| Cervical cancer is one of the most common malignancies in women.And it is the fourth pathogeny of cancer death in women.According to the latest global data released by the International Agency for Research on Cancer,there were 604,127 new cases and 341,831 deaths of cervical cancer worldwide in 2020.Although HPV(human papilloma virus)vaccine and cervical cancer screening have become effective strategies to prevent cervical cancer,high morbidity and mortality rates still exist in low-income and middle-income countries.Cervical adenocarcinoma(cervical AC)is the second most common histological type of cervical cancer,accounting for 20%to 25%of cervical cancer.In recent years,with the widespread application of cervical cytology and HPV screening,precancerous lesions and early cervical cancer have been diagnosed and treated in time,and the incidence and mortality of cervical squamous cell carcinoma(cervical SC)have decreased.However,the incidence of cervical AC has been reported to be on the rise,and the onset age is younger in recent years.More and more studies have confirmed that cervical AC is different from cervical SC in terms of epidemiology,treatment outcome and prognostic factors.About 15%to 20%of cervical AC is HPV negative,and HPV is not the driving factor for all cervical AC.In addition,the specific mechanism has not been clarified about the clearance of high-risk HPV infection and the occurrence and development of cervical AC.Therefore,the pathogenesis of cervical AC still needs to be explored.The surgery is the preferred treatment for early cervical cancer.The surgery can remove the primary lesion and evaluate the extent of tumor invasion and metastasis,which is conducive to select postoperative adjuvant therapy and prognosis evaluation.Lymph node metastasis(LNM)is one of the main metastasis routes and high risk factors of cervical cancer.Postoperative adjuvant therapy is required for patients with LNM,and the prognosis is poor.Most scholars believe that cervical AC is more prone to LNM and recurrence,and the survival time is shorter than that of cervical SC,which makes most cervical AC patients undergo radical surgery.Currently,lymphadenectomy is widely used to evaluate the status of lymph nodes in early cervical cancer.However,postoperative complications including lymphedema,urinary dysfunction and nerve damage,have been found to seriously affect the life quality of patients undergoing lymphadenectomy.According to WHO classification standards,cervical AC can be divided into more than ten pathological types.In addition to the subtype with a clear poor prognosis,other young cervical AC patients with a good prognosis also received radical surgery,which led to the loss of fertility.However,studies have found that only a few patients with early cervical AC have LNM.Although some evidence indicates that sentinel node biopsy can be used as a lymph node staging method for early cervical cancer,this technique has not been widely validated in clinical practice due to the limitation of developer and special equipment.Therefore,it is important to identify patients at low risk of LNM before lymphadenectomy.The occurrence and progression of cervical cancer is a complex process.Cervical cancer is mainly caused by some genetic changes and epigenetic changes.Data on multiple genomic and molecular characteristics of cervical cancer were published in the journal Nature in 2017.The data included the abnormal expression of miRNAs in cervical cancer.Studies have confirmed that miRNAs are a class of endogenous,single-stranded non-coding RNAs with regulatory functions found in eukaryotes,with a length of about 22 nucleotides.miRNAs destroy mRNA stability and inhibit mRNA translation by specifically binding to the 3’-untranslated region(3’-UTR)of the target gene messenger ribonucleic acid(mRNA).And then it plays a regulatory role on target genes.Therefore,miRNAs can regulate many important biological processes,including cell proliferation,apoptosis,autophagy,invasion,angiogenesis,and epithelial-mesenchymal transformation(EMT).Among them,miR-375 is abnormally expressed in human tumors and plays a role in promoting or suppressing cancer.Therefore,the abnormal expression of miR-375 is closely related to the tumorigenesis and tumor progression.It was found that miR-375 decreased significantly in cervical SC tissues,and the overexpression of miR-375 inhibited the proliferation,migration and invasion of cervical SiHa and CaSki cells.However,the role and regulatory mechanisms of miR-375 remain unclear in cervical AC.This project intends to study the expression of miR-375 in cervical AC,analyze the relationship between miR-375 and LNM of cervical AC,and explore the molecular mechanism in the progression of cervical AC,so as to provide a new target for the treatment of cervical AC.The main content of this study includes the following three parts.Part I:The expression of miR-375 and