Junfen Xu

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

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.

CUL2 overexpression driven by CUL2/E2F1/miR-424 regulatory loop promotes HPV16 E7 induced cervical carcinogenesis.

It has been shown that HPV16 E7, but not other genotypes, can bind to scaffold protein CUL2 during inducing cervical carcinogenesis, but the expression level, associated regulating mechanism, and potential carcinogenicity of CUL2 itself is still unknown as yet. Here, we demonstrated that CUL2 was specifically overexpressed in HPV16 positive cervical cancer cells and tissues, and CUL2 expression was significantly increased along with the cervical lesion progression and positively correlated with HPV16 E7. CUL2 knockdown slowed the growth of xenograft tumors in mouse models. Importantly, CUL2 specifically bound to HPV16 E7, but not HPV18 E7. Moreover, CUL2 acted as a direct target of miR-424, and reversely suppressed miR-424; E2F transcription factor 1 (E2F1) suppressed miR-424 expression; CUL2 bound to E2F1 and promoted E2F1 expression. Our results indicate the existence of a regulatory loop among CUL2, E2F1, and miR-424 in HPV16 positive cervical cancer cells. Our results suggest that E7 recruited CUL2, driven by CUL2/E2F1/miR-424 regulatory loop, is overexpressed and accelerates HPV16-induced cervical carcinogenesis. Our findings may serve as one of the explanations for a clinical phenomenon that HPV16 possesses the strongest cervical carcinogenicity among high-risk HPV genotypes.

miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

Micro ribonucleic acid‐93 promotes oncogenesis of cervical cancer by targeting RAB11 family interacting protein 1

Micro ribonucleic acid (RNA)‐93 (miR‐93) is a novel oncogenic miRNA dysregulated in many types of tumors. We aimed to further study the expression pattern and clinical significance of miR‐93 and its target, the RAB11 family interacting protein 1 (RAB11FIP1) gene, in cervical cancer.

A transcriptomic landscape of human papillomavirus 16 E6‐regulated gene expression and splicing events

The oncoprotein E6 of high‐risk human papillomavirus (HPV) is responsible for the initiation and progression of cervical cancer. In the present study, we performed RNA‐sequencing analysis in HPV16 E6‐expressing 293T cells and identified 56 differentially expressed genes (DEGs) and a number of cellular pathways significantly affected. We confirmed nine of these DEGs in both cell models and clinical tissue samples. Furthermore, we performed de novo transcriptome assembly of 1128 novel human transcripts and identified 22 that are differentially expressed in the presence of HPV16 E6. In addition, our analysis revealed distinct alternative splicing events in response to HPV16 E6 expression. Overall, the present study provides a comprehensive portrait of transcriptional and splicing signatures, as well as previously unknown genes differentially expressed in response to HPV16 E6, which prompts the need for a complete annotation of the HPV16 E6‐regulated transcriptome.