| BackgroundCervical cancer, with approximately0.5million new cases diagnosed and0.28million deaths every year, is the second most cancer among women worldwide. It has been well recognized that persistent infection with high-risk human papillomaviruses (HR-HPVs)such as HPV16,HPV18,HPV31, is the most important risk factor of cervical cancer and HR-HPVs have been reported to be detected in94%-100%of cervical adeno-and adenosquamous carcinomas and up to99.7%of cervical squamous cell carcinomas. HR-HPVs could integrated to host genome and encode two potent viral oncoproteins, E6and E7, which are often regarded as the oncoproteins and are crucial for host cell malignant transformation and immortalization. It is well characterized that HR-HPV E6is able to induce degradation of the tumor suppressor protein p53via the ubiquitin pathway. Besides, E6could promote tumor progression by interacting with other apoptosis related protein such as Bak, c-Myc, FADD and caspase8.HR-HPV E7inhibites pRb, leading to disruption of pRb/E2F complexes and the activation of E2F. E2F is able to enhance the expression of p16, c-Myc, cyclin A/E, p107, p130, CCNA2, MCM7, CCNB1, CCNB2, MSH6, resulting in malignant transformation. MicroRNAs (miRNAs) are a class of small non-coding endogenous RNAs in size of20-25nucleotides that regulate gene expression transcriptionally or post-transcriptionally through the RNAi pathway. Recent studies show that miRNAs mediate important biological activities such as cellular proliferation, differentiation and apoptosis and are involved in the development of a number of diseases. Expression of miRNAs is altered in different kinds of human cancer compared with the adjacent normal tissues by using miRNA microarray analysis. MiRNAs are involved in regulating proliferation, differentiation, apoptosis, cell cycle progression, and consequently they function as oncogenes or tumor suppressors, being related with tumor progression, metastasis and prognosis. Some miRNAs could be oncogenic in one cancer type but tumor suppressive in another type. Thus, miRNAs have been regarded as new tumor biomarkers nowadays.In many kinds of viral infection miRNAs may play a major role in regulating host gene expression or may conversely be used by host cells to control viral biological process. There is no evidence that HPVs produce their own viral miRNAs, however HR-HPVs are able to regulate the miRNAs of host cells, leading to the malignant transformation of cells. For instance, E6could inhibit the expression of miR-23b, miR-218and miR-34via the degradation of p53. And E7promotes the expression of miR-15b, miR-17-92, let-7a-d, let-7i and miR-106by activating E2F. However, the detailed roles of HR-HPV E6/E7to regulate miRNAs in cervical cancer progression need further studies.MiR-27b is a widely functional miRNA and is involved in diabetes, lipid metabolism, cardiovascular disease, virus infection and tumor progression. MiR-27b functions as a tumor suppressor in several kinds of cancers such as Colon cancer, non-small cell lung cancer, oralcancer, lymphoma and esophageal cancer. But in breast cancer and neuroglioma, miR-27b acts as an oncogene. Besides, miR-27b is related with therapy resistance. There are few reports about the roles of miR-27b in cervical cancer, and just one study shows the expression of miR-27b was higher in HR-HPV positive cervical cancer cell lines than in HPV negative cervical cancer cell lines. The mechanism of such aberrant expression is unclear.Peroxisome proliferator-activated receptor gamma (PPARy) is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily and is a reported target of miR-27b. PPARy is linked to diabetes, lipid metabolism, cardiovascular disease, virus infection and tumor progression. MiR-27b inhibits PPARy via its3’untranslated region in adipocytes, macrophages and neuroblastoma. It has been suggested that PPARy functions as a tumor suppressor because activation of PPARy exerts anti-tumor activity by promoting proliferation inhibition, apoptosis and differentiation in a variety of tumors, including cervical carcinoma. In addition, lower expression of PPARy has been detected in cervical carcinoma tissues than in normal cervical tissues.Na+/H+exchanger isoforml (NHE1), which is controlled by PPARy, is an integral membrane transport protein involved in regulating pH and in tumor cells. The activation of NHE1results in cytosolic alkalinization and then drives subsequent hallmark behaviors such as increased growth rate, glycolytic metabolism and both factor-independent and substrate-independent growth, which is essential for the development and maintenance of the transformed phenotype. A recent report has indicated that active PPARy inhibits growth of breast cancer cells by repressing NHE1expression.In