use of Nomogram to predict the risk of lymph node metastasis in cervical adenocarcinomaObject:1.The objective of this study was to detect the expression of miR-375 and explore its relationship with clinicopathological factors,especially LNM in patients with cervical AC.2.A Nomogram was constructed to predict the risk of LNM in cervical AC.Methods:1.The Cancer Genome Atlas(TCGA)database(https://portal.gdc.com)was used to analyze the expression difference of miR-375 in cervical AC and cervical SC.2.This study included 140 patients with cervical AC who underwent surgery in Qilu Hospital of Shandong University from 2009 to 2020,compared with 140 patients with cervical SC and 50 patients who underwent hysterectomy for benign uterine disease in the normal control group.None of the cervical cancer patients received preoperative radiotherapy,chemotherapy or other antitumor therapy.3.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect miR-375 expression.Logistic regression was used to analyze the ability of miR-375 to distinguish between cervical AC and SC.4.The relationship between miR-375 expression and clinicopathological factors was analyzed in patients with cervical AC.Kaplan-Meier method was used to analyze the relationship between miR-375 expression and survival prognosis of patients with cervical AC.5.Univariate logistic regression was used to analyze the clinicopathological factors affecting LNM of cervical AC.Multivariate logistic regression was used to construct a Nomogram model.Internal verification was used to predict the probability of LNM.Results:1.miR-375 was highly expressed in cervical ACThe results of TCGA database analysis showed that the expression of miR-375 in cervical AC was significantly higher than that in cervical SC.qRT-PCR results showed the expression of miR-375 in cervical AC tissues(10.20±13.36)was significantly higher than that in normal cervical tissues(1.64±1.75)(P<0.001).But the expression of miR-375 was significantly decreased in cervical SC tissues(0.79±1.14)(P=0.002).In addition,the expression of miR-375 also showed significant difference between cervical AC and cervical SC.And the expression of miR-375 in cervical AC tissue was significantly higher than that in cervical SC tissue(P<0.001),which was consistent with the results of TCGA database analysis.2.miR-375 had high sensitivity and specificity in distinguishing cervical AC from SCLogistic regression was used to analyze the ability of miR-375 to distinguish cervical AC from SC.The expression level of miR-375 was analyzed between the cervical AC and cervical SC tissue.The results showed that the area under(AUC)of receiver operating characteristic(ROC)curve was 0.865,the sensitivity was 87.1%,and the specificity was 66.4%(P<0.001).3.miR-375 was associated with LNM of cervical AC,but had no significant relationship with prognosisMiR-375 expression was significantly correlated with LNM(P=0.009),interstitial infiltration depth(P=0.006),and FIGO stage(P=0.001),but not with age,differentiation,lymphaticvascular space invasion(LVSI)and tumor size of cervical AC.Kaplan-Meier survival analysis showed that there was no significant difference in survival time between patients with high and low expression of miR-375(P>0.05).4.The predictors of LNMUnivariate analysis showed that miR-375,lymphovascular infiltration and interstitial infiltration depth were significantly correlated with LNM,while tumor grade,FIGO stage,vaginal involvement,patient age and laparoscopic surgery were not correlated with LNM.5.The Nomogram model to predict the risk of LNMBased on the results of this study,a Nomogram model was established using miR-375,LVSI and interstitial invasion as predictive variables.The Nomogram model was developed by combining these three prediction factors and had good prediction ability(AUC=0.779,sensitivity 85.3%,specificity 65.1%,P<0.001).Internal cross-validation yielded a consistency index(C-index)of 0.779.Decision curve analysis showed that this Nomogram model had high clinical practicability in predicting LNM.Conclusions:1.miR-375 is highly expressed in cervical AC tissues.MiR-375 has high sensitivity and specificity for distinguishing cervical AC from cervical SC,and is expected to be a molecular indicator for distinguishing the two.2.miR-375 is associated with LNM,the depth of interstitial invasion and FIGO stage of cervical AC,suggesting that miR-375 may be involved in the process of invasion and metastasis of cervical AC.3.A Nomogram model based on miR-375 and clinicopathological parameters is developed to effectively predict the risk of LNM in cervical AC.Part Ⅱ:Effects of miR-375 on biological behavior of cervical adenocarcinomaObject:The abnormal expression of miR-375 is closely related to the tumorigenesis and tumor progression in various tumors.But the expression level and function of miR-375 are not the same.Based on the first part of the study,we found that miR-375 was