HBXIP And KLK7 Represent The New Markers To Assessment Prognosis And Treatment Evaluation For Endometrial Adenocarcinoma And Cervial Cancer

Endometrial carcinoma is one of the three malignant tumors of female genital tract,accounted for 7% female malignant tumors.Studies have shown that there are two possible pathogenesis: estrogen dependent and estrogen independent.HBXIP is an important protein in the process of tumor.Hepatitis B X—interacting protein(HBXIP),which plays an important role in manycellular activities,such as apoptosis and mitosis,It was named according to that it cail bind to the C terminal of hepatitis B virus X protein(HBx)specifically.since HBXIP gene was first cloned in 1998,there are not many reports.Its physiological function is unclear.As the research develop thorough,studys focous on HBXIP.HBXIP gene is expressed almost all kinds of organizations of higher animals but high expressied in muscle and malignant tumor tissues.With baculovirus IAP(restrain apoptosis protein)HBXIP regulates cell division and Leads to apoptosis.Combined with a variety of proteins in the human body,HBXIP can also participate in cell apoptosis,proliferation,cell cycle progression,centrosome replication,and tumor cell migration process.Being a regulatory proteins,HBXIP,can impact gene expression by adjusting different transcription factors.HBXIP also participates in the m TORC1 signaling pathways and amino acid metabolism.HBXIP is high expressed in a wide variety of tumor cells.By inducing Hep G2 PI3K/AKT signal pathway activation and increasing the expression of Cyclin D1 in liver cancer cell,it can promote the growth of tumor cells.Being excessively expressed,HBXIP can cause the cancer angiogenesis.The expression of HBXIP and S100A4 was positively correlated,in breast cancer tissue.The study showes that HBXIP can promote the growth and metastasis of breast cancer cells by raiseing the expression of S100A4.In breast cancer cells,Through activation of transcription factor E2F1 and raiseing S period kinase associated protein 2(Skp2),HBXIP can promote tumor cell growth.By passing the activation TFIID and raiseing Lin28 B,HBXIP promotes breast tumor cell growth.Through activation of transcription factors Sp1 accidentally HBXIP can raise PDGFB,HBXIP can promote the growth ofbreast cancer cells.In addition,as survivin antiapoptotic cofactors,HBXIP can cause caspase-9 precursor activation inhibition.Currently The expression and mechanism of HBXIP in endometrial carcinoma is not reported.The formation and rapid progress of endometrial cancer significantly reduce the patient’s survival.In the process of identificating precancerous lesions and carcinoma,Definiting tumor pathological stage,guiding accurate treatment and valuating prognosis processes molecular markers which have been found constantly provide a strong support.Fei found that HBXIP could regulate the sensitivity of the Mc F-7 cell line to etoposide,which was one of the targets for human lung cancer treatment.I also found that kallikrein 7 was closely related to the surgical staging of cervical cancer,which may be one of the important markers of endometrial cancer.At present,the research of molecular markers of endometrial cancer is deepening,and new molecular markers are being discovered constantly.The PIK3 CA gene mutation leaded to the activation of PI3K/AKT/m TOR pathway in Progression of endometrial cancer.As one of the iconic antigens,Ki-67 exists commonly in the proliferating nuclei,which is an important marker of endometrial cance.Based on the molecular pathogenesis of endometrial cancer,Deep exploration molecular markers,can help us to further discuss the pathogenesis of endometrial carcinoma and the possibility of intervention in the critical steps.During evaluating the prognosis and treatment of endometrial carcinoma,it is an important task to study the molecular mechanism and to find molecular markers.Cervical cancer is the third most common cancer for women worldwide and the second most frequent cause of cancer-related death in many developing countries.In fact,the annual global statistics of WHO estimated 528,000 new cases and 266,000 deaths from cervical cancer around the year 2012.Although early detection and effective treatment for cervical cancer have been used and have been improved recently,the clinical outcome of patients remains unsatisfactory.The accurate diagnosis and prognosis in cervical cancer patients can make effective and timely decisions for appropriate therapy,and also reduce unnecessary costs associated with treatment.Therefore,it isnecessary to identify new genes and molecules directly associated with the progression