| Cervical cancer, the incidence of which in women is in the second place, is one of the highest degree of malignancy of the tumor. Nearly500,000women are diagnosed with cervical cancer each year in the world, of which nearly half of the patients died. Due to lack of universal screening, application of vaccines is limited because clinical trials in Chinese population have not yet been completed and the price is expensive, Chinese cases share a quarter of global morbidity and mortality. Thus cervical cancer is a serious threat to women’s health. For patients with advanced cervical cancer, there is no effective chemotherapy drugs, therefore looking for cancer therapeutic targets and developing specific molecular targeted drugs have important practical significance.It has been proved that high-risk HPV infection can be detected in99.7%of cervical squamous cell carcinoma and94%-100%of cervical adenocarcinoma. The most common type of infection are HPV16and HPV18. All HPV positive malignant cells comprising at least one copy of viral genome which is transcriptionally active. Only the viral DNA is random integrated into the host genome, E6and E7oncogenes maintain high expression, which is considered to be necessary and a rate-limiting step of cervical cancer. The dysregulation of E6and E7expression increase genomic instability, accumulate genetic and epigenetic changes in cells. Ultimately these changes can activate oncogenes, inactivate tumor suppressor genes, cause some cells selective growth and malignant transformation.High-risk HPV E6and E7could inactivate the tumor suppressor protein p53and pRb, respectively. E6can induce p53degradation by the ubiquitin pathway. E7can combine with Rb by conservative LxCxE motif, decompose Rb-E2F complexes through phosphorylation of Rb protein and release E2F1, which contribute to the trans-activation of c-Myc, cyclin A and E (cyclinA/E), in favor of intracellular DNA synthesis and cell transition from G1to S phase, promote cell proliferation.Recent studies show that non-coding microRNA (miRNA) are transcriptional or post-transcriptional regulated by E6and E7. miRNAs are first found in eukaryotes, endogenous, non-coding RNA with regulatory functions. The sizes of miRNAs are about18to25bases. By specific base pairing with the target mRNA, miRNAs can degrade, inhibit or activate mRNA. miRNAs expression have timing and tissue specificity and plays an important role in the organism growth, development, cell differentiation, proliferation and apoptosis. miRNA itself, like other RNA polymerase II transcription initiation, is transcriptional and post-transcriptional regulated. Many cell transcription factors, including c-Myc, p53and E2F, has been shown to regulate miRNA. Besides the proteins related to miRNA processing to be mature, including Drosha, DGCR8, exportin5, Dicer, TRBP and Ago2, can also influence the level of mature miRNAs.The abnormal expression of miRNAs is present in most human tumors. As onco-miR or tumor suppressor miR, miRNAs can regulate oncogenes and tumor suppressor gene mRNA, be involved in process of tumorigenesis. Since the environment and the target gene have difference, a number of miRNA play a tumor-promoting role in one type of tumor, and a suppressor role in another type. It is also found that oncogenic miRNAs (miR-21, miR-146a and miR-15/miR-16-1) increase and tumor suppressor miRNA (miR-143, miR-145, miR-218, miR-203, miR-23a/b and miR-34a) reduce in cervical cancer by miRNA chips or deep sequencing with cervical tissue or cell lines.Although HPV itself has not miRNA, due to the high expression of E6/E7in cervical cancer, which affect cell proliferation, apoptosis and invasion ability. It is significantly important to clarify the regulation functions of E6and E7to downstream miRNAs, interaction of miRNAs with their mRNA targets, and the role of miRNAs in the indirect interaction of proteins, to help us understand the occurrence, development and metastasis of cervical cancer and discover new therapeutic targets. Currently, a lot of evidence showed that high-risk HPV E6/E7regulate host cell miRNAs mainly through E6-p53and E7-pRb-E2F pathways.In the past, screen of the differential expressed miRNAs progress between cervical cancer and normal cervical tissue, or between HPV-positive cervical cancer cells and HPV-negative cervical cancer cells. Further experiments are required to verify whether the virus oncogenes E6/E7is linked to screened miRNA or not. Unfortunately, it is often found that the differentially expressed miRNAs is not subject to E6or E7regulation.Therefore, in this study we used siRNA to silence E6/E7in HPV16-positive cervical cancer cell lines, screen differences in miRNA expression profile with not silenced cells by miRNA array. Part of differentially expressed miRNAs and their relationship with E6or E7were ascertained in cells and tissue samples, which laid the foundation for further study of these miRNA role. Then the effect of miR-27b on cell proliferation, invasion and apoptosis