highly expressed in cervical AC,which was correlated with LNM,the depth of interstitial infiltration and FIGO stage.In this part,the influence of miR-375 was further explored on the biological behavior of cervical AC cells.Methods:1.qRT-PCR was used to detect the expression of miR-375 in cervical epithelial immortalized cells H8 and cervical cancer cell lines HeLa,SiHa and CaSki.2.HeLa of cervical AC cell line was to construct with stable transfection overexpression and interference of miR-375.3.CCK-8 assay and EdU assay were used to detect the effect of miR-375 on the proliferation of HeLa cells.4.Flow cytometry was used to detect the effect of miR-375 on apoptosis of HeLa cells.5.Scratch assay and Transwell assay were used to detect the effects of miR-375 on the migration and invasion ability of HeLa cells.6.Immunofluorescence and acridine orange staining were used to observe the effect of miR-375 on autophagy of HeLa cells.7.Western Blot was used to detect the effects of miR-375 on autophagy and EMT-related molecules.8.The effect of miR-375 on the growth of cervical AC line HeLa cell transplantation was verified by tumor formation in nude mice.Results:1.The expression difference of miR-375 in cervical cancer cellsThe results of qRT-PCR showed that miR-375 was significantly higher expressed(P<0.001)in HeLa(2.18±0.03)and significantly lower expressed(P<0.001)in CaSki(0.31±0.01)and SiHa(0.64±0.11)compared with H8 cells(1.00±0.03).This result was consistent with the expression pattern of miR-375 in cervical AC tissues and cervical SC tissues.Therefore,HeLa,a cervical AC cell line,was selected for follow-up experiments.2.miR-375 promoted the proliferation,migration and invasion of HeLa cellsBoth CCK-8 and EdU experiments showed that the overexpression of miR-375 significantly promoted HeLa cell proliferation,while the silencing of miR-375 significantly inhibited HeLa cell proliferation compared with the normal control group.Flow cytometry showed that miR-375 had no significant effect on HeLa cell apoptosis.Scratch test results showed that the migration area of HeLa cells was markedly greater than that of the normal control group after miR-375 overexpression.After miR-375 silencing,the migration area of HeLa cells was markedly less than that of the normal control group.Transwell assay showed that the subventricular HeLa cells were significantly more than the normal control group after miR-375 overexpression.After miR-375 silencing,the subventricular HeLa cells were significantly less than the normal control group.3.miR-375 promoted the autophagy of HeLa cellsImmunofluorescence results showed that the positive expression of LC3B was significantly higher than that of the normal control group after overexpression of miR-375.After miR-375 silencing,the positive expression of LC3B was significantly lower than that of the normal control group.Acridine orange staining showed that compared with the normal control group,the red fluorescence intensity of HeLa cells was enhanced after the overexpression of miR-375,indicating more acidic vesicle organelles and enhanced autophagy degree of HeLa cells.After miR-375 silencing,the red fluorescence intensity in HeLa cells was weakened,indicating that the acidic vesicle organelles were reduced and the autophagy of HeLa cells was weakened.4.miR-375 regulated the expression of proteins associated with autophagy and EMTWestern Blot results showed that after miR-375 overexpression,the expression of epithelial marker E-cadherin decreased significantly,while the expression of mesenchymal markers(Beclinl,ATG5,N-cadherin,MMP-2 and MMP-9)increased significantly.After miR-375 silencing,E-cadherin expression was significantly increased,while Beclinl,ATG5,N-cadherin,MMP-2 and MMP-9 expression were decreased to varying degrees.5.miR-375 promoted tumor growth in vivo experimentsSubcutaneous tumor formation in nude mice showed that compared with the normal control group,the tumor volume and weight were significantly increased in the miR-375 overexpression group,while significantly decreased in the miR-375 silencing group.Conclusions:MiR-375 promotes the proliferation,autophagy and invasion of cervical AC cells,and plays a role in promoting cancer AC.Part Ⅲ:Study on the mechanism of miR-375 promoting the progression of cervical adenocarcinomaObject:Epithelial-mesenchymal transition(EMT)plays an important role in tumor invasion and metastasis.Autophagy has been shown to be involved in the regulation of cell adhesion dynamics and EMT,which is involved in cell migration and tumor metastasis.MiRNAs are involved in multiple processes of autophagy regulation,and are also major regulators of EMTs.Based on the second part of the study,we found that miR-375 promoted the proliferation,autophagy and invasion of cervical AC cells,and plays a cancer-promoting role in cervical AC,but its specific mechanism of action remains unclear.In this part,the mechanism of