Study Of Notch Signaling Pathway On Carcinogenesis Of Cervical Cancer

Objective: Cervical cancer is the second most common cancer in women worldwide, with only a lower incidence of breast cancer. The reason of cervical cancer is relatively clear, and human papillomavirus(papillomavirus human, HPV), especially HPV16 and 18 has been identified as high risk factors. HPV E6 and E7 oncogenes integrate into host cells with p53 and p Rb binding respectively, which leads to the two kinds of anti oncogene inactivation, alters the cell growth cycle regulation, and causes the host cell immortalization, and eventually causes cervical cancer.It is found that the Notch signaling pathway plays a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and is related to the occurrence of human malignant tumors. Notch1, which was detected in the study of cervical cancer several decades ago, suggests that it may play a role in cervical cancer. Notch receptor and its ligand Jagged1 protein levels were increased in the process of cervical precancerous lesions to invasive cancer. Notch1 cooperate with HPV E6 and E7 gene to promote malignant squamous epithelial cells of cervix. Studies have also indicated that Notch1 may inhibit cervical cancer. The two different functions of Notch1 may be related to the concentration of Notch1 and the different stages of cervical lesions. Activation of Notch signal plays a key role in start of epithelial mesenchymal transition(EMT). Epithelial cells are ransformed into mesenchymal charactericstics in the process of EMT, which reduces intercellular adhesion ability and increases the movement ability with the results of increasing cell migration, inhibiting cell apoptosis and enhancing the invasion, and have the relationship among occurrence of tumor, invasion and metastasis closely.In addition, the Notch signal interact with other signals, such as Wnt/?-catenin, TGF-?, PI3K/Akt through different mechanisms to regulate the Notch signaling pathway.Methods:1 Establishing xenografts in nude mice. First, HPV16 positive SiHa cells were cultured in vitro. We amplified and extracted miRNA interference plasmids, including HPV 16E6-miRNA, HPV16 E7-miRNA interference plasmids and Neg-miRNA blank plasmids as negative control. The SiHa cells were inoculated into subcutaneous of the armpit near the outside of the right upper limb in nude mice. When the tumor volume was about 80~120mm3, 32 nude mice were divided into four groups according to the volume tumor, including two control groups, namely saline control group and the empty plasmid group(Neg-miRNA group) and two treatment groups, namely HPV16 E6 gene silencing group(HPV16 E6-miRNA group) and HPV16 E7 gene silencing group(HPV16 E7-miRNA group). Using transfection in vivo, xenografts of four groups in nude mice were respectively injected with normal saline, Neg-miRNA, HPV16 E6-miRNA, HPV16 E7-miRNA respectively every two days, at least 9 times in total. We observed xenografts growth and measured its volume before each injection. We killed nude mice 48 hours later when we gave the last injection, then measured the tumor volume size and weight and packed specimens. Then extracted RNA and detected the mRNA level of the target gene of xenografts using Real-Time PCR. Detect indicators including Notch1, Jagged1, PI3 K, p-AKT, NF-?B.2 Collecting cervical tissues from the gynecological patients who hospitalized in the Second Hospital of Hebei Medical University during March 2014 to December 2015 and underwent cervical biopsy or surgery because of cervical lesions, then picked the samples into 4% formaldehyde and placed into fridge. There were 43 preinvasive lesions, including 16 LSIL and 27 HSIL and 21 cervical cancer tissues without chemotherapy before surgery, including 18 squamous cell carcinoma(SCC) of the cervix and 3 adenocarcinoma(AC) of the cervix. There were also 8 cervical cancer tissues from patients who received neoadjuvant chemotherapy(NAC) before surgery, including 5 squamous cell carcinoma, 2 adenocarcinoma and 1 adenosquamous carcinoma of cervix. There were 6 patients diagnosed stage IA, 13 patients diagnosed stage IB, and 10 patients diagnosed stage IIA cervical carcinoma according to the 2009 FIGO. In the meantime, 10 gynecological patients who underwent hysterectomy because