The Mechanism Study Of ApoG2 Targeting Regulation DKK3 For Inhibiting The Biological Behavior Of Cervical Cancer

BackgroundCervical Cancer is one of the most common gynecological malignancies in women.According to reports,there were 604,000 new cases of cervical cancer and nearly 342,000 deaths worldwide in 2020.Both incidence rate(6.5%)and mortality rate(7.7%)rank fourth among female malignancies,mainly due to persistent infection with high-risk Human Papillomavirus(HPV).Although the application of HPV vaccines and cervical cancer screening has significantly reduced the incidence of cervical cancer,the number of new cases and deaths from cervical cancer is still high.In 2022,the Chinese Cancer Center released the latest statistical data:there were 119,300 new cases of cervical cancer and 17,000 deaths in China in 2016.Therefore,cervical cancer is still a major health challenge that poses a threat to women’s health in China and around the world.Currently,the treatment strategies for cervical cancer mainly focus on surgery,radiotherapy and chemotherapy.Although the causes of cervical cancer is clear,there are early screening methods and the effects of surgery and chemoradiotherapy are good,some patients still experience recurrence or metastasis after treatments.Drug resistance and toxic side effects are the main reasons for the recurrence and metastasis of patients.Studies have found that there are other factors involved in the occurrence and development of cervical cancer,in addition to persistent infection with high-risk HPV.Therefore,it is urgent to further understand the molecular mechanisms of the occurrence and development of cervical cancer and to seek effective drugs for the treatment of cervical cancer.Gossypol is a natural product extracted from the seeds,roots and leaves of cotton plants.It was later eliminated due to serious side effects,originally used as a male contraceptive.It was discovered that cottonseed phenol has inhibitory effects on colon cancer and melanoma in 1984.However,its adverse reactions have hindered the clinical application of cottonseed phenol,such as gastrointestinal reactions including hypokalemia,muscle weakness,anorexia,nausea,vomiting,as well as palpitations and liver dysfunction.The main reason is related to the two aldehyde groups in the chemical structure of cottonseed phenol.In recent years,researchers have modified the molecular structure of cottonseed phenol and synthesized a new derivative of cottonseed phenol called Apogossypolone-ApoG2,which has much higher anti-tumor activity and lower toxicity than cottonseed phenol.Studies have shown that ApoG2 can effectively inhibit the growth of nasopharyngeal carcinoma tumors and enhance the antitumor effect of cisplatin.ApoG2 can also inhibit the anti-apoptotic function of Bcl-2,Mcl-1,and Bcl-XL and induce apoptosis in pancreatic cancer cells.In addition,ApoG2 can inhibit the invasion and migration of tumor cells through the reverse epithelial-mesenchymal transition(EMT)process in pheochromocytoma.We searched the relevant literature and found that although ApoG2 has anti-tumor effects in diseases such as gastric cancer,colorectal cancer,nasopharyngeal carcinoma,breast cancer,liver cancer,prostate cancer and lymphoma,there is little research on its specific mechanism in cervical cancer.In recent years,studies have shown that the occurrence and development of cervical cancer are the results of the combined effects of multi-gene genetics and epigenetics.With the deepening of the study of epigenetic mechanisms,it has been discovered that more and more signaling pathways and genes have been proven to be related to the occurrence of cervical cancer.Studies have shown that the overactivation of the classic Wnt signaling pathway,the Wnt/β-catenin signaling pathway,is highly correlated with the pathogenesis of various human tumors.Dickkopf-related protein 3(DKK3),as an antagonist of the Wnt/β-catenin signaling pathway,can inhibit its nuclear translocation and reduce the expression of tumor proteins by inhibiting the activity of β-catenin.The DKK protein family encodes secreted glycoproteins,including five different proteins:DKK1,2,3,4,and Soggy(a unique DKK3-related protein).However,the DKK3 protein is significantly different from other members of the family.Structurally,in addition to the two cysteine-rich domains(DKK-N and colipase folding domain)that are shared among all family members,DKK3 also has a unique Soggy domain.Furthermore,the non-conserved linker region between the two domains in other members consists of 50-55 amino acids,the linker region in DKK3 is shorter,consisting of only 12 amino acids.Functionally,the ability of DKK3 to regulate the Wnt/β-catenin signaling pathway is controversial and it does not bind to lowdensity lipoprotein receptor-related proteins 5/6 or receptor Kremenl,unlike other members of the family.Due to these differences in structure and function,DKK3 exhibits biological characteristics that are distinct from other family members.The pathogenesis and development mechanism of cervical cancer is complex