The Expression Of CTHRC1 In Cervical Tissue With HPV16/18 And Its Functional Mechanism In Cervical Cancer Cell Lines

ObjectivesCervical cancer is one of the most common cancers worldwide,but its pathogenesisremains unclear.Extracellular secreted proteins servered as an important component of the tumor microenvironment andplay an important role in the tumor development and progression.In this study, we screened the populations in Shanghai suburbs to analyze the distribution of the high risk type of HPV. We further investigated how HPV infection affects tumor microenvironment of cervical cancer by analysis of the alteration of extracellular secreted proteins baseed on genome-wild transcriptomeanalysis.Among the deregulated extracellular secreted proteins, CTHRC1 is of particular interest to us. This study aims to analyze the expressions of CTHRC1 in cervical tissues with HPV 16/18 and its function in cervical cancer cells to investigated the biological functions and molecular mechanism of CTHRC1 intype HPV16/18 cervical cancer and hope it to become a new and potentialtherapeutic target in cervical cancer.Methods (1)Ten thousand women undergone cervical HPV typing, to ascertain its distribution features and high risk factors, as well as the distribution of high-risk HPV among the patients with cervical cancer and precancerous lesion. (2)To identify the extracellular secreted proteins induced by the viral oncogenes, we silenced endogenous E6 and E7 expression in cervical cancer cells withHPV16/18 by RNA interference (RNAi). (3)We compared the different expressions of CTHRC1 between cervical tissues with HPV16/18 and with HPV negative by RT-PCR. We further compared the different expressions CTHRC1 among the cervicitis, CIN and cervical cancer tissues with HPV 16/18 by RT-PCR and immunohistochemical methods.We analysed the correlationships between CTHRC1 protein expression and clinico-pathological features in cervical cancer patients.(4)CTHRC1 protein was purified by affinity chromatography. Stable CTHRC1 up-regulated and down-regulated expressionHPV positive cell lines were established by transfected with lentiviral vector. The changes in cell proliferation, invasion, migration and resistance to chemotherapy were detected respectively to ascertain the role of CTHRC1 on cervical cellular functions.Test the change in the migration function of cervical cancer cell treated with recombinational CTHRCl proteinafterusing anti-CTHRCl monoclonal antibody.(5)We constructed a luciferase gene reporting system and used phosphorylated signaling pathway microarray to detect the CTHRC1 protein effects on Wnt canonical pathway and non-classical pathway. (6)We detected CTHRC1 serum concentration in patients cervical cancer and CIN with HPV16/18 and evaluate its clinical diagnostic significance.Result(1)The five top HPV types are HPV 52,16,58,18 and 33 in 10,000 women and the majoy HPV types in women with CIN and cervical cancer among them are HPV 16,52 and 18.(2)The five top secreted proreins which are affected by the E6/E7 transcription regulation of microenvironment related significantly are GTF2I,INSL6, BIRC3,PTN,CTHRC1 in turn, wherein CTHRC1 associated with a variety of tumor development, we further validated correlation with cervical cancer, which is the study identified the gene.(3)The results of immunohistochemistry show that the high expression rate of CTHRC1 is far more in the cervical squamous carcinoma and cervical adenocarcinoma (50.49% and 68.96%) than the normal tissue (cervical squamous epithelium 3.33%and glandularepithelium 6.67%and CIN(21.0%) (P< 0.0001,.P< 0.0001,0.0181).In the cervical squamous carcinoma tissues,the expressions intensity of CTHRC1 are positively correlated with clinical stages(p=0.0021),tumor cell grade(p=0.0186),lymphatic node metastasis(p=0.0075), Lymphatic vascular invasion(LVI, p=0.0010), tumor diameter (P<0.0001) and the depth of invasion in cervical stromal (p=0.0001. (4) Wound healing assay showed that compared with control group,Lenti-CTHRC1 cells (HeLa and SiHa) can promote the cell migration,the CTHRC1 protein also significantly promote the cell migration,the migration ability of the lenti-shCTHRCl cell was significantly reduced, the P values were<0.001 there was statistical difference. In vitro invasion and migration assays showed that the number of Lenti-CTHRC1 cells passing through the Matrigel was significantly higher than that the negative control group,And the number of Lenti-shCTHRCl cells passing through the Matrigel per field was