Fenfen Wang

miR-375 Is Down-Regulated in Squamous Cervical Cancer and Inhibits Cell Migration and Invasion via Targeting Transcription Factor SP1

Pelvic lymph node metastases are regarded as the most important risk factor and a predictor of poor prognosis for patients with cervical cancer. Exploration of metastasis-related molecules is helpful toward improving the prognosis in cervical cancer. To identify the role of miR-375 in metastasis and progression of cervical cancer, we examined the expression of miR-375 in 170 cervical cancer tissues and 68 normal cervical tissues, using stem-loop quantitative PCR, and found that the expression of miR-375 in cervical cancer tissues was significantly decreased by 4.45-fold, compared with 68 normal tissues. A significant correlation existed between miR-375 expression and clinicopathologic parameters, including lymph node metastasis of cervical cancer. Overexpressed miR-375 suppressed cell proliferation, blocked G1-to-S cell-cycle transition, and inhibited cell migration and invasion in human cervical SiHa and CaSki cells. SP1, a potential target gene of miR-375, was inversely correlated with miR-375 expression in cervical cancer tissues. Moreover, SP1 was negatively regulated by miR-375, and knockdown of SP1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that down-regulated miR-375 promotes cell malignant behaviors via the target gene SP1 and may consequently contribute to the progression of cervical cancer.

Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29

miRNAs have the potential to act on diverse downstream genes, and miRNA signatures of HPV‐infected tissues may provide insight into HPV‐related carcinogenesis. We set out to profile miRNA expression in HPV‐infected samples and relate this to histological and grade‐specific alterations in the spectrum of cervical carcinogenesis in vivo. A total of 31 miRNAs showed significant and continuous expression along with the progression from normal cervical tissue to cancer, and six of them were validated in 133 samples. By bioinformatics analyses, we established a putative HPV‐associated miRNA–mRNA regulatory network, showing that miR‐29 is the most highly enriched. We also found that YY1 and CDK6 were both positively correlated with E6/E7 RNA expression and targeted by tumour‐suppressive miR‐29. Evidence of miR‐29 involvement in HPV infection was further verified in patient samples and by various experimental approaches. Taken together, our results suggest that HPVs have oncogenic properties at least in part by reshaping the milieu of cellular miRNAs. miR‐29 restrains cell cycle progression and induces apoptosis via YY1 and CDK6 promoting malignant transformation induced by HPV, although the abnormality of miR‐29 in HPV‐infected cells might be regulated in an indirect way. Copyright

Identification of suitable reference genes for measurement of gene expression in human cervical tissues.

For quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), the most commonly used normalization strategy is to select a stable reference gene. However, no suitable reference genes have been identified in cervical tissues to date. The aim of this study was to identify the most stable gene or a set of genes as reference genes for RT-qPCR analysis in cervical tissues from a panel of 12 candidates (ALAS1, PPIA, GAPDH, HBB, TBP, ACTIN, B2M, MBNL2, PGKL, RPLP0, RPL-4, and EEF1A1). In total, 20 normal and 20 cervical cancer specimens were examined. Gene expression data were analyzed using two different statistical models (geNorm and NormFinder). EEF1A1 was identified as the most stable and reliable reference gene, followed by GAPDH and RPLP0, whereas EEF1A1 and GAPDH were the best two-gene combination by NormFinder. The expression validity of EEF1A1 was further determined in 21 normal, 22 cervical intraepithelial neoplasia (CIN(2-3)), and 18 cancer tissues; no expression differences were found among normal, CIN(2-3), and cancer tissues (P>0.05). Our results suggested that EEF1A1 can be used as a reference gene for normalization in gene profiling studies in clinic cervical samples, and the combination of EEF1A1 and GAPDH could be recommended as a much more reliable normalization strategy.

Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n = 21; HR-HPV+ normal, n = 19; cervical intraepithelial neoplasia [CIN], n = 47; cancer, n = 21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant deregulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.

