Hesperidin - loaded PVA/alginate hydrogel: targeting NFκB/iNOS/COX-2/TNF-α inflammatory signaling pathway.

Introduction: Skin injuries represent a prevalent form of physical trauma, necessitating effective therapeutic strategies to expedite the wound healing process. Hesperidin, a bioflavonoid naturally occurring in citrus fruits, exhibits a range of pharmacological attributes, including antimicrobial, antioxidant, anti-inflammatory, anticoagulant, and analgesic properties. The main objective of the study was to formulate a hydrogel with the intention of addressing skin conditions, particularly wound healing. Methods: This research introduces a methodology for the fabrication of a membrane composed of a Polyvinyl alcohol - Sodium Alginate (PVA/A) blend, along with the inclusion of an anti-inflammatory agent, Hesperidin (H), which exhibits promising wound healing capabilities. A uniform layer of a homogeneous solution comprising PVA/A was cast. The process of crosslinking and the enhancement of hydrogel characteristics were achieved through the application of gamma irradiation at a dosage of 30 kGy. The membrane was immersed in a Hesperidin (H) solution, facilitating the permeation and absorption of the drug. The resultant system is designed to deliver H in a controlled and sustained manner, which is crucial for promoting efficient wound healing. The obtained PVA/AH hydrogel was evaluated for cytotoxicity, antioxidant and free radical scavenging activities, anti-inflammatory and membrane stability effect. In addition, its action on oxidative stress, and inflammatory markers was evaluated on BJ-1 human normal skin cell line. Results and Discussion: We determined the effect of radical scavenging activity PVA/A (49 %) and PVA/AH (87%), the inhibition of Human red blood cell membrane hemolysis by PVA/AH (81.97 and 84.34 %), hypotonicity (83.68 and 76.48 %) and protein denaturation (83.17 and 85.8 %) as compared to 250 µg/ml diclofenac (Dic.) and aspirin (Asp.), respectively. Furthermore, gene expression analysis revealed an increased expression of genes associated with anti-oxidant and anti-inflammatory properties and downregulated TNFα, NFκB, iNOS, and COX2 by 67, 52, 58 and 60%, respectively, by PVA/AH hydrogel compared to LPS-stimulated BJ-1 cells. The advantages associated with Hesperidin can be ascribed to its antioxidant and anti-inflammatory attributes. The incorporation of Hesperidin into hydrogels offers promise for the development of a novel, secure, and efficient strategy for wound healing. This innovative approach holds potential as a solution for wound healing, capitalizing on the collaborative qualities of PVA/AH and gamma irradiation, which can be combined to establish a drug delivery platform for Hesperidin.

Transcriptome-wide profiling identifies colon cancer-associated m6A transcripts and potential RNA methyl modifiers.

BACKGROUND: N6-methyladenosine (m6A) is a prevalent and crucial RNA methylation modification that plays a significant role in various biological and pathological processes. The dysregulation of m6A has been linked to the initiation, progression, and metastasis of several cancer types, including colon cancer. The transcriptome of colon cancer indeed provides insight into dysregulated coding and non-coding RNAs, but it does not reveal the mechanisms, such as m6A modifications, that determine post-transcriptional and pre-translational regulations. This study using MeRIP sequencing aims to explain the distribution of m6A modification across altered gene expression and its association with colon cancer. METHODS AND RESULTS: The levels of m6A in different colon cancer cell lines were quantified and correlated with the expression of m6A modifiers such as writers, readers, and erasers. Our results showed that global m6A levels in colon cancer were associated with METTL14, YTHDF2, and YTHDC1. We performed Epi-transcriptome profiling of m6A in colon cancer cell lines using Methylated RNA Immunoprecipitation (MeRIP) sequencing. The differential methylation analysis revealed 7312 m6A regions among the colon cancer cell lines. Our findings indicated that the m6A RNA methylation modifications were mainly distributed in the last exonic and 3' untranslated regions. We also discovered that non-coding RNAs such as miRNA, lncRNA, and circRNA carry m6A marks. Gene set enrichment and motif analysis suggested a strong association of m6A with post-transcriptional events, particularly splicing control. Overall, our study sheds light on the potential role of m6A in colon cancer and highlights the importance of further investigation in this area. CONCLUSION: This study reports m6A enrichment in the last exonic regions and 3' UTRs of mRNA transcripts in colon cancer. METTL14, YTHDF2, and YTHDC1 were the most significant modifiers in colon cancer cells. The functions of m6A-modified genes were found to be RNA methylation and RNA capping. Overall, the study illustrates the transcriptome-wide distribution of m6A and its eminent role in mRNA splicing and translation control of colon cancer.

Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis.

An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>-5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

Experimental and Computational Approaches for Non-CpG Methylation Analysis.

Cytosine methylation adjacent to adenine, thymine, and cytosine residues but not guanine of the DNA is distinctively known as non-CpG methylation. This CA/CT/CC methylation accounts for 15% of the total cytosine methylation and varies among different cell and tissue types. The abundance of CpG methylation has largely concealed the role of non-CpG methylation. Limitations in the early detection methods could not distinguish CpG methylation from non-CpG methylation. Recent advancements in enrichment strategies and high throughput sequencing technologies have enabled the detection of non-CpG methylation. This review discusses the advanced experimental and computational approaches to detect and describe the genomic distribution and function of non-CpG methylation. We present different approaches such as enzyme-based and antibody-based enrichment, which, when coupled, can also improve the sensitivity and specificity of non-CpG detection. We also describe the current bioinformatics pipelines and their specific application in computing and visualizing the imbalance of CpG and non-CpG methylation. Enrichment modes and the computational suites need to be further developed to ease the challenges of understanding the functional role of non-CpG methylation.

Alteration of miR-362-5p and miR-454-3p expression elicits diverse responses in breast cancer cell lines.

BACKGROUND: The heterogeneity of breast tumors presents a challenge in disease management, necessitating an understanding of the molecular mechanisms driving breast tumorigenesis. Aberrant expression of microRNAs is known to promote tumor growth and progression. Our previous RNA-sequencing dataset revealed the upregulation of miR-362-5p and miR-454-3p in breast tumors. We investigated potential role of miR-362-5p and miR-454-3p in breast cancer using MDAMB231 and MCF7 cell lines. METHODS AND RESULTS: The expression of miR-362-5p and miR-454-3p were altered in MCF7 and MDAMB231 using mimics and inhibitors. The effect on cell viability, cell cycle progression and migration was assessed using Alamar blue assay, flow cytometry and wound healing assay. Further, the expression of potential target genes were measured using real-time PCR. Our results indicated that an increased expression of miR-362-5p promoted cell growth and survival in MCF7, but decreased cell migration. In contrast, miR-362-5p overexpression reduced cancer cell growth, survival and migration in MDAMB231. Overexpression of miR-454-3p was oncogenic in both cell lines but suppressed migration in the aggressive cell line MDAMB231. CONCLUSION: Two microRNAs, miR-362-5p and miR-454-3p, were evaluated for functional activity in breast cancer cell lines and they showed increased proliferative signals and tumorigenic properties.

Identification of novel dysregulated circular RNAs in early-stage breast cancer.

Breast cancer is a major cause of cancer-related death in women worldwide. Non-coding RNAs are a potential resource to be used as an early diagnostic biomarker for breast cancer. Circular RNAs are a recently identified group of non-coding RNA with a significant role in disease development with potential utility in diagnosis/prognosis in cancer. In this study, we identified 26 differentially expressed circular RNAs associated with early-stage breast cancer. RNA sequencing and two circRNA detection tools (find_circ and DCC) were used to understand the circRNA expression signature in breast cancer. We identified hsa_circ_0006743 (circJMJD1C) and hsa_circ_0002496 (circAPPBP1) to be significantly up-regulated in early-stage breast cancer tissues. Co-expression analysis identified four pairs of circRNA-miRNA (hsa_circ_0023990 : hsa-miR-548b-3p, hsa_circ_0016601 : hsa_miR-1246, hsa_circ_0001946 : hsa-miR-1299 and hsa_circ_0000117:hsa-miR-502-5p) having potential interaction. The miRNA target prediction and network analysis revealed mRNA possibly regulated by circRNAs. We have thus identified circRNAs of diagnostic implications in breast cancer and also observed circRNA-miRNA interaction which could be involved in breast cancer development.

