Xiaoyun Wan

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

miR-375 is upregulated in acquired paclitaxel resistance in cervical cancer

Background:Chemo-resistance is one of the key causal factors in cancer death and emerging evidences suggest that microRNAs (miRNAs) have critical roles in the regulation of chemo-sensitivity in cancers. Cervical cancer is one of the most common malignancies in women and insensitive to chemotherapy clinically.Methods:The differentially expressed miRNAs in cervical squamous cell carcinoma tissues were screened by using a microarray platform (μParaflo Sanger miRBase release 13.0). The expression of miR-375 was determined by stem-loop RT–PCR using 23 clinical cervical cancer samples and 2 cervical cancer cell lines. We exogenously upregulated miR-375 expression in SiHa and Caski cells using a pre-miRNA lentiviral vector transfection and observed its impact on paclitaxel sensitivity using MTS. The cells that stably overexpressed miR-375 were subcutaneously injected into mice to determine tumour growth and chemo-sensitivity in vivo.Results:Twenty-one differentially expressed miRNAs were found by miRNA microarray between pro- and post-paclitaxel cervical cancer tissues. Of those, miR-375 showed consistent high expression levels across paclitaxel-treated cervical cells and tissues. Paclitaxel induced upregulated miR-375 expression in a clear dose-dependent manner. Forced overexpression of miR-375 in cervical cancer cells decreased paclitaxel sensitivity in vitro and in vivo.Conclusion:Collectively, our results suggest that miR-375 might be a therapeutic target in paclitaxel-resistant cervical cancer.

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.

The association of XRCC1 gene single nucleotide polymorphisms with response to neoadjuvant chemotherapy in locally advanced cervical carcinoma.

BackgroundPlatinum-based neoadjuvant chemotherapy (NAC) is new therapeutic strategy for locally advanced cervical carcinoma, but the variables used to predict NAC response are still infrequently reported. The aim of our study was to investigate the association between XRCC1 gene single nucleotide polymorphisms (SNPs) and NAC response.MethodsSeventy patients with locally advanced cervical carcinoma who underwent NAC were collected. SNPs of XRCC1 (at codon 194 and 399) and XRCC1 protein expression were detected. The association of XRCC1 gene SNPs and protein expression with NAC response were analyzed.ResultsResponse to NAC was not statistically significant in three genotypes, Arg/Arg, Arg/Trp, Trp/Trp of XRCC1 at codon 194(X2 = 1.243, P = 0.07), while responses were significantly different in genotypes Arg/Arg, Arg/Gln, Gln/Gln of XRCC1 at codon 399 (X2 = 2.283, P = 0.020). The risk of failure to chemotherapy in the patients with a Gln allele(Arg/Gln+Gln/Gln) was significantly greater than that with Arg/Arg(OR = 3.254, 95%CI 1.708 ~ 14.951). The expression level of XRCC1 protein was significantly associated with response to NAC. Moreover, the genotype with the Gln allele(Arg/Gln+Gln/Gln) at codon 399, but not codon at 194, presented a significantly higher level of XRCC1 protein expression than that with Arg/Arg genotype (F = 2.699, p = 0.009).ConclusionSNP of XRCC1 gene at codon 399 influences the response of cervical carcinoma to platinum-based NAC. This is probably due to changes in expression of XRCC1 protein, affecting response to chemotherapy.

EMA-CO chemotherapy for high-risk gestational trophoblastic neoplasia: a clinical analysis of 54 patients

This study was designed to analyze the outcomes of chemotherapy for high-risk gestational trophoblastic neoplasia (GTN) with EMA-CO regimen as primary and secondary protocol in China. Fifty-four patients with high-risk GTN received 292 EMA/CO treatment cycles between 1996 and 2005. Forty-five patients were primarily treated with EMA-CO, and nine were secondarily treated after failure to other combination chemotherapy. Adjuvant surgery and radiotherapy were used in the selected patients. Response, survival and related risk factors, as well as chemotherapy complications, were retrospectively analyzed. Thirty-five of forty-five patients (77.8%) receiving EMA-CO as first-line treatment achieved complete remission, and 77.8% (7/9) as secondary treatment. The overall survival rate was 87.0% in all high-risk GTN patients, with 93.3% (42/45) as primary therapy and 55.6% (5/9) as secondary therapy. The survival rates were significantly different between two groups (χ2= 6.434, P = 0.011). Univariate analysis showed that the metastatic site and the number of metastatic organs were significant risk factors, but binomial distribution logistic regression analysis revealed that only the number of metastatic organs was an independent risk factor for the survival rate. No life-threatening toxicity and secondary malignancy were found. EMA-EP regimen was used for ten patients who were resistant to EMA-CO and three who relapsed after EMA-CO. Of those, 11 patients (84.6%) achieved complete remission. We conclude that EMA-CO regimen is an effective and safe primary therapy for high-risk GTN, but not an appropriate second-line protocol. The number of metastatic organs is an independent prognostic factor for the patient with high-risk GTN. EMA-EP regimen is a highly effective salvage therapy for those failing to EMA-CO.

