The Roles And Mechanisms Of MiR-424and Cul2/E2F1/miR-424Feedback Circuitry In The Development Of Cervical Cancer

Cervical cancer (CC) ranks as the third most common malignancy in women worldwide. More than85%of the global burden occurs in the developing world, where, in most countries, cervical cancer is the most prevalent cause of cancer-related deaths in women. Infection with high-risk human papillomavirus (HR-HPV) is a necessary factor in the development of cervical cancer. The life-time risk of cervical HPV infection can be as high as80%, up to90%are cleared within2years, only5-10%persist and less than1%develop cervical cancer. In addition, a large number of studies have shown that only targeting HR-HPV key oncogenic protein E6/E7expression could not completely deter the malignant phenotype of cancer cells, and so far no therapeutic HPV vaccine is available. Thus, The outcome of HR-HPV infection depends on both viral and host. A clear understanding of the interaction between HPV oncoprotein and key host cellular molecular is essential for developing effective therapeutic approaches for this disease.MicroRNAs (miRNAs) are small noncoding single-stranded RNAs and key regulators of gene expression and modulate up to one-third of all genes though they constitute only about1%of human genomes. miRNAs negatively regulate gene expression at the posttranscriptional level by base pairing with the3’untranslated region (UTR) of their target mRNAs. It is believed that miRNAs participate almost all the important physiological processes, such as development, differentiation, metabolism and death. In parallel, much hard genetic evidence has showed that many miRNAs are strongly implicated in cancers, either as tumor suppressors or oncogenes,and also paly key roles in viral infection, clearance and replication. Compared with gene and protein network, miRNAs, a new powerful tool, have more unique advantages in clarifying the pathogenesis and identifying therapeutic targets of cancers. Therefore, identifying the roles and mechanisms of certain miRNA is likely to bring new breakthroughs to reveal the molecular mechanisms in the development of HR-HPV-induced cervical cancer.Given the importance of miRNAs in cancers, our previous study identified a characteristic miRNA expression profile in cervical carcinoma, in which miR-424was one of the most obviously downregulated miRNAs in the cervical cancer compared with normal cervical tissues. It has been shown that miR-424controls many crucial biological activities, including cellular differentiation and proliferation, cell-cycle progression and angiogenesis, all of which are often perturbed in malignancies. These data highlight the potential pivotal roles of miR-424both in the development and progression of malignancy.In the present study, we explored the tumor-suppressive significance of miR-424both in cervical cancer tissues and cells; further identified the direct functional target which mediates multiple suppressive actions of miR-424on the progression of cervical cancer; then determined the gene or maybe a regulatory loop as the cause of derepression of miR-424; finally investigated the relationship between miR-424and HPV16oncoprotein in cervical cancer. The aim of our study is to provide insight of new mechanism of the development of cervical cancer and obtain experimental evidence for exploring new strategy for cervical cancer therapy and prevention. Part I The expression and role of miR-424in cervical cancerObjectiveTo verify the decreased expression of miR-424in cervical cancer tissues; to analyze the associations of miR-424expression with clinicopathological parameters; and to investigate the biological actions of miR-424in cervical cancer cells.MethodsExpression of miR-424was examined in74cervical normal tissues and147cervical cancer using quantitative real-time RT--PCR (qRT--PCR). The associations of miR-424expression with clinicopathological parameters, including age, clinical stage, pathological grade, SCC-Ag, tumor size, lymph node metastasis, vascular involvement and stromal invasion, were further analyzed in147cervical cancer patients. miR-424mimic or negative control was transfected into two human cervical carcinoma cell lines, Siha and CaSki cells. Cell proliferation experiments were carried out by MTT assay. Cell cycle and apoptosis analysis were conducted by BrdU&7AAD kit and Annexin-V&PI kit, respectively. Cell migration and invasion were detected by wound healing assay and transwell assay.Results1. Expression of miR-424was frequently downregulated in cervical cancer tissues compared with cervical normal tissues.2. Low miR-424level was significantly associated with advanced FIGO stage, poor tumor differentiation, nodal metastasis, vascular involvement and deep stromal invasion.3. Enforced expression of miR-424functionally suppressed cell proliferation, induced apoptosis, blocked G1/S transition, reduced migration and invasion in cervical cancer SiHa and CaSki cells.Conclusions1. Expression of miR-424is frequently downregulated in cervical cancer tissues and associated with clinicopathological characters.2. Enforced