Transcriptional gene silencing of HPV16 E6/E7 induces growth inhibition via apoptosis in vitro and in vivo

OBJECTIVE Transcriptional silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression. Our objective was to estimate the effective value of E6/E7 specific siRNA-induced transcriptional gene silencing as a potential therapeutic strategy for cervical cancer. METHODS In vitro studies were performed by employing two categories of siRNA targeting promoter of E6/E7 gene and E7 transcript, respectively, and inhibitory effect of both siRNAs was further observed in vitro and on xenograft in BALB/c mice that were inoculated with siRNA transfected SiHa cells and parental SiHa cells followed by siRNA intratumoral injection in vivo. Tumor volume and growth curves were assessed. Furthermore, cellular proliferation and apoptosis of inoculated tumors were determined by immunohistochemistry staining and TUNEL assay. RESULTS The two most active siRNA sequences specifically knockdown E6/E7 expressions at mRNA level in HPV16 positive Siha cells, increased p53 and decreased p16 expressions at protein level, inhibited cell proliferation, and induced cell apoptosis in vitro. Furthermore, both siRNAs effectively inhibited tumor formation and growth no matter in mice with siRNA transfected cells in vitro or with siRNA intratumoral injection in vivo. TUNEL staining and FCM assay consistently showed that tumor retardation was through induction of cellular apoptosis. CONCLUSION RNAi targeting the promoter of HPV16 E6/E7 acts effectively in vitro and in vivo, especially through intratumoral delivery, and may be a candidate therapeutic strategy for cervical cancer.

HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo.

PurposeCadherin switch, as a key hallmark of epithelial–mesenchymal transition (EMT), is characterized by reduced E-cadherin expression and increased N-cadherin or P-cadherin expression, and has been implicated in many aggressive tumors, but the importance and regulatory mechanism of cadherin switch in cervical cancer have not been investigated. Our study aimed to explore the role of cadherin switch by regulation of HPV-16 E6/E7 in progression and metastasis of cervical cancer.MethodsThe expressions of E-cadherin and P-cadherin were examined by immunohistochemical staining in 40 cases of high-grade cervical lesions with HPV-16 infection only in which HPV-16 E6 and E7 expression had been detected using qRT-PCR method. Through modulating E6 and E7 expression using HPV-16 E6/E7 promoter-targeting siRNAs or expressed vector in vitro, cell growth, migration, and invasion were separately tested by MTT, wound-healing and transwell invasion assays, as well as the expressions of these cadherins by western blot analyses. Finally, the expressions of these cadherins in cancerous tissues of BALB/c-nu mouse model inoculated with the stable HPV-16 E6/E7 gene silencing Siha and Caski cells were also measured by immunohistochemical staining.ResultsPearson correlation coefficient analyses showed the strongly inverse correlation of E-cadherin expression and strongly positive correlation of P-cadherin expression with E6/E7 level in 40 cases of high-grade cervical lesions. Furthermore, the modulation of HPV-16 E6/E7 expression remarkably influenced cell proliferation, migration, and invasion, as well as the protein levels of E-cadherin and P-cadherin in cervical cell lines. Finally, the reduction of HPV-16 E6/E7 expression led to up-regulated expression of E-cadherin and down-regulated expression of P-cadherin in BALB/c-nu mouse model in vivo assay.ConclusionsOur results unraveled the possibility that HPV-16 E6/E7 could promote cell invasive potential via regulating cadherin switching, and consequently contribute to progression and metastasis of cervical cancer.

MicroRNA Detection in Cervical Exfoliated Cells as a Triage for Human Papillomavirus–Positive Women

Background Papanicolaou (Pap) triage, with high specificity, has been recommended for primary Human papillomavirus (HPV) testing but is flawed by poor sensitivity and cytologist dependence. We evaluated the potential role of microRNA (miRNA) detection in cervical exfoliated cells in HPV-positive women from a clinic-based population. Methods Primary HPV testing as well as Pap test were performed on all eligible women. Six miRNAs (miR-424/miR-375/miR-34a/miR-218/miR-92a/miR-93) were detected by RT-qPCR in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. Mann–Whitney U test, the receiver operating characteristic curve, logistic regression, and Pearson’s Chi-square were used to assess data. All tests of statistical significance were two-sided. Results A total of 1021 eligible HPV-positive women were enrolled. The expression of miR-424/miR-375/miR-34a/miR-218 in high-grade cervical intraepithelial neoplasia (CIN) and abnormal cytology was statistically significantly lower than that in low-grade CIN and normal cytology, respectively (all P < .05). Compared with the Pap test, both miR-424 and miR-375 detection achieved higher sensitivity (76.0% and 74.9% vs 63.8%, P < .05), higher negative predictive value (NPV) (85.7% and 85.4% vs 79.3%, P < .05), and comparable specificity while identifying CIN2 or worse (CIN2+). Similar results were achieved while identifying CIN3+. Multi-marker panels based on miR-424, miR-375, and miR-218 further improved the performance over any single miRNA test or Pap test. Conclusion Single miR-424 or miR-375 detection and miR-424/miR-375/miR-218–based multimarker panels in cervical exfoliated cells show superior performance over Pap triage for high-grade CIN identification in a clinic-based population. Detection of miRNA may provide a new triage option for HPV-positive women.