Outcomes of conservative therapy for young women with early endometrial adenocarcinoma

Four of six patients with endometrial cancer had initial response to progesterone therapy and obtained complete response; three among them had successful pregnancies with three live births. The results suggest that progesterone therapy, combined with assisted reproductive technology, provides more chance of carrying a full-term pregnancy for patients with endometrial adenocarcinoma.

Relapsed or refractory gestational trophoblastic neoplasia treated with the etoposide and cisplatin/etoposide, methotrexate, and actinomycin D (EP-EMA) regimen

Objective To evaluate the effectiveness of the etoposide and cisplatin/etoposide, methotrexate and actinomycin D (EP‐EMA) regimen in patients with gestational trophoblastic neoplasia who had been successfully treated with the etoposide, methotrexate, and actinomycin D/cyclophosphamide and vincristine (EMA‐CO) regimen but experienced a relapse, or who became refractory to EMA−CO treatment. Methods From January 1999 to December 2005, 18 patients with gestational trophoblastic neoplasia who had been successfully treated with the EMA‐CO regimen but sustained a relapse (n = 7) or who became refractory to it (n = 11) were treated with the EP‐EMA regimen. The effectiveness, adverse effects, and tolerated dose intensity of the EP‐EMA regimen were retrospectively analyzed. Results The 18 patients received a total of 74 cycles of the EP‐EMA regimen and 12 (66.7%) achieved complete remission. Nine of the 11 patients (81.8%) apparently resistant to the EMA‐CO regimen achieved complete remission. However, only 3 of the 7 patients (42.9%) who experienced a relapse after treatment with the EMA‐CO regimen achieved complete remission. The main adverse effects of the EP‐EMA regimen were myelosuppression and gastrointestinal problems. Because of myelosuppression and hepatotoxicity, only 56.8% of the patients could be treated with the planned dose intensity. Conclusion EP‐EMA may be an effective option for the treatment of gestational trophoblastic neoplasia in patients resistant to treatment with the EMA‐CO regimen. However, it does not seem to benefit all the patients who experienced a relapse after treatment with the EMA‐CO regimen.

miR-375 mediated acquired chemo-resistance in cervical cancer by facilitating EMT.

Acquired chemo-resistance is one of the key causal factors in cancer death. Emerging evidences suggest that miRNA and epithelial–mesenchymal transition play critical roles in the chemo-resistance in cancers. Here, we showed the association of paclitaxel-resistance with miR-375 over-expression and epithelial–mesenchymal transition inducement in cervical cancer. Using different cervical cancer cell models, we found that paclitaxel transiently induced up-regulation of miR-375 expression, proliferation inhibition, transition from epithelial to mesenchymal phenotype, and consequently impaired paclitaxel sensitivity. Forced over-expression of miR-375 may suppress Ecadherin expression by a directly targeting pathway, which led to paclitaxel resistance. Contrarily, re-expression of Ecadherin partly reversed epithelial–mesenchymal transition phenotype and miR-375 induced paclitaxel-resistance. Our findings suggest that paclitaxel-induced miR-375 over-expression facilitates epithelial–mesenchymal transition process via directly targeting Ecadherin, proliferation inhibition, and consequently results in chemo-resistance in cervical cancer cells. A reversion of miR-375 or Ecadherin expression may be a novel therapeutic approach for overcoming chemo-resistance in cervical cancer.

Factors associated with positive margins in patients with cervical intraepithelial neoplasia grade 3 and postconization management

To evaluate the risk factors for positive margins in cervical intraepithelial neoplasia (CIN) grade 3 and the outcomes of postconization management.