miR-375 was further explored related to autophagy and EMT in cervical AC.Methods:1.The potential targets of miR-375 were predicted using Targetscan、miRWalk、miRanda and RNAhybrid,and verified by double luciferase assay.2.qRT-PCR was used to detect the expression of miR-375 and CDH1 in 30 cervical AC tissues and normal cervical tissues,as well as the expression of CDH1 in HeLa cells.Pearson correlation was used to analyze the relationship between CDH1 and miR-375 expression in cervical AC.qRT-PCR was used to detect the expression of CDH1 in HeLa cells after miR-375 overexpression and silencing.3.HeLa cell lines were stably transfected by overexpression and interference with miR-375 and CDH1.In order to determine whether CDH1 can reverse the biological behavior changes of HeLa cells induced by miR-375,HeLa cells were further transfected with miR-375 overexpression vector and CDH1 overexpression vector.In order to study the effect of autophagy,the autophagy inhibitor 3-MA was added to the miR-375 overexpression group and the CDH1 silencing group,respectively.Therefore,the experiment was divided into five groups of cells:miR-375 overexpression group(miR-375),miR-375 overexpression+CDH1 overexpression group(miR-375+CDH1),miR-375+3-MA,CDH1 silencing group(CDH1 shRNA)and CDH1 shRNA+3-MA.4.CCK-8 and EdU assay were used to detect the effects of miR-375 on the proliferation ability of HeLa cells.Scratch assay and Transwell assay were used to detect the effects of miR-375 on the migration and invasion ability of HeLa cells.Immunofluorescence and acridine orange staining were used to observe the effects of miR-375 on the autophagy of HeLa cells.5.The Rescue experiment was verified that the effect of miR-375 on cervical AC was to realize the regulation of autophagy and EMT-related molecules expression through CDH1.6.The effect of miR-375 on the growth of cervical AC was verified by tumor formation in nude mice.Immunohistochemistry was used to detect the expressions of Ki67 and LC3B in tumor tissues.Western Blot was used to detect the effects of miR-375 on autophagy and EMT-related molecules in tumor tissues.7.HeLa cells overexpressing miR-375 were co-incubated with proteasome inhibitor(MG132)and lysosome inhibitor chloroquine(CQ),respectively,and then treated with protein synthesis inhibitor cycloheximide(CHX).Western Blot was used to detect E-cadherin expression.8.HeLa cells overexpressing miR-375 were transfected with small interfering ATG5(si-ATG5)to verify the effect of ATG5 on E-cadherin autophagic lysosome pathway degradation.Results:1.CDH1 was the downstream target gene of miR-375The downstream target gene CDH1 of miR-375 was screened by Targetscan,miRWalk,miRanda and RNAhybrid databases.The predicted results showed that miR-375 had a binding site to the 3’-UTR of CDH1 gene.The results of double luciferase experiment showed that compared with 3’-UTR-NC+miR-375 group(1.00±0.02)and mutant CDH1 3’-UTR+miR-375 group(0.80±0.01),the luciferase activity in cells transfected with wild-type CDH1 3’-UTR and miR-375 plasmid was significantly decreased(0.57±0.02,P<0.001).The results indicated that CDH1 was a downstream target gene of miR-375.QRT-PCR results showed that CDH1 expression was low in cervical AC tissues(0.34±0.28,P<0.001)compared with normal cervical tissues(1.12±0.53).In addition,CDH1 was significantly lower expression in HeLa cells(0.76±0.02,P=0.001)compared with H8 cells(1.00±0.05).There was a negative correlation between CDH1 and miR-375 expression in cervical AC tissues(r=0.943,P<0.001).The results of qRT-PCR showed that compared with the normal control group(1.00±0.01),the expression of CDH1 was significantly down-regulated(0.41±0.01,P<0.001)after miR-375 overexpression in HeLa cells.Compared with the normal control group(1.02±0.03),the expression of CDH1 was significantly up-regulated(7.40±0.07,P<0.001)after silencing miR-375.2.miR-375 promoted the proliferation,migration and invasion of HeLa cells through CDH1CCK-8,EdU,scratches and Transwell experimental results showed that the proliferation,migration and invasion ability of HeLa cells were significantly decreased compared with miR-375 overexpression group when CDH1 expression was further up-regulated on the basis of miR-375 overexpression.The proliferation,migration and invasion of HeLa cells were significantly decreased when 3-MA was added to miR-375 overexpression group and CDH1 silencing group,respectively.3.miR-375 promoted the autophagy of HeLa cells through CDH1Immunofluorescence and acridine orange staining results showed that the overexpression of miR-375 could promote LC3B expression and HeLa cell autophagy in the second part of the experiment.When CDH1 expression was further up-regulated on the basis of miR-375 overexpression,LC3B positive expression rate was significantly decreased,the acidic vesicle organelles were reduced,and the autophagy degree of HeLa cells was weakened compared with miR-375 