of uterus myoma and/or adenomyosis were collected as the control group with negative results of TCT and HPV before surgery and no abnormal results after pathological test. All cases were treated with Roche Cobas 4800 HPV detection system, by Real-Time PCR of 14 kinds of high-risk HPV DNA for amplification and detection, including HPV16, 18 and the rest of the 12 kinds of subtype(31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). The basic information, HPV results, and the complete pathological results of all patients were collected. The protein expression of the target gene was detected by immunohistochemical method. Detection indicators include Notch1, Jagged1, PI3 K, p-AKT and NF-?B. Then we analyzed the effects of Notch signaling pathway on the pathoge- nesis of EMT and cervical cancer.Results: 1 Successful establishment of xenografts of nude mice 1.1 Nude mice all survived. The tumorigenic incubation was about 30 days when the volume of xenografts reached about 80~120mm3. We could see obvious xenografts with irregular shape,and individual tumor could appear ulceration and bleeding. Then we peeled the tumor easily and saw hard tumor. White section of xenografts showed necrotic foci, and no obvious vessels. The tumor rate was 100%. 1.2 HPV16 E6 and E7 genes were silenced by transfection in vivo. The tumor volume and weight of the two treatment groups in the nude mice were less than two control groups(P<0.05). 1.3 Expression of mRNA of Notch1, Jagged1, PI3 K, p-AKT and NF-?B of xenograftsWe detected the expression of mRNA in 4 groups of xenografts by Real-Time PCR. The expression of Notch1, Jagged1, PI3 K, p-Akt and NF-?B mRNA in HPV16 E6 gene silencing group and HPV16 E7 gene silencing group were all downregulated comparing with the empty vector group and saline control group(P<0.05). Compared with the saline control group, the expression of Notch1, Jagged1, PI3 K, p-Akt and NF-?B mRNA of empty vector group has no significant difference(P>0.05). The Notch signal transduction pathway was inhibited by HPV16E6/E7 gene silencing in xenografts of nude mice. 2 Analysis of human cervical samples 2.1 The rate of high-oncogenic risk HPV(hr-HPV) infectionThe rate of hr-HPV increased in the progression of cervical lesions. Normal control group had no HPV infection; the rate of hr-HPV infection in LSIL was 1.25%(2/16); the rate of hr-HPV infection in HSIL was 88.89%(24/27); and the rate of hr-HPV infection in cervical carcinoma was 100%(29/29). The hr-HPV infection was mainly HPV16 positive(45/55), secondly HPV18(4/55). 2.2 Expression of human cervical tissue samples Notch1, Jagged1, PI3 K, p-AKT and NF-?BThe expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B protein in normal and LSIL was less than HSIL and cervical carcinoma. The difference of expression of those protein was significant(P<0.05). The expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B increased in the process of development of cervical lesions and cervical cancer.The difference of expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B between SCC with NAC and without NAC were significant(P<0.05). We could not analyse the difference of expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B between AC with NAC and without NAC because the number of AC with NAC was too small, only 2 cases.Those results might explain that the expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B were related to chemo among SCC, but had no effect on AC.Conclusions:1 Establish xenografts of nude mice by SiHa cells successfully and prove that HPV16 E6-miRNA and HPV16E7-miRNA can effectively inhibit the growth of xenografts. RNA interference(RNAi) can be used for studies of cervical cancer in vivo.2 Down-regulating of HPV16 E6/E7 gene can inhibit Notch signal pathway through RNAi. Notch signal and HPV16 E6/E7 gene has a synergistic effect.3 The expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B increased in the process of development of cervical lesions and cervical cancer. Notch signal pathway is involved in the occurrence of cervical cancer.4 Preoperative neoadjuvant chemotherapy can reduce the expression of Notch1, Jagged1, PI3 K, p-AKT and NF-?B proteins in SCC tissues, but may have no significant effect on the expression of those proteins in AC tissues.