of miRNA-135a-5p on STMN1 and its effect on proliferation and migration of ovarian cancer cells.Methods:1.MiRNAs that may regulate STMN1 were predicted by Target Scan Human and Star Base prediction software.The miRNAs were screened by TCGA database and bioinformatics analysis method to obtain the miRNA that most closely related to STMN1.The miRNA was miRNA-135a-5p.The expression of miRNA-135a-5p in different ovarian cancer cell lines was verified by qRT-PCR.2.In ovarian cancer A2780 and SKOV3 cell lines,overexpression or interference of miRNA-135a-5p was induced by transfection of miRNA-135a-5p-mimic or miRNA135a-5p-inhibitor.The effect of miRNA-135a-5p on the biological function of ovarian cancer cells was verified by CCK-8 assays,colony formation assays,wound healing assays and Transwell assays.3.The expression of STMN1 was detected by Western Blotting after transfection of miRNA-135a-5p-mimic and miRNA-135a-5p-inhibitor to induce miRNA-135a-5p overexpression or interference.Dual luciferase assay was conducted to verify the direct regulatory effect of miRNA-135a-5p on STMN1.4.After transfection of miRNA-135a-5p-mimic and STMN1 overexpression plasmid,the expression of STMN1 was detected by Western Blotting,and the effect on the biological function of ovarian cancer cells were verified by CCK-8 assays,colony formation assays and transwell assays.5.Set the cisplatin concentration gradient to explore the effect of miRNA-135a-5p expression on ovarian cancer cells subjected to cisplatin.6.The effect of miRNA-135a-5p expression on ovarian cancer cells subjected to cisplatin was investigated in vivo.Results:1.The expression of miRNA-13 5a-5p is lower in ovarian cancer tissue compared with normal ovary.And its expression is negatively correlated with STMN1.The expression of miRNA-135a-5p in ovarian cancer cell lines A2780 and SKOV3 was lower than that in human normal ovarian epithelial cell line IOSE.And the expression of miRNA-135A-5P in A2780 was lower than that in SKOV3.2.Ovarian cancer cell line A2780,transfected with miRNA-135a-5p-mimic,decreased cell proliferation and migration compared with that transfected with miRNA135a-5p-mimic-NC.3.Ovarian cancer cell line SKOV3,transfected with miRNA-135a-5p-inhibitor,accelerated cell proliferation and migration compared with that transfected with miRNA-135a-5p-inhibitor-NC.4.Dual luciferase assay confirmed that miRNA-135a-5p binds to STMN1 3’UTR.Moreover,the expression of STMN1 in SKOV3 cells was increased after transfection with miRNA-135a-5p-inhibitor.On the contrary,the expression of STMN1 in A2780 cells was decreased after transfection with miRNA-135a-5p-mimic.5.Ovarian cancer cell line A2780 was transfected with miRNA-135a-5p-mimic and STMN1 overexpression plasmid,and the expression of STMN1 was detected.The expression of STMN1 was significantly higher than that of miRNA-135a-5p-mimic group.The effects of miRNA-135a-5p-mimic on cell proliferation and migration could be reversed.6.The sensitivity of A2780 ovarian cancer cells to cisplatin was increased after transfection with miRNA-135a-5p-mimic.When transfected with miRNA-135a-5pmimic and STMN1 overexpression plasmid,the sensitivity to cisplatin was decreased compared with that of transfected with miRNA-135a-5p-mimic alone.7.In vivo experiments confirmed that the high expression of miRNA-135a-5p may play a synergistic role with cisplatin to increase the response of ovarian cancer cells to cisplatin.Conclusions:1.miRNA-13 5a-5p is low expressed in ovarian cancer and can be targeted to regulate STMN1,thus affecting the proliferation and migration of ovarian cancer cells and the sensitivity of cisplatin to chemotherapy.2.miRNA-135a-5p/STMN1 affects the proliferation and migration of ovarian cancer cells by regulating HIF1α.3.miRNA-135a-5p,STMN1 and HIF1α may be meaningful molecular targets for the future treatment of ovarian cancer and increase the sensitivity of chemotherapy.