overexpression group.Adding 3-MA to the miR-375 overexpression group and the CDH1 silencing group,respectively,the expression of LC3B significantly decreased and HeLa cell autophagy was inhibited.4.miR-375 regulated the expression of autophagy and EMT-related proteins through CDH1Rescue experiment was used to further verify whether miR-375 could regulate the expression levels of autophagy and EMT-related proteins through CDH1.HeLa cells were transfected with miR-375 and CDH1 overexpression or interference vectors,and were divided into four groups of cells:miR-375+/CDH1+5 miR-375+/CDH1-,miR-375-/CDH1+,and miR-375-/CDH1-.The results showed that E-cadherin expression was significantly decreased,while the expressions of N-cadherin,MMP-2,MMP-9,Beclinl and ATG5 were significantly increased when CDH1 was down-regulated in miR-375 overexpressed cells.When CDH1 was up-regulated in miR-375 down-regulated cells,E-cadherin expression was significantly increased,while the expressions of N-cadherin,MMP-2,MMP-9,Beclinl and ATG5 were significantly decreased.These results indicate that miR-375 can regulate CDH1-mediated autophagy and EMT-related proteins expression.In addition,adding 3-MA to miR-375 overexpression group and CDH1 silencing group,respectively,the expression of E-cadherin significantly increased and the expression of N-cadherin,MMP-2,MMP-9,Beclinl and ATG5 significantly decreased.5.miR-375 promoted tumor growth through CDH1 in vivo experimentsThe results of subcutaneous tumor formation in nude mice were as follows.When CDH1 expression was further up-regulated on the basis of miR-375 overexpression,tumor volume and weight,Ki67 and LC3B expression rates were significantly decreased compared with miR-375 overexpression group.Adding 3-MA to miR-375 overexpression group and CDH1 silencing group.respectively.tumor growth was inhibited and the expression of Ki67 and LC3B significantly decreased in tumor tissues.The results of Western Blot analysis were as follows.Compared with the normal control group.miR-375 overexpression could significantly reduce the expression of E-cadherin and increase the expression of N-cadherin.MMP-2,MMP-9.Beclin1 and ATG5 in tumor tissues.When CDH1 expression was further up-regulated on the basis of miR-375 overexpression.the expression of E-cadherin was significantly increased,while the expressions of N-cadherin.MMP-2.MMP-9.Beclinl and ATG5 were significantly decreased.Adding 3-MA to miR-375 overexpression group and CDH1 silencing group,respectively,the expression of E-cadherin significantly increased and the expression of N-cadherin,MMP-2,MMP-9,Beclinl and ATG5 were inhibited.6.miR-375 regulated the autophagic lysosomal degradation of E-cadherinCompared with the normal control group,the level of E-cadherin was still significantly decreased in the presence of CHX(a protein synthesis inhibitor)in HeLa cells overexpressing miR-375.HeLa cells overexpressed with miR-375 were incubated with MG132(a proteasome inhibitor)and DMSO(dimethyl sulfoxide)for 2 h respectively,and then treated with CHX.There was no significant difference in E-cadherin expression between the MG132 group and the DMSO group.HeLa cells overexpressing miR-375 were incubated with CQ(a lysosome inhibitor)and PBS(phosphate buffer saline)for 12 h respectively,and then treated with CHX.The expression of E-cadherin showed significant differences between the CQ group and the PBS group.The results showed that miR-375 accelerated the degradation of E-cadherin through the autophagic lysosome pathway.7.miR-375 regulated the autophagic lysosomal degradation of E-cadherin through the ATG5 pathwayThe second part of the experiment confirmed that miR-375 could promote the expression of ATG5 in HeLa cells.HeLa cells with stable overexpression of miR-375 were transfected with si-ATG5.Western Blot analysis showed that compared with the miR-375 overexpression group,the expression of ATG5 were significantly decreased and the expression of E-cadherin were significantly increased after further knockdown of ATG5.In addition,HeLa cells overexpressed with miR-375 and HeLa cells transfected with miR-375 and si-ATG5 were treated with CHX,respectively.Western Blot analysis showed that in the presence of CHX,the expression of ATG5 was significantly decreased and the expression of E-cadherin was significantly increased after further knockdown of ATG5 in HeLa cells overexpressing miR-375.Experiments showed that miR-375 could regulate the autophagic lysosomal degradation of E-cadherin through ATG5 pathway.Conclusions:1.miR-375 promotes the proliferation,autophagy and invasion of cervical AC cells through negative regulation of CDH1 gene.2.miR-375 promotes autophagic lysosomal degradation of E-cadherin through the ATG5 pathway.3.miR-375 plays a cancer-promoting role in cervical AC through EMT and can be used as a potential therapeutic target for cervical AC. |
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