and prognosis of cervical cancer.Human tissue kallikrein(KLK1)and the kallikrein-related peptidases(KLK2–15)are secreted serine proteases,encoded by a group of genes tandemly localized on chromosome 19q13.3-4.Kallikrein are mainly secreted by epithelial cells and are involved in numerous physiological processes,including extracellular-matrix remodel-ling,skin shedding,prohormone processing,neural plasticity,activation of growth factors,digestion of growth factor binding proteins,and electrolyte balance.Ample evidence suggests that members of the kallikrein family members are differentially produced in several cancer types.Moreover,expression of kallikrein family members are associated with clinicopathological parameters of patients with ovarian,breast,gastric,colorectal,pancreatic,hepatic,and esophageal cancer.In particular,numerous clinical studies have linked the differential expression of KLK family members to their potential roles as cancer biomarkers.Human tissue kallikrein 7(KLK7),also known as human stratum corneum chymotryptic enzyme,was first identified in human skin extracts.Although it has been shown that KLK7 is involved in the degradation of several components of the extracellular matrix(ECM),its roles in tumor remain unclear.To date,accumulating evidence demonstrated that KLK7 transcript and/or KLK7 protein are overexpressed in a variety of tumors including breast,ovarian,and cervical cancer.Talieri M,et al.reported that breast cancer patients with KLK7 positive tumors have relatively shorter disease-free survival(DFS)and overall survival(OS)than patients with KLK7 negative tumors.Shan SJ,et al.suggested that KLK7 is associated with some unfavorable characteristics of ovarian cancer in univariate analysis.Reportedly,a significantly higher KLK7 expression rate was observed in cervical cancer compared to normal endocervical glands,and might play an important role in cervical cancer development.The main results were followed:Paper OneTo detect the expression of HBXIP in fresh tissues of endometrialObjective: To detect the expression of HBXIP in fresh tissues of endometrialadenocarcinoma.To investigate the expression of human HBXIP in endometrial adenocarcinoma and its influence on prognosis.To analyze the relationship between the expression of HBXIP and FIGO stage,survival time and clinical pathological data,so as to provide new thoughts for improving prognosis of endometrial carcinoma.Construction of HBXIP gene eukaryotic expression plasmid pc DNA3.1-myc-his A-HBXIP was transfected into human endometrial carcinoma HEC-1-A cells,a stable cell line expression of HBXI P protein,and detection of HBXIP gene in human endometrial carcinoma HEC-1-A cells.Methods: Western blotting were used to detect the expression of HBXIP in 30 cases of endometrial carcinoma,10 cases of atypical endometrical hyperplasia and 20 cases with normal endometrium.Immunohistochemical methods were used to detect the expression of HBXIP in 136 cases of endometrial adenocarcinoma,10 cases of atypical endometrical hyperplasia and 20 cases with normal endometrium.(Endometrial adenocarcinoma period Ⅰ 56 cases,period Ⅱ 43,periodⅢ 27 cases and period Ⅳ 10 cases)(Myometrial invasion ≥1/2 58 cases,Myometrial invasion <1/2 78 cases),Immunohistochemical methods and Analyze HBXIP expression and correlation of endometrial carcinoma.Clinicpatiohological information of cases were collected and survival analysis was analyzed combining follow-up data.The relationship between the expression of HBXIP and prognosis of endometrial carcinoma was analyzed.Before operation,the patients had not been treated with radiotherapy,chemotherapy and hormonal therapy.Specimens were diagnosed by pathology review.Full-length HBXI fragment was amplified by polymerase chain reaction(PCR),it was subcloned into theeukaryotic expression vector pc DNA3.1-myc-his A to construct recombinant plasmid pc DNA3.1-myc-his A-HBXIP.Analysis nucleotide sequence and DNAsequencing confirmed that the recombinant plasmid pc DNA3.1-myc-his A-HBXIP insert the correct nucleotide sequence of HBXIP.Liposomepc DNA3.1-myc-A-HBXIP transfected HEC-1-A cells and used Westem blot to detect protein expression of HBXIP in human endometrial carcinoma HEC-1-Acells.Cell migration and invasion was determinedby the transwell migration and invasion assays respectively.Result: The difference of the high expression rate of HBXIP in normal endometrium,atypical endometrical hyperplasia and endometrial adenocarcinoma was Statistical significance(P<0.0 01)To detect the expression of HBXIP in 8 cases of fresh tissue of endometrial adenocarcinoma,8 cases of fresh tissue of atypical endometrical and normal endometrium,7 cases of HBXIP expression in cancerous tissue increased obviously.The high expression rate of HBXIP in normal endometrium,atypical endometrical hyperplasia and endometrial adenocarcinoma was 20%,60%,84% respectively and the difference was significant(χ2=11.013 P=0.002).High expression of HBXIP was positively correlated with FIGO stage,histological grade,Lymph nodes metastasis and Myometrial invasion of endometrial adenocarcinoma,the difference was significant(P<0.0 01);but not correlated with age.