Inhibitory Effect Of Polypeptide LSSDR412 On Cervical Cancer Via CLDN4/Wnt/β-catenin Signaling Pathway

Background:Cervical cancer is a kind of malignant tumor that occurs in the female lower reproductive tract,with a high incidence,showing an obvious younger trend in recent years,seriously endangering the health of the majority of women.At present,it has been confirmed that persistent high-risk human papillomavirus infection is directly related to the pathogenesis of cervical cancer,and the early screening and reasonable treatment of infection has important clinical significance.At present,the treatment of cervical cancer is mainly based on clinical stages.Early cervical cancer is mainly treated by surgery and postoperative adjuvant therapy,and advanced cervical cancer is mainly treated by concurrent radiotherapy and chemotherapy.The prognosis of patients with advanced cervical cancer is directly related to their sensitivity to radiotherapy and chemotherapy.At present,the most commonly used chemotherapy regimen for cervical cancer is cisplatin-based single-drug chemotherapy or combined chemotherapy,but platinum drugs are prone to drug resistance,and clinically need drugs that can improve the drug resistance of cervical cancer.Bee venom has been proved to have broadspectrum antibacterial and anticancer activity,and because its main way of action is to cause cell death by punching holes in the cell membrane,it does not rely on proprietary targets,resulting in low drug resistance.However,the clinical application of bee venom is limited because of its non-specific cell killing ability and hemolysis.Our group analyzed the main functional components of bee venom and modified it by chemical synthesis.The polypeptides that can target HPV virus were added to the front end to form a new fusion peptide LSSDR412 containing 38 amino acids.The mechanism of anti-cervical cancer against LSSDR412 and the safety of its application in vivo are not clear.Objective:By studying the effects of polypeptide LSSDR412 on the malignant biological behavior of cervical cancer,including proliferation,migration and invasion in vitro and in vivo,to explore the way of cell death,and then to study the regulation mechanism of its anti-cervical cancer effect,so as to provide a theoretical basis for the clinical transformation of polypeptide LSSDR412.Methods:Part 1: The effect of LSSDR412 on malignant biological behavior,HPVE6,E7 protein and apoptosis of cervical cancer.1.Effect of LSSDR412 on malignant biological behavior of cervical cancer(1)The effect of LSSDR412 on the malignant biological behavior of cervical cancerCCK8 was used to detect the cell survival rate of different concentrations of LSSDR412 on different cervical cancer cell lines(Caski,Hela,Si Ha,C33A)and human keratinocytes after Ha Ca T24 hours and 48 hours,and the 50% inhibitory concentration(IC50)of LSSDR412 was calculated,and the cell lines Caski and Hela with similar IC50 were selected and treated with LSSDR412 of 0-30 μ g / ml,respectively,and the effect of drug treatment on the clone formation ability of cervical cancer cells was detected.(2)Effect of LSSDR412 on the proliferation of cervical cancer in vivoFirstly,the blood of mice was collected for hemolysis test of LSSDR412 and melittin in vitro.Then the subcutaneously transplanted tumor model of cervical cancer in nude mice was established by using Hela cells and randomly divided into two groups.LSSDR412 and saline were injected intraperitoneally every 48 hours at a dose of8mg/kg.A total of 8 times of administration.During this period,the general state of nude mice and the changes of subcutaneous transplanted tumor volume were observed,and the body weight change curve and tumor growth curve were drawn.The nude mice were killed 48 hours after treatment,and the tumor was completely stripped and weighed and embedded in paraffin and OCT.The expression level of Ki67 in control group and LSSDR412 group was detected by immunofluorescence.The liver and kidney of nude mice were dissected completely and stained with HE to observe the effect of drug treatment on liver and kidney.(3)Effect of LSSDR412 on migration and invasion of cervical cancer cells in vitroCaski and Hela were treated with LSSDR412 at the concentration of 0-30 μg/ml,respectively.The changes of cell migration distance at 24 hours after drug treatment were detected by scratching,and the cell penetration ability was detected by Transwell chamber,so as to reflect the effect of LSSDR412 on the migration and invasion of cervical cancer cells.The inhibitory effect on the invasion of cervical cancer cells was verified by Western Blot to detect the effect of LSSDR412 on EMT-related proteins.