significantly lower than that in the lenti-shRNA negative control group,all of the P values were 0.001,there were significantly statistical difference beteween the two groups(the lenti-CTHRC1 groups and the lenti-shCTHRC1 groups). These results suggest that CTHRCl can significantly promote the in vitro invasion and migration ability of cervical cance cells. (5)Two minites after SiHa cellstreated with anti-CTHRC1 function-blocking antibody,we also treated SiHa cells with 20nM CTHRC1 recombinant protein and performedtranswell migration analysis. The result was that the number of cells passing through the transwell chamber was significantly reduced(p<0.001) suggesting that anti-CTHRC1 monoclonal antibody can reverse the role of recombinant CTHRC1 protein in promoting cervical cancer cell migration.(6)MTT assay showed that compared with control group,the cell viability of Lenti-CTHRC1 cells and the Lenti-shCTHRC1 cell had no significantly difference in the presence ofcisplatin or paclitaxel,there was no statistical difference.CCK8 assay showed that the growth rate of the cervical cance cells(HeLa and SiHa)transfected with CTHRC1 and the negeative control group was nostatistical difference, MS751 cell transfected with lenti-shCTHRC1 and the negeative control group was also no statistical difference. Howevre,thetumor formation in vivo showed that the weight volume of the tumor in lenti-CTHRC1 group(0.634±0.327)g were higher than that of control group(0.226±0.160)g, the volume of the tumor in lenti-CTHRC1 group0.340±0.21 (cm3) were aslo higher than that of control group0.13±0.08(cm3),the P values were 0.044 and 0.028 respectively,there were significantly statistical difference beteween the two groups. (7)In the Wnt pathway classic report system,compared with control group, the TOP activities in Hela cells groups which were treated by CTHRC1 proteins of 10 nm,20 nm CTHRC1 protein was obviously reduced,there are significant difference between them(p=0.023and 0.0057,respectively);The similar results were found in SiHa cells groups(p=0.035and 0.0068,respectively).In non-classical Wnt pathway reporting system, compared with control group, the ATF2 activities in Hela cells groups which were treated by CTHRC1 proteins of 10 nm,20 nm CTHRC1 protein was obviously increased.,there are significant difference between them(p=0.0095and 0.0056,respectively).The same phenomenon was observed in SiHa cells while recombinant CTHRC1 proteins treated SiHa cells for 30,120 and 720 min,compared with control group,the expression of Phosphorylated JNKwere increased; on the contrary, GSK3α/β phosphorylation were reduced, there are significant difference statistically (p<0.05).(8)In the group of healthy human, patients with CIN and patients with cervical cancer, the serous CTHRC1 average concentration detected by ELISA were3.21±1.70ng/ml(1.42to7.31ng/m),4.70±3.12ng/ml(1.26to14.15ng/ml)and 7.32±3.51ng/ml (2.97-16.44ng/ml),respectively.There was significantly difference among healthy human, patients with CIN and the cervical cancer group,P value <0.0001 to healthy group and 0.00053 to patients with CIN group, respectively. In the group of cervical cancer group, The preoperative serum CTHRC1 average concentration was 10.36±2.70ng/ml, and ten cases of postoperative serum CTHRC1 concentration was 4.21±1.78 ng/ml. There was significant difference between the two subgroups (P<0.001).The results of ROC curve displayed that if we diagnose high risk cervical cancer by serum CTHRC1 concentration, the Area Under Curve (AUC) was 0.890±0.048. The critical value of diagnosis was4.20ng/ml determined by Youden’s index, with 84% and 80% of sensitivity and specificity, respectively.Conclusions(1)People with type HPV16 and 18 in this district are the key object forthemanagement and research.(2)CTHRC1 is an important secreted protein modulated by HPVE6/E7 in cervical cancer microenvironment.(3)CTHRC1 is closely related with poor prognostic factors in HPV16/18 positive squamous cervical cancer, such as clinical stage, lymphatic metastasis, vessel infiltration, tumor grade, cervical infiltration depth and tumor size. It might be a promising biomarker for cervical cancer prognosis and recurrence. (4)CTHRC1 can promote the invasion and migration of tumor cells in cervical cancer.(5)It may be CTHRC1 in an important regulation mechanism of cervical cancer that It inhibits the canonical Wnt signaling pathway and promote the non-classic Wnt signaling pathway.(6)CTHRC1 might be a potential theraputic target for cervical cancer.