were studied, its target gene PLK2was found through software prediction and experimental validation. The direct effect of miR-27b on PLK2mRNA3’UTR was confirmed by dual luciferase report experiment. Afterwards, PLK2was silenced or over-expressed in cervical cancer cells to study how the regulation of miR-27b on PLK2affect cell phenotype. Finally, the mechanism of E7up-regulation of miR-27b was studied preliminarily.Targeted silence HPV16oncogenes e6/e7in CaSki cells, which was compared with control group by miRNA array to discover differentially expressed miRNAs. It’s found that38miRNAs were down-regulated and6miRNAs were up-regulated in the samples of E6and E7silenced. According to fold change of differences and literature reports associated with E6or E7downstream pathways, five miRNAs (hsa-miR-20a-5p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-93-5p, hsa-miR-106b-5p) were screened out, which were verified further in miRNA chip samples and SiHa cells with QPCR relative quantification. It was showed that the five miRNAs were down-regulated in silenced group, consistent with the microarray results.To understand which oncogene, E6or E7, affect the expression levels of these five miRNA, we constructed e6and e7gene over-expression plasmids, which were transfected into SiHa cells. It is showed that the five miRNAs increased in different degrees in e6or e7overexpression group, corresponding to the results in e6/e7silenced samples. Both E6and E7could up-regulate miR-20a-5p, miR-93-5p and miR-106b-5p increase, E7could increase miR-27b-3p and E6could improve miR-24-3p. It is further proved that the levels of E6, E7mRNA and the five miRNAs were significantly higher in HPV16-infected cervical squamous cancer samples than those in adjacent tissues.Previous studies also found that the five miRNAs increased in cervical cancer tissue or cells, some of which were studied to discover their target genes, however, the up-regulation mechanism of the miRNAs were not been involved. The present study provided the direct evidence that HPV viral oncogene can affect the levels hsa-miR-20a-5p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-93-5p and hsa-miR-106b-5p. These miRNAs may directly or indirectly regulated by the E6or E7as carcinogenesis factors, and play a role in tumorgenesis, invasion and metastasis.In order to understand how viral oncogenes regulate the expression of miRNAs and how the miRNAs affect tumor progression, we selected miR-27b which was reported fewer in cervical cancer to study in-depth.It was confirmed that miR-27b could promotes cancer cell proliferation and invasion, inhibition of apoptosis by over-expression or inhibition of miR-27b in CaSki cell with CCK-8cell proliferation assay, transwell chamber cell invasion assay and caspase-3activity of paclitaxel-induced apoptosis detection experiment. The results indicat that miR-27b has a cancer-promoting role in cervical cancer, which is consistent with the role in breast cancer and gliomas. To understand how miR-27b contribute to carcinogenesis, we predict its target genes with three online miRNA analysis software, Targetscan, Microcosm and Miranda, from the intersection of which, tumor suppressor were selected and their relevant to miR-27b were ascertained with experiments in miR-27b mimics or inhibitor transfected CaSki and SiHa cells. The results showed that the mRNA and protein levels of tumor suppressor PLK2were negatively correlated with miR-27b, suggesting that miR-27b may influence PLK2through post-transcriptional regulation.Prediction results indicated that PLK2mRNA3’UTR does exist target region of miR-27b. To verify the direct effect of miR-27b and seed area, we constructed PLK2mRNA3’UTR region of wild-type plasmid and seed area whole mutant plasmid, which were transfected into cells with miR-27b mimics to perform double luciferase report experiment. It is revealed that miR-27b significantly reduced fluorescence of the cells transfected with wild-type plasmid, but have no effect on that of mutant plasmids, suggesting that miR-27b may act on the mutation regions of PLK2mRNA3’UTR to inhibit PLK2at post-transcriptional level.By comparison of miR-27b and PLK2expression levels in HPV16-positive cervical carcinoma tissues and adjacent tissues, we found that miR-27b was significantly higher in tumor tissues than the adjacent tissues, on the contray PLK2was lower in tumor tissues. This discover supports the negative regulation of miR-27b to PLK2found in cell experiments.In order to understand how PLK2influence the role of miR-27b to cervical cancer cells, we studied PLK2’s effect on cell proliferation, invasion and paclitaxel-induced apoptosis in PLK2over-expressed or silenced SiHa cells. The results indicated that, PLK2could inhibit cell proliferation, reduce cell viability with paclitaxel irritated, promote paclitaxel-induced apoptosis, but has nothing to do with cell invasiveness.Previous studies have found that in primary B-cell tumors and ovarian cancer, PLK2promoter CpG was methylated