(4)Effect of LSSDR412 on metastasis of cervical cancer in vivoThe lung metastasis model of cervical cancer in tail vein of nude mice was established by injecting Hela cells into the tail vein of nude mice.The nude mice were treated with intraperitoneal injection of LSSDR412 and normal saline respectively.8mg LSSDR412 was injected once in 48 hours for a total of 16 times.48 hours after treatment,the lung tissues of nude mice were dissected and paraffin embedded and analyzed by HE staining.2.Effect of LSSDR412 on the expression of E6 and E7 proteins in HPVCaski and Hela were treated with LSSDR412 at the concentration of 0-30μg/ml,respectively.The expression of HPV E6 and E7 protein was detected by Western Blot method.The best concentration of the drug was 30μg/ml,and then the Caski and Hela cells were treated with the drug.The expression of HPV E6 and E7 protein was detected at different time points(0,0.5,1,2,4,8 h).3.Effect of LSSDR412 on apoptosis of cervical cancer cells(1)The effect of LSSDR412 on apoptosis of cervical cancer cells in vitro.Caski and Hela were treated with LSSDR412 at the concentration of 0-30 μ g / ml,respectively.Genomic DNA breaks in the late stage of apoptosis were labeled by TUNEL to detect the difference in the number of DNA breaks at different concentrations.Pan-casepase inhibitor Z-VAD-FMK and LSSDR412 were used in the culture of Hela and Caski cells,and the changes of cell proliferation were detected by CCK8 assay.The mitochondrial apoptosis pathway related proteins Bax and Bcl-2,and apoptosis key execution protease Cleaved-casepase3 were detected by Western Blot.(2)Effect of LSSDR412 on apoptosis of cervical cancer cells in vivoWestern Blot was used to detect the expression of mitochondrial apoptotic pathway related proteins Bax,Bcl-2 and Cleaved-casepase3 in the subcutaneously transplanted tumors of the two groups.Part 2: Study on the mechanism of LSSDR412 against cervical cancer X1.Effects of LSSDR412 on Wnt/β-catenin signaling pathway and CLDN4expressionThe expression of key proteins of Wnt pathway β-catenin,Active β-catenin,pGSK β,GSK β,MMP9 and c-Myc in Hela and Caski cells treated with LSSDR412 were detected by Western Blot.The changes of m RNA levels of β-catenin target genes TCF and LEF after treatment with LSSDR412 were detected by RT-q PCR.The expression of β-catenin and MMP9 in LSSDR412 group and control group was detected by immunohistochemical method.The expression of tight junction protein CLDN4 after the treatment of LSSDR412 was detected by Western Blot.2.Study on the mechanism of CLDN4 in cervical cancer(1)the pathological sections of cervical cancer and paracancerous tissues collected in clinic were stained with CLDN4 by immunohistochemical method,and its expression was analyzed.By summarizing the prognosis information of patients,the survival curve was drawn,and the relationship between CLDN4 protein and prognosis was analyzed.The expression of CLDN4 in different cervical cancer tissues and adjacent tissues and different cervical cancer cell lines was detected by Western Blot,and the expression of CLDN4 in different cervical cancer cell lines was detected by q PCR.(2)Lentivirus was used to construct CLDN4 knockdown sh RNA vector.Caski and Hela cell lines with high expression of CLDN4 were selected as transfection cell lines with low CLDN4.Plate cloning assay,cell scratch assay,cell Transwell assay and Western Blot assay were used to investigate the effects of CLDN4 knockdown on the proliferation,migration and invasion of cervical cancer cells.(3)Western Blot was used to detect the changes of β-catenin,Active β-catenin,GSK3 β,p-GSK3 β and MMP9 after CLDN4 knockdown.CO-IP assay was used to detect the interaction between AXIN and β-catenin after CLDN4 knock down.3.Study on the target of LSSDR412CO-IP assay was used to detect the change of the interaction between CLDN4 andβ-catenin after LSSDR412 treatment.