miR-17 ~ 92 suppresses proliferation and invasion of cervical cancer cells by inhibiting cell cycle regulator Cdt2.

Cervical cancer (CC) is the 4th most leading cause of death among women worldwide, and if diagnosed in late stages the treatment options are almost negligible. 99% of CC is caused by high-risk human papilloma viruses (HR-HPV). Upon integration into human genome, the encoded viral proteins mis-regulate various onco-suppressors and checkpoint factors including cell cycle regulators. One such protein is cell cycle S phase licensing factor, CDC-10 dependent transcript-2 (Cdt2) which has been reported to be highly upregulated in various cancers including CC. Also, in CC cells, several tumor suppressor miRNAs are suppressed, including miR-17 ~ 92 cluster. In this study, we report that miR-17 ~ 92 directly recruits to 3'UTR of Cdt2 and downregulates this oncogene which suppresses the proliferation, migration and invasion capabilities of the CC cell lines without affecting non-cancerous cells. We further show that suppression of Cdt2 by miR-17 ~ 92, blocks the cancerous cells in S phase and induces apoptosis, eventually leading to their death. Hence, our work for the first time, mechanistically shows how miR-17 ~ 92 could work as tumor suppressor in cervical cancer cells, opening up the potential of miR-17 ~ 92 to be used in developing therapy for cervical cancer treatment.

Ab initio modelling of an essential mammalian protein: Transcription Termination Factor 1 (TTF1).

Transcription Termination Factor 1 (TTF1) is an essential mammalian protein that regulates transcription, replication fork arrest, DNA damage repair, chromatin remodelling etc. TTF1 interacts with numerous cellular proteins to regulate various cellular phenomena which play a crucial role in maintaining normal cellular physiology, and dysregulation of this protein has been reported to induce oncogenic transformation of the cells. However, despite its key role in many cellular processes, the complete structure of human TTF1 has not been elucidated to date, neither experimentally nor computationally. Therefore, understanding the structure of human TTF1 is crucial for studying its functions and interactions with other cellular factors. The aim of this study was to construct the complete structure of human TTF1 protein, using molecular modelling approaches. Owing to the lack of suitable homologues in the Protein Data Bank (PDB), the complete structure of human TTF1 was constructed by ab initio modelling. The structural stability was determined with molecular dynamics (MD) simulations in explicit solvent, and trajectory analyses. The frequently occurring conformation of human TTF1 was selected by trajectory clustering, and the central residues of this conformation were determined by centrality analyses of the Residue Interaction Network (RIN) of TTF1. Two residue clusters, one in the oligomerization domain and other in the C-terminal domain, were found to be central to the structural stability of human TTF1. To the best of our knowledge, this study is the first to report the complete structure of this essential mammalian protein, and the results obtained herein will provide structural insights for future research including that in cancer biology and related studies.Communicated by Ramaswamy H. Sarma.

Purification and Structural Characterization of N-Terminal 190 Amino Acid Deleted Essential Mammalian Protein; Transcription Termination Factor 1.

The mammalian transcription termination factor 1 (TTF1) is an essential protein that plays diverse cellular physiological functions like transcription regulation (both initiation and termination), replication fork blockage, chromatin remodeling, and DNA damage repair. Hence, understanding the structure and mechanism conferred by its variable conformations is important. However, so far, almost nothing is known about the structure of either the full-length protein or any of its domains in isolation. Since the full-length protein even after multiple attempts could not be purified in soluble form, we have codon optimized, expressed, and purified the N-terminal 190 amino acid deleted TTF1 (ΔN190TTF1) protein. In this study, we characterized this essential protein by studying its homogeneity, molecular size, and secondary structure using tools like dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, Raman spectroscopy, and atomic force microscopy (AFM). By CD spectroscopy and DLS, we confirmed that the purified protein is homogeneous and soluble. CD spectroscopy also revealed that ΔN190TTF1 is a helical protein, which was further established by analysis of Raman spectra and amide I region deconvolution studies. The DLS study estimated the size of a single protein molecule to be 17.2 nm (in aqueous solution). Our structural and biophysical characterization of this essential protein will open avenues toward solving the structure to atomic resolution and will also encourage researchers to investigate the mechanism behind its diverse functions attributed to its various domains.

A study for evaluating clinical relevance of circulating cell-free DNA in cervical cancer.

Introduction: Recent techniques available for the detection of cervical cancer (CC) are highly invasive and costly, which makes it a rate-limiting step toward early diagnosis of this fatal disease. Evaluation of circulating cell-free DNA (ccfDNA) through liquid biopsy is a minimally invasive and cost-effective method that may serve as a unique tumor marker for early detection, treatment monitoring, the status of residual disease, and distant tumor metastasis in CC patients. Materials and Methods: In this study, initially, ccfDNA was measured in serum samples from 11 histopathologically proven cervix carcinoma patients and 8 controls. On successful screening, it was further extended to 2 more patients with a series of serum samples extracted at 3 different phases of the concurrent chemoradiotherapy (i.e., before, during, and after 6 months of follow-up). Results: Agarose gel electrophoresis profile for ccfDNA of CC patients showed that of 11 patients, 4 patients had a comparatively higher tumor burden (ccfDNA) than the other 7 patients. Notably, during concurrent chemoradiotherapy, ccfDNA load disappeared and, after 6 months of follow-up, appeared back due to distant metastasis. Conclusion: Hence, we propose that this method could be an affordable and reliable way to diagnose/screen CC.

miR-34a negatively regulates cell cycle factor Cdt2/DTL in HPV infected cervical cancer cells.

MicroRNAs have emerged as an important regulator of cell cycle and various other cellular processes. Aberration in microRNAs has been linked with development of several cancers and other diseases but still very little is known about the mechanism by which they regulate these cellular events. High risk human papilloma virus (HR HPV) is the causative agent of 99% of cervical cancer cases which attenuates multiple tumor suppressors and checkpoint factors of the host cell. The viral proteins also stabilize many oncogenic factors, including an essential cell cycle regulator Cdt2/DTL which in turn promotes cell transformation and proliferation. In this study, we report that a micro-RNA, miR-34a by suppressing HPV E6 protein, destabilizes Cdt2/DTL protein level in HPV infected cervical cancer cell lines. Destabilization of Cdt2 stabilizes pro-apoptotic and onco-suppressor proteins like p21 and Set8 and suppresses cell proliferation, invasion and migration capabilities of the HPV positive cervical cancer cells. Overexpression of either HPV E6 or Cdt2 genes along with miR-34a restored back the suppressed proliferation rate. This study is the first-ever report to show that miR-34a regulates cell cycle factor Cdt2 by suppressing viral E6 protein level, thus opening up the possibility of exploring miR-34a as a specific therapy for cervical cancer treatment.