and transcription was suppressed, which are associated with tumorigenesis, recurrence, insensitive to chemotherapeutics. Over-expression of PLK2could promote apoptosis, increase sensitivity to chemotherapy. PLK2also interact with TSC proteins, influencing mTOR signaling, inhibiting tumor growth and reducing cell viability.This study is the first report of tumor suppressor PLK2regulated at post-transcriptional level by miRNA, is related to the effects of miR-27b promoting cell proliferation and affecting cell chemosensitivity. Initially demonstrated that the existence of regulatory pathways E7-miR-27b-PLK2in cervical cancer. Then how could E7up-regulate miR-27b? we tried to understand the mechanism from miRNA processing. DGCR8, a double strands RNA binding protein, plays an important role in the biosynthesis pathway of the miRNA. It combines with RNase Ⅲ enzyme, Drosha, to form a complex in the nucleus, which can process primary miRNA (pri-miRNA) into precursor miRNA (pre-miRNA). It is found that the mRNA of DGCR8increased in a variety of tumors, including hepatocellular carcinoma, colorectal cancer, basal cell carcinoma, squamous cell carcinoma and pleomorphic adenocarcinoma with QPCR or cDNA microarray, subsquently the levels of mature miRNAs were changed.We found that DGCR8mRNA were up-regulated in HPV16infected cervical cancer tissues which showed a positive correlation with virus oncogene E7. It is further confirmed that E7can up-regulate DGCR8by over-expression or silencing of E7in SiHa cells. When DGCR8was silenced using targeted siRNA in SiHa cells, miR-27b decrease. The outcome suggest in HPV16-infected cervical cancer cells, oncogene E7may promote miR-27b mature by up-regulating DGCR8.Guo et al also found that inhibition of the expression of DGCR8in ovarian cancer cell SKOV3made miRNA expression abnormal and miR-27b significantly reduced. Over-expressing of miR-27b in SKOV3cells could significantly promote cell colony formation. Targeted silencing of Drosha and DGCR8in breast cancer cell induced growth inhibition, where screened out miRNAs related to maintain growth, including miR-20a and miR-27b. The two studies above were consistent with what we found in cervical cancer. Interestingly, it was reported that silence of DGCR8in colorectal cancer cells HCT116down-regulated miR-20a, miR-93and miR-106b, which were also differential expressed in our microRNA array, and validated by QPCR, were relevant to E6and E7. They are worthy of further study that whether HPV oncogenes affect miRNAs by regulating DGCR8is the principal pathway in cervical cancer, and whether most of differential expressed miRNA in our miRNA array reduced in E6E7silenced group was related to DGCR8decrease.Besides post-transcriptional processing of miR-27b through DGCR8, are there other ways E7regulate miR-27b? It has shown that miR-27b was up-regulated in breast cancer by abnormal histone acetylation, and E7could not only combine with histone deacetylase and inhibit its binding to the promoter E2F2, hence promote E2F2transcriptional activation, but also interact with the histone deacetylase HDAC1, HDAC4, and HDAC7, enhancing HIF-la-mediated transcriptional activation. Therefore, it is also worth discussing whether E7could regulate miR-27b by histone deacetylase regulation in cervical cancer.In addition, there are binding sites of transcription factor C/EBP alpha at the promoter of C9orf3, which is miR-27b host gene. CSF-3induced C/EBP alpha increasing, and promote C9orf3and miR-27b transcriptional activation in the process of original granulocyte differentiation into granulocytes. HER2/neu (ERBB2), EGF and TNF-α promote miR-27b trans-activation by AKT/NF-κB pathway in breast cancer. Thus it is worthy of further study whether E7could activate the transcription of miR-27b by transcription factors in cervical cancer.Through the above study, we proved it is a feasible strategy to discover regulated miRNA by targeted silence of potentially regulatory genes. We obtained and verified five miRNAs, hsa-miR-20a-5p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-93-5p and hsa-miR-106b-5p, which are regulated by HPV16oncogene E6/E7in cervical cancer. Next, we selected miR-27b to study in depth and firstly reported that tumor suppressor PLK2regulated by miRNA at the post-transcriptional level. We found miR-27b can negatively regulate PLK2mRNA3’UTR, promoting cell proliferation and cell sensitivity to chemotherapeutic drugs, which demonstrates that an E7-miR-27b-PLK2regulatory pathway related to cell proliferation and apoptosis exists in cervical cancer. Finally, we revealed that E7could up-regulate miR-27b via regulation of DGCR8, a key enzyme of miRNA maturity, from the perspective of post-transcriptional processing. It remains to be further studied whether E7could play a role in transcription factor or histone deacetylase regulation to up-regulate miR-27b. This study supports miR-27b could be a promising target for cancer therapy. |
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