and it is a multi-factor,multi-gene,multi-step collaborative process.The detailed mechanism of DKK3 involved in cervical cancer is not yet fully understood.After reviewing relevant literature,it was found that there is little research on the role of ApoG2 in cervical cancer both domestically and internationally,and currently,no studies have found whether the regulation of cervical cancer by ApoG2 is related to DKK3 expression.Therefore,this paper aims to study the effects and biological functions of ApoG2 on cervical cancer in vivo and in vitro,explore whether it is related to the expression of DKK3 and attempt to discover its mechanism of action,providing new ideas for the drug treatment of cervical cancer.The specific research content includes the following three parts:Part Ⅰ:Study on the effects and biological functions of ApoG2 on cervical cancerResearch objectives:1.To investigate the effects of ApoG2 on the biological functions of cervical cancer cells through in vitro experiments;2.To study the effects of ApoG2 on nude mice cervical cancer xenografts through in vivo experiments.Research methods:1.CCK-8 cell proliferation assay,cell cloning formation assay,flow cytometry,Transwell invasion assay,and Western Blot were used to detect the effects of different concentrations(0,2.5μmol/L,5μmol/L,10μmol/L and 20μmol/L)of ApoG2 on the biological behavior of cervical cancer cell including proliferation,apoptosis,invasion and migration.2.To construct a BALB/c-nu nude mouse cervical cancer transplantation tumor model and study the effects of ApoG2 on cervical cancer transplantation tumors in nude mice.Results:1.CCK-8 assay and cell clone formation assay showed that ApoG2 could reduce the viability of CaSki and HeLa cells.With the increase of drug exposure time and concentration,cell proliferation was inhibited in a time-and dose-dependent manner.Flow cytometry analysis showed that ApoG2 promoted apoptosis of CaSki and HeLa cells in a dose-dependent manner.Transwell invasion assay and Western Blot confirmed that the migration and invasion ability of CaSki and HeLa cells were decreased after ApoG2 treatment.2.Nude mice were randomly divided into experimental group and control group.The experimental group received intraperitoneal injection of ApoG2 100mg/kg and the control group received 9%DMSO.The experimental results showed that:the tumor growth rate was significantly inhibited by ApoG2.Compared with the control group,ApoG2 can significantly slow down the growth of tumors in nude mice.By measuring the volume and weight of subcutaneous transplanted tumors in nude mice,it can be verified that ApoG2 has an inhibitory effect on cervical cancer transplanted tumors.Conclusions:1.ApoG2 can inhibit the proliferation,invasion and migration of cervical cancer cells,promote apoptosis and has tumor suppressor effect.2.In vivo experiments confirmed that ApoG2 has tumor suppressor effect.Part Ⅱ:The roles of DKK3 in cervical cancer and the correlations between ApoG2 and DKK3Research objectives:Through the first part of in vivo and in vitro experiments,we have confirmed that ApoG2 has a tumor suppressor effect in cervical cancer,so is the effect of ApoG2 related to DKK3 molecules?Are the two related?In this part,we aim to explore the correlation between ApoG2 and the expression of DKK3,as well as further investigate the role of DKK3 in cervical cancer.Research methods:1.The expression of DKK3 in cervical cancer tissues and adjacent tissues was detected by immunohistochemical method and the relationship between DKK3 and clinicopathological factors and clinical prognosis was analyzed.2.Different concentrations of ApoG2(0,2.5μmol/L,5μmol/L,10μmol/L and 20μmol/L)were applied to cervical cancer cells,and the Western Blot method is used to detect the changes of DKK3 expression in CaSki cells and HeLa cells.3.A lentiviral vector for overexpression of DKK3 was constructed to up-regulate the expression of DKK3 molecules in cervical cancer cells.CCK-8 proliferation assay,cell clone formation assay,flow cytometry,Transwell invasion assay and Western Blot assay were used to detect the effects of DKK3 overexpression on the proliferation,apoptosis,invasion and migration of cervical cancer cells,and to explore the role of DKK3 in cervical cancer.4.Reverse experiments were conducted to further explore whether the inhibitory effect of ApoG2 on cervical cancer cells is related to the DKK3 molecule.Results:1.Immunohistochemical analysis showed that DKK3 was lowly expressed in cervical cancer tissues and highly expressed in adjacent tissues.The expression level of DKK3 was related to cervical cancer FIGO staging and clinical prognosis.2.After different concentrations of ApoG2 treated on CaSki cells and HeLa cells,with the increase of ApoG2 drug concentration,the expression of DKK3 protein increased in a dosedependent manner.3.Results from CCK-8 proliferation assay and cell clone formation assay showed that overexpression of DKK3 significantly reduced the proliferation activity of CaSki and HeLa cells.Flow cytometry and Transwell assay further confirmed