our study, we silenced the expression of HPV16E6/E7in HPV16positive cervical cancer cell lines via RNAi technology and found that the expression of miR-27b was downregulated by miRNA micoarray technology. The results showed it is E7that regulates miR-27b-PPARy-NHE1pathway to influence cervical cancer progession. The aim of this study is to provide fresh molecular insights into new biomarker or therapy of cervical cancer.Part I Identification of miRNAs regulated by E6/E7using microarray technologyObjectiveTo identify miRNAs which are regulated by E6/E7using microarray technology.Methods1. HPV16E6/E7in CaSki cells were silenced by siRNA. To test the effect of siRNA, total RNA and protein were extracted followed by Real-time PCR and Western Blot.2. Microarray was used to identify miRNAs which were regulated by HPV16E6/E7.3. The results of microarray were confirmed by Real-time PCR.4. HPV16E6/E7were overexpressed by E6or E7plasmids to further confirm the results of microarray.5. Test the expression of selected miRNAs in CaSki, SiHa cells (HPV16+) and C33A(HPV-) cells by Real-time PCR.Results1. The expression of miR-27b was downregulated after silencing HPV16E6/E7.2. The expression of miR-27b was upregulated after transfecting HPV16E7plasmids.3. The basal level of miR-27b was higher in CaSki, SiHa cells than in C33A cells.Conclusions1. HPV16E7upregulates the expression of miR-27b. 2. The basal level of miR-27b is higher in HPV16positive cell line than HPV negative cell line.Part Ⅱ HPV16E7regulates miR-27b to promote proliferation and invasion of cervical cancerObjective1. To identify the target gene and downstream pathway of miR-27b.2. To investigate the function of miR-27b in cervical cancer cells.3. To explore the mechanism that HPV16E7regulated miR-27b in cervical carcmogenesis.4. To examine altered expression of miR-27b and its downstream genes in HPV16positive cervical carcinoma tissues and para-carcinoma tissues.Methods1. Cervical cancer cells were transfected by miR-27b mimics and inhibitors followed by Real-time PCR to test the expression of miR-27b.2. Cervical cancer cells were transfected by miR-27b mimics or inhibitors followed by Real-time PCR and Western Blot to test the expression of PPARγ and NHE1.3. Cervical cancer cells were transfected by miR-27b mimics or inhibitors followed by CCK8assay to test the proliferation of the cells.4. Cervical cancer cells were transfected by miR-27b mimics followed by transwell invasion assay to test the invasion of the cells.5. Cervical cancer cells were transfected by si-PPARy followed by Real-time PCR and Western Blot to test the expression of PPARy and NHE1.6. Cervical cancer cells were transfected by si-PPARy followed by CCK8assay to test the proliferation of the cells. 7. Cervical cancer cells were transfected by si-PPARy followed by transwell invasion assay to test the invasion of the cells.8. Cervical cancer cells were transfected by HPV16E7plasmids followed by Real-time PCR and Western Blot to test the expression of PPARy and NHE1.9. Cervical cancer cells were co-transfected by HPV16E7plasmids and miR-27b inhibitors followed by Real-time PCR and Western Blot to test the expression of PPARy and NHE1.10. The expression of HPV16E7, miR-27b, PPARy and NHE1in6HPV16positive cervical carcinoma tissues and para-carcinoma tissues were detected by Real-time PCR.Results1. After transfecting miR-27b mimics, the expression of PPARy was inhibited and the expression of NHE1was promoted, while transfection of miR-27b inhibitors resulted in the upregulation of PPARy and downregulation of NHE1.2. The expression of NHE1was enhanced after silencing PPARy.3. The overexpression of HPV16E7led to the inhibition of the expression of PPARy and promotion of NHE1. After co-transfected HPV16E7and miR-27b inhibitors, the weakened effects of E7to PPARγ and NHE1were observed.4. The overexpression of miR-27b and the underexpression of PPARy resulted in the promotion of cell proliferation while the underexpression of miR-27b led to the inhibition of cell proliferation.5. The overexpression of miR-27b and the underexpression of PPARy resulted in the promotion of cell invasion.6. The expression of HPV16E7, miR-27b and NHE1were higher in HPV16positive cervical carcinoma tissues than that in para-carcinoma tissues. Conclusions1. miR-27b inhibits the expression of its target gene PPARy and promotes the expression of NHE1.2. PPAR.y inhibits the expression of NHE1.3. HPV16E7downregulates the expression of PPARy and upregulation of NHE1via the promotion of miR-27b4. miR-27b promotes the proliferation and invasion of cervical cancer cells.5. PPARγ inhibits the proliferation and invasion of cervical cancer cells.6. HPV16E7, miR-27b and NHE1may acts as oncogenes while PPARy functions as a tumor suppressor. |
