A comprehensive review on the functional role of miRNA clusters in cervical cancer.

Cervical cancer (CC) poses a significant health threat in women globally. MicroRNA clusters (MCs), comprising multiple miRNA-encoding genes, are pivotal in gene regulation. Various factors, including circular RNA and DNA methylation, govern MC expression. Dysregulated MC expression correlates strongly with CC development via promoting the acquisition of cancer hallmarks. Certain MCs show promise for diagnosis, prognosis and therapy selection due to their distinct expression patterns in normal, premalignant and tumor tissues. This review explains the regulation and biological functions of MCs and highlights the clinical relevance of abnormal MC expression in CC.

Cervical cancer is a major global health concern, mostly caused by human papillomavirus infection, resulting in a high number of new cases and fatalities annually. This review examines the role of specific genetic variables known as microRNA clusters (MCs) in the development and progression of cervical cancer. The MCs harbor many microRNAs that control genes related to tumor proliferation, infiltration and dissemination. Understanding the functioning of these MCs can aid in early diagnosis of cervical cancer and predicting its patterns of behavior. We explore the potential benefits of assessing MC expression levels in cancer staging and prognosis, and the development of diagnostic tools and treatments. Targeting these molecular targets could provide interesting opportunities for future cancer treatments.

Human papillomavirus-driven repression of NRF2 signalling confers chemo-radio sensitivity and predicts prognosis in head and neck squamous cell carcinoma.

PURPOSE: To investigate the role of NRF2 signalling in conferring superior prognosis in patients with HPV positive (HPV+ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV-ve) HNSCC and develop molecular markers for selection of HPV+ve HNSCC patients for treatment de-escalation trials. METHODS: NRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ve HNSCC and HPV-ve HNSCC in prospective and retrospective tumor samples as well as from TCGA database were compared. Cancer cells were transfected with HPV-E6/E7 plasmid to elucidate if HPV infection represses NRF2 activity and sensitizes to chemo-radiotherapy. RESULTS: Prospective analysis revealed a marked reduction in expression of NRF2, and its downstream genes in HPV+ve tumors compared to HPV-ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ve HNSCC compared to HPV-ve HNSCC and revealed that HPV+ve HNSCC patients with 'low NQO1' expression showed better overall survival compared to HPV+ve HNSCC patients with 'high NQO1' expression. Ectopic expression of HPV-E6/E7 plasmid in various cancer cells repressed constitutive NRF2 activity, reduced total GSH, increased ROS levels, and sensitized the cancer cells to cisplatin and ionizing radiation. CONCLUSION: Low constitutive NRF2 activity contributes to better prognosis of HPV+ve HNSCC patients. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV + ve HNSCC patients for de-escalation trials.

Integrated Bioinformatics Analysis Identifies Crucial Biochemical Processes Shared between Pancreatitis and Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with rapid progression and an abysmal prognosis. Previous research has shown that chronic pancreatitis can significantly increase the risk of developing PDAC. The overarching hypothesis is that some of the biological processes disrupted during the inflammatory stage tend to show significant dysregulation, even in cancer. This might explain why chronic inflammation increases the risk of carcinogenesis and uncontrolled proliferation. Here, we try to pinpoint such complex processes by comparing the expression profiles of pancreatitis and PDAC tissues. METHODS: We analyzed a total of six gene expression datasets retrieved from the EMBL-EBI ArrayExpress and NCBI GEO databases, which included 306 PDAC, 68 pancreatitis and 172 normal pancreatic samples. The disrupted genes identified were used to perform downstream analysis for ontology, interaction, enriched pathways, potential druggability, promoter methylation, and the associated prognostic value. Further, we performed expression analysis based on gender, patient's drinking habit, race, and pancreatitis status. RESULTS: Our study identified 45 genes with altered expression levels shared between PDAC and pancreatitis. Over-representation analysis revealed that protein digestion and absorption, ECM-receptor interaction, PI3k-Akt signaling, and proteoglycans in cancer pathways as significantly enriched. Module analysis identified 15 hub genes, of which 14 were found to be in the druggable genome category. CONCLUSION: In summary, we have identified critical genes and various biochemical processes disrupted at a molecular level. These results can provide valuable insights into certain events leading to carcinogenesis, and therefore help identify novel therapeutic targets to improve PDAC treatment in the future.

Detection of mitochondrial dysfunction in vitro by laser-induced autofluorescence.

Mitochondrion plays a significant role in a variety of biological functions. Because of their diverse character and location in the cellular systems, mitochondria commonly get exposed to various extrinsic and intrinsic cellular stresses. The present study reports a novel approach to detection of mitochondrial dysfunction based on tryptophan autofluorescence of its proteins in mouse liver, using laser-induced fluorescence (LIF) as a tool. Mitochondria, isolated from the mouse liver, were initially tested for purity and integrity using lactate dehydrogenase and succinate dehydrogenase (SDH) assays. Mitochondrial stress was induced by treating the isolated mitochondria with heavy metals at 10 and 0.01 mM for sodium arsenite and mercuric chloride, respectively. Upon treatment with the heavy metal, tryptophan autofluorescence quenching was recorded at 281 nm excitation. The functional integrity of the mitochondria treated with heavy metals was evaluated by measuring SDH and cytochrome c oxidase activities at various concentrations of mitochondria, which showed impaired activity as compared to control upto a concentration of 6.25 µg. A significant shift was also observed in the autofluorescence of proteins upto the level below 1 µg, suggesting their conformational change and hence altered structural integrity of mitochondria. Circular dichroism spectroscopy data of the mitochondrial proteins treated with heavy metals further validates their conformational change as compared to untreated control. The present study clearly shows that the LIF can be a novel detection tool to detect altered structural integrity of cellular mitochondria upon stress, and it also possesses the potentiality to combine with other interdisciplinary modalities.