(P >0.001).Five-year progession free survival(PFS)rate of the high HBXIP expression group was lower than the low HBXIP expression group with significant difference(P<0.001);High expression of HBXIP,FIGO stage and Myometrial invasion depth was statistically significant influenced on Over survival(P<0.001);Myometrial invasion depth was more closely with OS;The influence of Different Lymph node metastasis on OS had statistical significance.(P=0.001).In the extraction process total RNA of HBXIP gene did not occurred significant degradation,the total degradation of NA integrity and non—degradable.It showd that total RNA and Mrna of HBXIP gene are integrated.Product of PCR showed a clear specific band at 276 bp and there was the correct size consistented with the length to the estimated c DNA.The eukaryotic expression plasmid of pc DNA3.1-myc-his A-HBXIP was successfully constructed.HBXIP gene fragmented in eukaryotic expression vector pc DNA3.1-myc-his A-HBXIP was corrected by digestionand PCR.To detect the expression of HBXIP protein in human endometrial carcinoma HEC-1-A cells by Western blot the pc DNA3.1-myc-his A-HBXIP group and the group pc DNA3.1-myc-his A,relative expression of HBXIP protein,the differencewas statistically significant(P<0.05).Transwell migration assay showed ectopic expression of HBXIP enhanced cell migration;The invasive capacity of HEC-1-A cells was significantly enhanced upon transfection with HBXIP plasmid compared to control and untreated groups;Used HBXIP si RNA to transfect HEC-1 A cell line,Western blotting tested the expression levels of HBXIP and MMP9,After HBXIP gene was silenced,Expression of MMP9 lessed,Overexpressed HBXIP enhanced the expression of MMP9.Conclusion: From normal endometrium to the progress of the endometrial adenocarcinoma.the expression of HBXIP intensity gradually increased.High expression of HBXIP in endometrial adenocarcinoma was possibly correlated differentiation and local invasion of cancer cells.High expression indicated early recurrence.HBXIP may be used as a marker to predict early recurrence of endometrial adenocarcinoma.HBXIP gene was successful constructed and transformed into the eukaryotic expression vector pc DNA3.1-myc-his A plasmid cloning site 2.recombinant plasmid pc DNA3.1-myc-his A-HBXIP constructed with HBXIP gene transfected HEC-1-A cells.The stablely expression in HEC-1-A cells made the further study of the foundation for HBXIP 3.HBXIP could suppress cell migratory and invasive ability of endometrial carcinoma cells 4.HBXIP could regulate the expression of MMP9,Through MMP9,HBXIP may be play an important role in promoting the invasion and metastasis.Paper Two:Objective: Our study was aimed to investigate the diagnostic andprognostic performance of serum human tissue kallikrein 7(KLK7)in cervicalcancer.Methods: The study comprised of 78 patients with cervical cancer,36 patients with benign cervical diseases,and 50 healthy controls.Serum KLK7 levelswere determined using ELISA method.Receiver operating characteristic curve wasperformed to assess the diagnostic performance of serum KLK7.Survival curves bythe Kaplan–Meier method were plotted to display overall survival distributions.Univariable and multivariable Cox regression analysis were performed to assessindependent prognostic factors for overall survival in cervical cancer.Results: Serum KLK7 levels in cervical cancer patients were 3.13 ± 1.99 lg/ml,significantly higher than the values obtained from patients with benign cervicaldisease(1.40 ± 1.14 lg/ml;P < 0.001)and healthy controls(1.27 ± 0.73 lg/ml;P <0.001),respectively.The sensitivity,specificity,positive and negative predictivevalues of serum KLK7 for cervical cancer were 75.6,80.2,77.6,and 78.4 %,respectively.Serum KLK7 levels in patients with cervical cancer were significantlycorrelated with FIGO stage,lymph node metastasis,and stromal invasion.Whenanalyzed with a survival curve,high serum KLK7 level was correlated with poorprognosis.Conclusion: Our findings suggest that serum KLK7 may be a valuablediagnostic biomarker for cervical cancer,and may help to determine the individualprognosis of these patients.