β-catenin activator SKL2001 and LSSDR412 were used to act on cervical cancer cells.The changes of proteins related to Wnt/ β-catenin signal pathway were detected by Western Blot to identify the target of LSSDR412.Part 3: In vivo experiment of LSSDR412 against cervical cancerThe mouse cervical cancer in situ model was established by injecting U14 cells into the cervical surface of mice.The mice were randomly divided into four groups:U14-Mock group,LSSDR412+U14-Mock group,U14-sh Cldn4 group and LSSDR412+U14-sh Cldn4 group.Seven days after the establishment of the model,the drug was given to the rats.LSSDR412 was given every 48 hours at a dose of 8mg/kg.U14-Mock group and U14-sh Cldn4 group were given the same dose and frequency of saline for a total of 10 times.After treatment,the mice were killed,and the uterus and bilateral appendages were dissected completely,as well as the mesentery with tumor metastasis,and then the cervical tumor was stained with Ki67 immunohistochemical staining.Results:Part 1: Effects of peptide LSSDR412 on malignant biological behavior,HPVE6,E7 protein and apoptosis of cervical cancer1.Effect of LSSDR412 on malignant biological behavior of cervical cancer(1)LSSDR412 inhibited the proliferation and clonogenesis of cervical cancer cells Hela and Caski.Inhibition of proliferation was time-and dose-dependent.However,the effect of LSSDR412 on cervical cancer cells C33 A and Si Ha was small,which was similar to that on normal keratinocyte Ha Ca T.(2)LSSDR412 can significantly inhibit the growth of subcutaneously transplanted tumor in nude mice after treatment,and the tumor weight is lower in LSSDR412 group.LSSDR412 group had a significantly lower percentage of Ki67 positive cells than control group.Compared with the control group,there was no significant change in the structure of liver and kidney in LSSDR412 treatment group.The results show that LSSDR412 is safe to use in vivo.(3)After the treatment of LSSDR412,the migration and invasion ability of cervical cancer cells decreased significantly,and the degree of weakening was positively correlated with the dose of the drug.(4)LSSDR412 can inhibit the invasion and metastasis of cervical cancer by inhibiting EMT process in vitro.XII(5)LSSDR412 treatment can significantly inhibit the number and size of pulmonary metastases in the caudal vein lung metastasis model of cervical cancer in nude mice.2.The effect of LSSDR412 on the expression of HPV E6 and E7 proteinWhen the concentration of LSSDR412 was 30μg/ml for more than 4 hours,LSSDR412 could significantly inhibit the expression of E6 and E7 protein in Hela and Caski cells.3.Effect of LSSDR412 on apoptosis of cervical cancer cellLSSDR412 could significantly promote the apoptosis in cervical cancer cells.It is mainly through casepase-dependent mitochondrial apoptosis pathway into the apoptosis of cervical cancer cells,which has also been confirmed in animal experiments in vivo.Part 2: Study on the mechanism of LSSDR412 against cervical cancer1.Effects of LSSDR412 on Wnt/β-catenin signaling pathway and CLDN4expressionLSSDR412 can significantly inhibit the expression of Wnt/β-catenin pathway related proteins in Hela and Caski cells.The expression and transcriptional activity ofβ-catenin were significantly inhibited.The expression level of CLDN4 was inhibited by LSSDR412.2.Study on the mechanism of CLDN4 in cervical cancer(1)CLDN4 is highly expressed in cervical cancer and is associated with poor prognosis.Knocking down CLDN4 can inhibit the proliferation,migration,invasion and EMT process in cervical cancer.(2)In cervical cancer cells,CLDN4 can bind to β-catenin and reduce the binding of ubiquitin proteasome complex composed of β-catenin and AXIN,resulting in the decrease of the degradation of β-catenin,thus stabilizing β-catenin,increasing the activeβ-catenin in the nucleus and promoting cancer.3.Study on the target of LSSDR412CLDN4 is the target of LSSDR412.After the action of LSSDR412,the binding between CLDN4 and β-catenin is reduced,which plays an anticancer role.Part 3: In vivo experiment of LSSDR412 against cervical cancerLSSDR412 can inhibit the proliferation of cervical cancer in mice with normal immune function,and knocking down CLDN4 has the same effect,and there is a synergistic effect between them in vivo.Conclusion:1.LSSDR412 can inhibit the proliferation,invasion and metastasis of cervical cancer in vitro and in vivo,and inhibit the expression of HPVE6 and E7 protein.It mainly induces apoptosis of cervical cancer cells through endogenous casepasedependent mitochondrial pathway.No obvious side effects were found in the application of LSSDR412 in vivo.2.LSSDR412 can down-regulate the expression of CLDN4 in cervical cancer cells.CLDN4 is highly expressed in cervical cancer,which is associated with poor prognosis.Knocking down CLDN4 can inhibit the proliferation,migration and invasion of cervical cancer.3.CLDN4 is the target of LSSDR412.After the action of LSSDR412,the binding between CLDN4 and β-catenin is reduced,which plays an anticancer role.4.LSSDR412 can inhibit the proliferation of cervical cancer in mice with normal immune function,and knocking down CLDN4 has the same effect,and there is a synergistic effect between them in vivo.