Clustered miRNAs and their role in biological functions and diseases.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs known to regulate expression of protein-coding genes. A large proportion of miRNAs are highly conserved, localized as clusters in the genome, transcribed together from physically adjacent miRNAs and show similar expression profiles. Since a single miRNA can target multiple genes and miRNA clusters contain multiple miRNAs, it is important to understand their regulation, effects and various biological functions. Like protein-coding genes, miRNA clusters are also regulated by genetic and epigenetic events. These clusters can potentially regulate every aspect of cellular function including growth, proliferation, differentiation, development, metabolism, infection, immunity, cell death, organellar biogenesis, messenger signalling, DNA repair and self-renewal, among others. Dysregulation of miRNA clusters leading to altered biological functions is key to the pathogenesis of many diseases including carcinogenesis. Here, we review recent advances in miRNA cluster research and discuss their regulation and biological functions in pathological conditions.

DNA methylation regulated microRNAs in human cervical cancer.

Regulation of miRNA gene expression by DNA promoter methylation may represent a key mechanism to drive cervical cancer progression. In order to understand the impact of DNA promoter methylation on miRNAs at various stages of cervical carcinogenesis, we performed DNA methylation microarray on Normal Cervical Epithelium (NCE), Cervical Intraepithelial Neoplasia (CIN I-III) and Squamous Cell Carcinoma (SCC) tissues to identify differentially methylated miRNAs followed by validation by bisulfite sequencing. Further, expression of miRNAs was analyzed by qRT-PCR in clinical tissues and cervical cancer cell lines. Transcriptional activity was determined by luciferase assay. We identified a total of 69 hypermethylated and hypomethylated miRNA promoters encompassing 78 CpG islands in all except Y chromosome, among the three groups. The candidate DNA promoters of miR-424 were significantly hypermethylated and miR-200b and miR-34c were significantly hypomethylated in SCC compared to NCE (P < 0.05). Expression of miR-424, miR-200b, and miR-34c were inversely correlated with promoter DNA methylation in tissue samples. Treatment of cell lines with 5-aza-2'-deoxycytidine showed differential expression in all three miRNAs. We observed a decrease in miRNA promoter activity following in vitro SssI methylase treatment of miR-424, miR-200b, and miR-34c. Luciferase assay demonstrated that miR-200b and miR-424 functionally interacts with 3'-UTR of HIPK3 and RBBP6 respectively and decreased their activity in presence of miR-200b and miR-424 mimics transfected in SiHa cells. Taken together, we have identified deregulation of miRNAs by aberrant DNA promoter methylation, leading to its transcriptional silencing during cervical carcinogenesis, which can be potential targets for diagnosis and therapy.

Age dependent neuroprotective effects of medhya rasayana prepared from Clitoria ternatea Linn. in stress induced rat brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Indian traditional medicinal system in Ayurveda suggests several preparations, known as medhya rasayanas, of diverse plant origin to enhance the health in general, reduce stress and improve brain function in particular during ageing. These effects in the context of contemporary knowledge and the underlying mechanisms are not clearly understood. Autophagy and DNA damage induced repair are inter-related quintessential pathways and are significantly altered during stress and ageing. Hence, medhya rasayana prepared from Clitoria ternatea (locally known as shankhpushpi) was used to test these effects in Wistar rat model of various age groups upon stereotaxic mediated kainic acid induced brain injury. MATERIALS AND METHODS: The rodent experiments were carried out in one, twelve and eighteen months old male Wistar rats. The rats were orally fed with medhya rasayana prepared from Clitoria ternatea (3g per kg body weight/day) for 60 days. Stereotaxic mediated kainate stress to the hippocampus was performed on day 61. The rats were sacrificed on 66th day and the brain tissues were analyzed histologically and measured for autophagy, base excision repair and antioxidant enzyme activities. In addition, cognitive functions were analyzed by employing novel object recognition task and Morris water maze tests. The gene expression profile of hippocampus was assessed by microarray hybridization and two genes are validated. RESULTS: Our study showed significant decrease of autophagy by medhya rasayana in both 12 and 18 months old rats. The hippocampal CA3 cellularity were increased in stereotaxic mediated stressed rats by medhya rasayana. There were no significant differences in constitutive base excision repair and antioxidant enzyme activities. Medhya rasayana treatment also significantly increased episodic memory in rats. Microarray experiments for pathway specific gene expression analysis showed altered expression of genes of long-term potentiation, axon guidance, neuroactive ligand-receptor interaction, regulation of autophagy, lysosome, homologous recombination and nucleotide excision repair pathways in adult rats by medhya rasayana. CONCLUSIONS: In the present study, we show that reduction in autophagy is crucial for medhya rasayana induced protection of rat hippocampal cells and that artificially enhanced autophagy protects the brain cell damage by maintaining the selective DNA damage repair pathway and removal of reactive oxygen species to inhibit apoptosis. These findings suggest autophagy directed pathways by medhya rasayana prepared from C. ternatea protects the brain cells from stress induced injury.