Perspectives of long non-coding RNAs in cancer.

A recent advance in transcriptomics has spawned the 'Decade of non-coding RNAs' by potentiating the growing numbers of long non-coding RNA in cancer. LncRNA involvement in cancer denotes its significance beyond our perception as they participate in tumor suppression and promoting oncogenesis, which raises them as a mighty class of effectors or regulators. Aberrantly expressed lncRNAs interact with major protein and coding partners, which ultimately deregulate normal cellular processes and drive the cell towards malignant state. Identification of theses interactions are utmost important as lncRNAs can be ideal targets for therapy. Dysregulation of lncRNAs by genomic alterations like single nucleotide variations and gene fusions are also potential modulators of their secondary structure. In this review, we discuss the various molecular interactions of lncRNAs with major bio-molecules and genetic variations in lncRNA genes and their importance in cancer. This systematic review outlines the vivid role of lncRNAs in cancer context and opens up future conceptual applications.

Analysis of APOBEC3A/3B germline deletion polymorphism in breast, cervical and oral cancers from South India and its impact on miRNA regulation.

Breast cancer and cervical cancer are the leading causes of death in women worldwide as well as in India, whilst oral cancer is the top most common cancer among Asian especially in Indian men in terms of both incidence and mortality rate. Genetic factors determining the predisposition to cancer are being explored to identify the signature genetic variations associated with these cancers. Recently, a germline deletion polymorphism in APOBEC3 gene cluster which completely deletes APOBEC3B coding region has been studied for its association with cancer risk. We screened the germline deletion polymorphism in 409 cancer patients (224 breast cancer, 88 cervical cancer and 97 oral cancer samples), 478 controls and 239 cervical cancer tissue DNAs of South Indian origin. The results suggest that the APOBEC3A/3B deletion polymorphism is not significantly associated with cancer risk in our study population (OR 0.739, 95 % CI, p value 0.91457). Considering the viral restriction property of APOBEC3s, we also screened cervical cancer tissue DNAs for the human papilloma virus infection. We observed a gradual increase in the frequency of HPV16 infection from AA/BB cases (66.86 %) to AA/-- cases (71.43) which signifies the impact of this deletion polymorphism in HPV infection. In addition, we performed in silico analysis to understand the effect of this polymorphism on miRNA regulation of the APOBEC3A/3B fusion transcript. Only 8 APOBEC3B targeting miRNAs were observed to regulate the fusion transcript of which miR-34b-3p and miR-138-5p were found to be frequently downregulated in cancers suggesting miRNA-mediated deregulation of APOBEC3A expression in cancer patients harbouring this particular deletion polymorphism.

TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas.

Squamous cell carcinoma (SCC) of the uterine cervix and oral cavity are most common cancers in India. Telomerase reverse transcriptase (TERT) overexpression is one of the hallmarks for cancer, and activation through promoter mutation C228T and C250T has been reported in variety of tumors and often shown to be associated with aggressive tumors. In the present study, we analyzed these two hot spot mutations in 181 primary tumors of the uterine cervix and oral cavity by direct DNA sequencing and correlated with patient's clinicopathological characteristics. We found relatively high frequency of TERT hot spot mutations in both cervical [21.4 % (30/140)] and oral [31.7 % (13/41)] squamous cell carcinomas. In cervical cancer, TERT promoter mutations were more prevalent (25 %) in human papilloma virus (HPV)-negative cases compared to HPV-positive cases (20.6 %), and both TERT promoter mutation and HPV infection were more commonly observed in advanced stage tumors (77 %). Similarly, the poor and moderately differentiated tumors of the uterine cervix had both the TERT hot spot mutations and HPV (16 and 18) at higher frequency (95.7 %). Interestingly, we observed eight homozygous mutations (six 228TT and two 250TT) only in cervical tumors, and all of them were found to be positive for high-risk HPV. To the best of our knowledge, this is the first study from India reporting high prevalence of TERT promoter mutations in primary tumors of the uterine cervix and oral cavity. Our results suggest that TERT reactivation through promoter mutation either alone or in association with the HPV oncogenes (E6 and E7) could play an important role in the carcinogenesis of cervical and oral cancers.