that overexpression of DKK3 significantly inhibited the invasion ability and promoted apoptosis of CaSki and HeLa cells.After DKK3 overexpression,the expression of E-cadherin,an EMT-related protein,increased,while the levels of N-cadherin and Vimentin decreased.The expression of anti-apoptotic molecule Bcl-2 decreased,while the expression of Cl-Caspase 3 increased in CaSki and HeLa cells.4.DKK3 was knocked down or overexpressed,followed by treatment with 20μmol/L ApoG2 for 24h.Results from CCK-8 and cell clone formation assay showed that overexpression of DKK3 significantly enhanced the inhibitory effect of ApoG2 on CaSki cells,while interfering with DKK3 expression reversed the inhibitory activity of ApoG2 on CaSki cells at the same concentration.Flow cytometry and Transwell assay showed,compared with the control group,DKK3 knockdown could enhance the invasive ability of ApoG2 on CaSki cells,while this ability was reversed after DKK3 overexpression.The results of Western Blot experiments showed that after down-regulating the expression of DKK3 in CaSki cells and adding ApoG2 drug treatment for 24 hours,the expression levels of E-cadherin and Cl-Caspase 3 decreased,and the protein expression levels of N-cadherin,Vimentin and Bcl-2 increased;the results after overexpression of DKK3 on the contrary.Conclusions:1.DKK3 is low expressed in cervical cancer tissue and high expressed in adjacent normal tissue,which is associated with FIGO staging and lymph node metastasis.2.ApoG2 has an anti-cancer effect,which may be achieved by upregulating DKK3.3.DKK3 has a tumor suppressor effect in cervical cancer,can inhibit the proliferation,migration and invasion of CaSki cells and HeLa cells and promote cell apoptosis.Part Ⅲ:ApoG2 inhibits the occurrence and development of cervical cancer by upregulation of DKK3 and related mechanismsResearch objectives:The first two parts of the experiment have verified the inhibitory effect of ApoG2 on cervical cancer,which is closely related to the expression of the DKK3 molecule.Regulating the expression of DKK3 in cells can help reduce the risk of cervical cancer and inhibit its progression.However,the specific mechanism of ApoG2 on cervical cancer is still unclear.In this part,we attempted to explore the specific mechanism of ApoG2 regulating the DKK3 molecule.Research methods:1.Firstly,we used Western Blot method to detect the changes of PI3K/AKT pathway related molecules after ApoG2 drug treatment to verify the correlation between ApoG2 and PI3K/AKT signaling pathway.In the first part,we have already verified that ApoG2 can upregulate the expression of DKK3.In this part of the experiment,we explored the relationship between DKK3 and PI3K/AKT signaling pathway by up-regulating and decreasing the expression of DKK3 molecules in cells,and further verified that ApoG2 may inhibit the activity of PI3K/AKT signaling pathway by up-regulating DKK3.2.The Rescue experiment was used to detect the changes of molecules related to the PI3K/AKT signaling pathway,to verify that DKK3 may play a role through the PI3K/AKT signaling pathway,after adding AKT inhibitors interfered by DKK3 to CaSki cells and HeLa cells.3.After adding an AKT inhibitor to inhibit the PI3K/AKT signaling pathway,to detect the effect on the cell biological function regulated by DKK3.Results:1.The Western Blot results showed that the protein expression levels of p-AKT and pPI3K were significantly decreased after the addition of 20μmol/L ApoG2 drug treatment in CaSki cells and HeLa cells for 24h.It showed that ApoG2 could inhibit PI3K/AKT signaling pathway activity.Further interference and upregulation of DKK3 were performed,and changes in the PI3K/AKT signaling pathway were observed:After interfering with DKK3 expression,the expression levels of p-AKT and p-PI3K proteins increased significantly,and the expression levels of p-AKT and p-PI3K proteins decreased after DKK3 was up-regulated,while the expression levels of unphosphorylated AKT and PI3K proteins had no significant changes.2.The AKT inhibitor MK-2206 was added to DKK3-interfering CaSki cells and HeLa cells.The results of Western Blot experiments showed that the up-regulation of p-AKT caused by DKK3 interference could be reversed after adding the AKT inhibitor,and there was no significant change in unphosphorylated AKT.3.The results of CCK-8 cell proliferation experiments,cell clone formation experiments and Transwell invasion experiments showed that inhibiting the AKT signaling pathway can reverse the cell proliferation,invasion and migration promoted by interfering with DKK3;Western Blot experiments further verified that inhibiting the AKT signaling pathway can reverse the effects of interfering with DKK3 on the expression of EMT-related proteins and cell migration.Conclusions:1.ApoG2 may play a tumor suppressor role by inhibiting the activity of PI3K/AKT signaling pathway.2.DKK3 may be the upstream regulatory gene of the PI3K/AKT signaling pathway.ApoG2 inhibits the proliferation,invasion and migration of cervical cancer cells and promotes apoptosis by up-regulating DKK3.