Inhibition of cervical cancer cell growth in vitro and in vivo by lentiviral-vector mediated shRNA targeting the common promoter of HPV16 E6 and E7 oncogenes.

Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. Small interfering RNAs (siRNA) or double-stranded RNAs can knock down target genes effectively through siRNA-induced transcriptional gene silencing (TGS). Here, we established lentiviral-vector mediated shRNA (LV-shRNA) targeting common promoter of HPV16 E6/E7 and targeting E6 transcript, transduced the lentiviral construct into cervical HPV16-positive cell lines Siha and Caski, then selected and established stably transduced monoclonal cell lines. The results showed that LV-shRNA targeting promoter, as well as targeting E6 transcript, effectively knocked down E6 and E7 expression, resulted in accumulation of p53 and pRB protein and decrease of MCM7 and p16 protein, and consequently remarkably reduced the abilities of proliferation and invasiveness of cervical cancers cells in vitro. Then we inoculated subcutaneously those monoclonal cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth, as well as prolonged survival time of mice incubated by cells with LV-shRNA targeting promoter and E6 transcript. Our results may provide evidence for application of LV-shRNA targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy.

Human Papillomavirus Type 58 Genome Variations and RNA Expression in Cervical Lesions

ABSTRACT Human papillomavirus type 58 (HPV58) is relatively prevalent in China and other Asian countries. In this study, the HPV58 genome in cervical lesions was decoded from five grade 2 or 3 cervical intraepithelial neoplasia lesion (CIN2/3) samples and five cervical cancer tissues using rolling-circle amplification of total cell DNA and deep sequencing and verified by whole-genome cloning and sequencing. HPV58 isolates from China feature a total of 52 nucleotide substitutions (0.66%) from the reference HPV58 sequence, which appear mainly in two regions, with 12 from nucleotides (nt) 3430 to 4136 covering the E2/E4/E5 open reading frames (ORFs) and 13 from nt 4621 to 5540 covering the L2 ORF; these could be grouped as HPV58 Chinese Zhejiang-1, -2, and -3 (CNZJ-1, -2, and -3) according to their sequence similarities and restriction enzyme digestion. Phylogenetically, CNZJ-3 is similar to the reference HPV58 sublineage A1 sequence. The other two are close to sublineage A2. Analysis of cervical lesion-derived RNA revealed abundant HPV58 early transcripts spliced at the E6 and E1/E2 ORFs, where two 5′ splice sites at nt 232 and nt 898 and two 3′ splice sites at nt 510 and nt 3355 can be identified. Thus, our study represents the first genome-wide analysis of HPV58 and its expression in cervical lesions.

Expression of E-, P- and N-Cadherin and Its Clinical Significance in Cervical Squamous Cell Carcinoma and Precancerous Lesions

Aberrant expression of classical cadherins has been observed in tumor invasion and metastasis, but its involvement in cervical carcinogenesis and cancer progression is not clear. We investigated E-, P- and N-cadherin expression and its significance in cervical squamous cell carcinoma (SCC) and cervical intraepithelial neoplasia (CIN). This retrospective study enrolled 508 patients admitted to Womens Hospital, School of Medicine, Zhejiang University with cervical lesions between January 2006 and December 2010. Immunochemical staining was performed in 98 samples of normal cervical epithelium (NC), 283 of CIN, and 127 of early-stage SCC. The association of cadherin staining with clinical characteristics and survival of the patients was evaluated by univariate and multivariate analysis. We found gradients of decreasing E-cadherin expression and increasing P-cadherin expression from NC through CIN to SCC. Aberrant E-cadherin and P-cadherin expression were significantly associated with clinical parameters indicating poor prognosis and shorter patient survival. Interestingly, we found very low levels of positive N-cadherin expression in CIN and SCC tissues that were not related to CIN or cancer. Pearson chi-square tests showed that E-cadherin expression in SCC was inversely correlated with P-cadherin expression (E-P switch), and was not correlated with N-cadherin expression. More important, patients with tissues exhibiting an E-P switch in expression had highly aggressive phenotypes and poorer prognosis than those without E-P switch expression. Our findings suggest that E-cadherin and P-cadherin, but not N-cadherin staining, might be useful in diagnosing CIN and for predicting prognosis in patients with early-stage SCC.