expression of miR-424inhibited cell growth by both enhancing apoptosis and blocking G1/S transition, and suppressed cell migration and invasion in human cervical cancer cell lines, implying that miR-424functions as a tumor suppressor in the development of cervical cancer. Part Ⅱ miR-424participate in the progression of cervical cancer by targeting checkpoint kinase1(Chkl) and phosphorylated Chkl (p-Chkl)ObjectiveTo identify the target gene of miR-424and to reveal the mechanism of miR-424in cervical cancer progression.MethodsFirstly, miR-424mimic or negative control was transfected into two human cervical carcinoma cell lines, Siha and CaSki cells. qRT-PCR was used to detect Chkl mRNA level, and western blot was used to detect Chkl and p-Chkl protein level. Luciferase reporter assay combined with site-directed mutagenesis method were used to determine Chkl as the direct target of miR-424. Secondly, knock down Chkl using specific RNAi in SiHa and CaSki cells. Western blot analysis was used to detect Chkl Chk1, p-Chkl and MMP9protein levels. MTT assay was used to examine cell proliferation. Flow cytometry analysis was used to determine cell cycle and apoptosis. Wound healing assay and transwell assay were used to detect cell migration and invasion. Finally, levels of Chkl and p-Chkl expression were examined by immunohistochemistry (IHC) staining in147cervical cancer and74normal tissues that were the same cases for miR-424detection, and the association of Chkl and p-Chkl with clinicopathological parameters and miR-424expression in cervical cancer patients were analyzed, respectively. Concomitantly, expression of Chkl and p-Chkl were also examined by western blot in an extended panel of40cervical cancer samples and20normal tissues. We then analyzed the associations of Chkl, p-Chkl and MMP9with metastasis in four cervical cancer tissues with lymph node metastasis and eight without by western blot.Results1. Transfection with miR-424mimic significantly decreased the levels of Chkl and p-Chkl protein in both SiHa and CaSki cells, but Chkl mRNA level was not changed.2. The pmiRGLO dual-luciferase reporter vector containing fragments of the Chkl3’-UTR was cotransfected with miR-424mimic or miRNA-negative control. A significant reduction of luciferase activity was observed in SiHa cells with miR-424transfection, compared with the negative control. Mutation of the predicted-binding site of miR-424on the Chkl3’-UTR rescued the luciferase activity.3. The siRNA specific for Chkl significantly decreased the level of MMP9protein expression compared with negative controls in both cell lines. siRNA against Chkl produced an anti-proliferative effect compared with non-targeting siRNA control. Moreover, both SiHa and Caski cells transfected with Chkl siRNA presented enhanced cell apoptosis, accompanied by a cell-cycle arrest at the Gl/S transition. In addition, abolition of Chkl expression inhibited the migratory potential of both cell lines. Similarly, siRNA specific for Chkl suppressed invasive behavior of both cell lines.4. IHC staining and western blot showed that Chkl and p-Chkl protein levels were visibly elevated in cervical cancer tissues compared with the normal tissue, respectively. The expression of Chkl or p-Chkl protein was inversely correlated with miR-424expression in221cervical tissues. Increased expressions of Chkl and p-Chkl were positively correlated with poor prognostic factors in cervical cancer patients, as similar as miR-424low expression. The expression of Chkl, p-Chkl and MMP9proteins exhibited a significant increase in cervical cancer tissues with lymph node metastasis compared with tissues without metastasis, and the expression of Chkl and p-Chkl proteins were positively correlated to MMP9expression, respectively.Conclusions1. Overexpression of miR-424inhibits the expression of protein Chkl and p-Chkl at residues Ser345. 2. Chkl is a direct functional target mediating multiple suppressive actions of miR-424on the progression of cervical cancer.3. The tumor oncogenic role of Chkl in the progression of cervical cancer might via upreglating the expression of p-Chkl and MMP9. Part Ⅲ The feedback loop involving Cul2, E2F1, and miR-424is required for the maintenance of miR-424supression in cervical cancerObjectiveHow is miR-424suppression triggered and maintained during the development of cervical cancer? To validate the existence of Cul2/E2F1/miR-424regulatory feedback loop and identify the regulatory feedback, loop that maintains miR-424supression.MethodsE2F1siRNA or siRNA negative control was transfected into SiHa cell, miR-424expression was detected by qRT-PCR. An area of2kb upstream of miR-424containing two E2F1binding sites was cloned into PGL3-Basic vector. SiHa cells were transfected with a firefly luciferase reporter gene construct containing the miR-424regulatory area (wild-type or deletion mutant in the E2F1binding sites) and were lysed and the luciferase activity was measured using the Dual Luciferase Reporter Assay System. In addition, E2F1siRNA or siRNA-NC and the firefly luciferase reporter gene construct containing the miR-424regulatory