MiR-21 Regulates Growth and Migration of Cervical Cancer Cells by RECK Signaling Pathway.

Expression of miR-21 has been found to be altered in almost all types of cancers, and it has been classified as an oncogenic microRNA. In addition, the expression of tumor suppressor gene RECK is associated with miR-21 overexpression in high-grade cervical lesions. In the present study, we analyze the role of miR-21 in RECK gene regulation in cervical cancer cells. To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression using siRNAs. We analyzed the expression of miR-21 and RECK, as well as functional effects on cell proliferation and migration. We found that in cervical cancer cells, there was an inverse correlation between miR-21 expression and RECK mRNA and protein expression. SiRNAs to miR-21 increased luciferase reporter activity in construct plasmids containing the RECK-3'-UTR microRNA response elements MRE21-1, MRE21-2, and MRE21-3. The role of miR-21 in cell proliferation was also analyzed, and cancer cells transfected with siRNAs exhibited a markedly reduced cell proliferation and migration. Our findings indicate that miR-21 post-transcriptionally down-regulates the expression of RECK to promote cell proliferation and cell migration inhibition in cervical cancer cell survival. Therefore, miR-21 and RECK may be potential therapeutic targets in gene therapy for cervical cancer.

The Autophagy Process in Cervical Carcinogenesis: Role of Non-Coding-RNAs, Molecular Mechanisms, and Therapeutic Targets.

Autophagy is a highly conserved multistep lysosomal degradation process in which cellular components are localized to autophagosomes, which subsequently fuse with lysosomes to degrade the sequestered contents. Autophagy serves to maintain cellular homeostasis. There is a close relationship between autophagy and tumor progression, which provides opportunities for the development of anticancer therapeutics that target the autophagy pathway. In this review, we analyze the effects of human papillomavirus (HPV) E5, E6, and E7 oncoproteins on autophagy processes in cervical cancer development. Inhibition of the expression or the activity of E5, E6, and E7 can induce autophagy in cells expressing HPV oncogenes. Thus, E5, E6, and E7 oncoproteins target autophagy during HPV-associated carcinogenesis. Furthermore, noncoding RNA (ncRNA) expression profiling in cervical cancer has allowed the identification of autophagy-related ncRNAs associated with HPV. Autophagy-related genes are essential drivers of autophagy and are regulated by ncRNAs. We review the existing evidence regarding the role of autophagy-related proteins, the function of HPV E5, E6, and E7 oncoproteins, and the effects of noncoding RNA on autophagy regulation in the setting of cervical carcinogenesis. By characterizing the mechanisms behind the dysregulation of these critical factors and their impact on host cell autophagy, we advance understanding of the relationship between autophagy and progression from HPV infection to cervical cancer, and highlight pathways that can be targeted in preventive and therapeutic strategies against cervical cancer.

Transregulation of microRNA miR-21 promoter by AP-1 transcription factor in cervical cancer cells.

BACKGROUND: Gene expression profiles have demonstrated that miR-21 expression is altered in almost all types of cancers and it has been classified as an oncogenic microRNA. Persistent HPV infection is the main etiologic agent in cervical cancer and induces genetic instability, including disruption of microRNA gene expression. In the present study, we analyzed the underlying mechanism of how AP-1 transcription factor can active miR-21 gene expression in cervical cancer cells. METHODS: To identify that c-Fos and c-Jun regulate the expression of miR-21 we performed RT-qPCR and western blot assays. We analyzed the interaction of AP-1 with miR-21 promoter by EMSA and ChIP assays and determined the mechanism of its regulation by reporter construct plasmids. We identified the nuclear translocation of c-Fos and c-Jun by immunofluorescence microscopy assays. RESULTS: We demonstrated that c-Fos and c-Jun proteins are expressed and regulate the expression of miR-21 in cervical cancer cells. DNA sequence analysis revealed the presence of AP-1 DNA-binding sites in the human miR-21 promoter region. EMSA analyses confirmed the interactions of the miR-21 upstream transcription factor AP-1. ChIP assays further showed the binding of c-Fos to AP-1 sequences from the miR-21 core promoter in vivo. Functional analysis of AP-1 sequences of miR-21 in reporter plasmids demonstrated that these sequences increase the miR-21 promoter activation. CONCLUSIONS: Our findings suggest a physical interaction and functional cooperation between AP-1 transcription factor in the miR-21 promoter and may explain the effect of AP-1 on miR-21 gene expression in cervical cancer cells.

Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells.

BACKGROUND: Expression of the microRNA miR-21 has been found to be altered in almost all types of cancers and it has been classified as an oncogenic microRNA or oncomir. Due to the critical functions of its target proteins in various signaling pathways, miR-21 is an attractive target for genetic and pharmacological modulation in various cancers. Cervical cancer is the second most common cause of death from cancer in women worldwide and persistent HPV infection is the main etiologic agent. This malignancy merits special attention for the development of new treatment strategies. In the present study we analyze the role of miR-21 in cervical cancer cells. METHODS: To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression in a cervical intraepithelial neoplasia-derived cell lines using siRNAs. The effect of miR-21 on gene expression was assessed in cervical cancer cells transfected with the siRNA expression plasmid pSIMIR21. We identified the tumor suppressor gene PTEN as a target of miR-21 and determined the mechanism of its regulation throughout reporter construct plasmids. Using this model, we analyzed the expression of miR-21 and PTEN as well as functional effects such as autophagy and apoptosis induction. RESULTS: In SiHa cells, there was an inverse correlation between miR-21 expression and PTEN mRNA level as well as PTEN protein expression in cervical cancer cells. Transfection with the pSIMIR21 plasmid increased luciferase reporter activity in construct plasmids containing the PTEN-3'-UTR microRNA response elements MRE21-1 and MRE21-2. The role of miR-21 in cell proliferation was also analyzed in SiHa and HeLa cells transfected with the pSIMIR21 plasmid, and tumor cells exhibited markedly reduced cell proliferation along with autophagy and apoptosis induction. CONCLUSIONS: We conclude that miR-21 post-transcriptionally down-regulates the expression of PTEN to promote cell proliferation and cervical cancer cell survival. Therefore, it may be a potential therapeutic target in gene therapy for cervical cancer.

Utility of microRNAs and siRNAs in cervical carcinogenesis.

MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells.

HPV-Based Screening, Triage, Treatment, and Followup Strategies in the Management of Cervical Intraepithelial Neoplasia.

Cervical cancer is the second most common cause of death from cancer in women worldwide, and the development of new diagnostic, prognostic, and treatment strategies merits special attention. Many efforts have been made to design new drugs and develop immunotherapy and gene therapy strategies to treat cervical cancer. HPV genotyping has potentially valuable applications in triage of low-grade abnormal cervical cytology, assessment of prognosis and followup of cervical intraepithelial neoplasia, and in treatment strategies for invasive cervical cancer. It is known that during the development of cervical cancer associated with HPV infection, a cascade of abnormal events is induced, including disruption of cellular cycle control, alteration of gene expression, and deregulation of microRNA expression. Thus, the identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis, identifying cervical cancer molecular markers, and developing specific targeting strategies against tumor cells. Therefore, in this paper, we discuss the main diagnostic methods, management strategies, and followup of HPV-associated cervical lesions and review clinical trials applying gene therapy strategies against the development of cervical cancer.

Targeted treatments for cervical cancer: a review.

Cervical cancer is the second most common cause of cancer death in women worldwide and the development of new diagnosis, prognostic, and treatment strategies merits special attention. Although surgery and chemoradiotherapy can cure 80%-95% of women with early stage cancer, the recurrent and metastatic disease remains a major cause of cancer death. Many efforts have been made to design new drugs and develop gene therapies to treat cervical cancer. In recent decades, research on treatment strategies has proposed several options, including the role of HPV E6 and E7 oncogenes, which are retained and expressed in most cervical cancers and whose respective oncoproteins are critical to the induction and maintenance of the malignant phenotype. Other efforts have been focused on antitumor immunotherapy strategies. It is known that during the development of cervical cancer, a cascade of abnormal events is induced, including disruption of cellular cycle control, perturbation of antitumor immune response, alteration of gene expression, and deregulation of microRNA expression. Thus, in this review article we discuss potential targets for the treatment of cervical cancer associated with HPV infection, with special attention to immunotherapy approaches, clinical trials, siRNA molecules, and their implications as gene therapy strategies against cervical cancer development.