area were co-transfected into SiHa cells, cell extracts were prepared24h after transfection and the luciferase activity was measured. Chromatin immunoprecipitation (ChIP) was further determined E2F1directly bind miR-424promoter area. Briefly, the chromatin fragments, derived from pcDNA3.1+/E2F1or pcDNA3.1+treated SiHa cells, were immunoprecipitated with5ug of antibody against E2F1. DNA extraction was performed using Pierce ChIP kit. RT-PCR analysis was performed for E2F1binding sites in miR-424regulatory areas using the specific primers. miR-424mimic or negative control was transfected into SiHa cells, Cul2mRNA and protein were detected by qRT-PCR and western blot. Luciferase reporter assay combined with site-directed mutagenesis method were used to determine Cul2as the direct target of miR-424. Transient inhibit Cul2by siRNA or transient over-express Cul2by Cul2plasmid in SiHa cells. miR-424expression was determined by qRT-PCR, and E2F1was determined by western blot. HA-tagged Cul2plasmid was transiently transfected into293T cells, and co-immunoprecipitation(Co-IP) was done to verify the binding of Cul2and E2F1protein. SiHa cells were transfected with E2F1siRNA or E2F1expression plasmid, western blot was conducted to confirm E2F1indirectly affected Cul2expression through regulating miR-424level. SiHa cells were transfected with miR-424mimic, qRT-PCR and western blot detection were used to confirm E2F1expression levels.SiHa cells were transfected with Cul2siRNA, Cul2expression plasmid, E2F1siRNA, or E2F1expression plasmid, respectively, the impact of Cul2and E2F1on cell proliferation was tested by CCK8method and ki-67immunofluorescence, and cell cycle was tested by flow cytometryResults1. E2F1suppression by E2F1siRNA strongly reduced miR-424expression level. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) analysis in SiHa cells revealed that, E2F1bound in miR-424promoter regions.2. Upon overexpression of miR-424, Cul2mRNA and protein levels were reduced. Luciferase reporter assay was confirmed that Cul2is a direct target of miR-424. Transient inhibition of Cul2by siRNA resulted in downregulation of E2Fland upregulation of miR-424in SiHa cells. We also identified increased expression of Cul2in SiHa cells resulted in overexpression of E2F1and suppression of miR-424. Co-IP showed Cul2can combine with E2F1. 3. Inhibition of E2F1expression resulted in decreased Cul2. Upregulation of E2F1resulted in increased Cul2expression. miR-424suppressed E2F1expression by affecting the Cul2levels.4. SiHa cells were transiently transfected with the respective E2F1siRNA, E2F1expression plasmid, Cul2siRNA or Cul2expression plasmid. Overexpression of Cul2or E2F1promoted cell proliferation, increased ki-67expression, and induced cell cycle Gl-S transition.Conclusions1. E2F1is a direct regulator of miR-424expression and suppresses miR-424expression.2. The feedback loop involving Cul2, E2F1, and miR-424is required for the maintenance of miR-424supression in SiHa cells. Part IV Exploring the relationship between miR-424and high-risk human papillomavirus16early oncoprotein E7in cervical cancerObjectiveTo explore the possible mechanism through which HPV16E7regulates miR-424.MethodsExpression of miR-424and HPV16E7were detected in cervical normal tissues without HR-HPV infection, cervical normal tissues, CIN II-III, and Cervical cancer tissues with HPV16infection based on qRT-PCR. Correlation between miR-424and HPV16E7was then analyzed. We assessed HPV16E7expression level in SiHa cells transfected with miR-424mimic by qRT-PCR; also miR-424expression level in SiHa cells transfected with siRNAs targeting HPV16E7or with HPV16E7expression plasmid.A plasmid expressing HA-HPV16E7protein was transiently transfected into293T cells. Selective precipitation from the cell lysate of HA protein using HA-tagged Co-IP kit was to test whether HPV-16E7binds to E2F1. Plasmid expressing HA-HPV16E7protein was transiently co-transfected with a Cul2expression plasmid into293T cells, and co-precipitation of HA-HPV16E7protein and Cul2protein was tested by HA-tagged Co-IP kit. E2F1and Cul2levels were measured upon overexpression of HPV16E7or inhibition of HPV16E7expression in SiHa cells.Results1. In four groups of cervical tissues, a gradually decreased expression of miR-424and increased expression of HPV16E7were observed from HPV-/cervical normal throughout HPV16+/cervical normal and HPV16+/CINII-III to HPV16+/cervical cancer tissues. Correlation analysis showed that the expression of miR-424was negatively correlated with HPV16E7.2. Ectopic expression of miR-424could not influence HPV16E7mRNA level, While overexpression or inhibition of HPV16E7could change miR-424expression level.3. HPV16E7directly bound to E2F1and increase E2F1expression.4. HPV16E7bound to Cul2, but did not regulate the expression of Cul2.Conclusions1. miR-424expression is negatively correlated with HPV16E7expression during HPV16induced cervical carcinogenesis.2. miR-424could not change the expression of HPV16E7in cervical cancer SiHa cells.3. HPV16E7binds to E2F1and Cul2, and consequently indirectly alteres expression of miR-424.