LncRNAs: the good, the bad, and the unknown.

Long non-coding RNAs (lncRNAs) are significant contributors in maintaining genomic integrity through epigenetic regulation. LncRNAs can interact with chromatin-modifying complexes in both cis and trans pathways, drawing them to specific genomic loci and influencing gene expression via DNA methylation, histone modifications, and chromatin remodeling. They can also operate as building blocks to assemble different chromatin-modifying components, facilitating their interactions and gene regulatory functions. Deregulation of these molecules has been associated with various human diseases, including cancer, cardiovascular disease, and neurological disorders. Thus, lncRNAs are implicated as potential diagnostic indicators and therapeutic targets. This review discusses the current understanding of how lncRNAs mediate epigenetic control, genomic integrity, and their putative functions in disease pathogenesis.

Breaking the aging epigenetic barrier.

Aging is an inexorable event occurring universally for all organisms characterized by the progressive loss of cell function. However, less is known about the key events occurring inside the nucleus in the process of aging. The advent of chromosome capture techniques and extensive modern sequencing technologies have illuminated a rather dynamic structure of chromatin inside the nucleus. As cells advance along their life cycle, chromatin condensation states alter which leads to a different epigenetic landscape, correlated with modified gene expression. The exact factors mediating these changes in the chromatin structure and function remain elusive in the context of aging cells. The accumulation of DNA damage, reactive oxygen species and loss of genomic integrity as cells cease to divide can contribute to a tumor stimulating environment. In this review, we focus on genomic and epigenomic changes occurring in an aged cell which can contribute to age-related tumor formation.

Oncogenic lncRNAs alter epigenetic memory at a fragile chromosomal site in human cancer cells.

Chromosome instability is a critical event in cancer progression. Histone H3 variant CENP-A plays a fundamental role in defining centromere identity, structure, and function but is innately overexpressed in several types of solid cancers. In the cancer background, excess CENP-A is deposited ectopically on chromosome arms, including 8q24/cMYC locus, by invading transcription-coupled H3.3 chaperone pathways. Up-regulation of lncRNAs in many cancers correlates with poor prognosis and recurrence in patients. We report that transcription of 8q24-derived oncogenic lncRNAs plays an unanticipated role in altering the 8q24 chromatin landscape by H3.3 chaperone-mediated deposition of CENP-A-associated complexes. Furthermore, a transgene cassette carrying specific 8q24-derived lncRNA integrated into a naïve chromosome locus recruits CENP-A to the new location in a cis-acting manner. These data provide a plausible mechanistic link between locus-specific oncogenic lncRNAs, aberrant local chromatin structure, and the generation of new epigenetic memory at a fragile site in human cancer cells.

Centromeric Transcription: A Conserved Swiss-Army Knife.

In most species, the centromere is comprised of repetitive DNA sequences, which rapidly evolve. Paradoxically, centromeres fulfill an essential function during mitosis, as they are the chromosomal sites wherein, through the kinetochore, the mitotic spindles bind. It is now generally accepted that centromeres are transcribed, and that such transcription is associated with a broad range of functions. More than a decade of work on this topic has shown that centromeric transcripts are found across the eukaryotic tree and associate with heterochromatin formation, chromatin structure, kinetochore structure, centromeric protein loading, and inner centromere signaling. In this review, we discuss the conservation of small and long non-coding centromeric RNAs, their associations with various centromeric functions, and their potential roles in disease.

Association between functional TERT promoter polymorphism rs2853669 and cervical cancer risk in South Indian women.

A single nucleotide polymorphism (SNP) rs2853669 (A>G) in the telomerase reverse transcriptase (TERT) promoter has recently been reported in chr5:1,295,349 T>C (T349C), and was shown to be associated with increased cancer risk and poor survival in a specific population. However, at present, the role of this particular SNP with TERT promoter driver mutations and its genetic association with human papilloma virus (HPV) in patients with cervical cancer has not been determined. In the present study, the genetic association of the functional SNP rs2853669 in the presence/absence of TERT promoter hotspot mutations and HPV in patients with cervical cancer of South Indian origin was evaluated. To understand and compare the frequency of the variant allele and its risk association in different cancer types of various populations, the SNP was genotyped in 257 cervical cancer samples and 295 controls, and its associations with TERT promoter hotspot mutations and HPV were analyzed. Furthermore, an extensive search of previously published articles in PubMed, Embase and Web of Science was conducted; a meta-analysis was carried out to elucidate the association of the SNP with different cancer types in global populations. The SNP analysis showed significantly high frequency (41%) of homozygous variant allele rs2853669 (GG) in patients with cervical cancer compared with control samples [Recessive allele model odds ratio (OR)=1.71; 95% CI=1.20-2.43; P=0.003]. No significant interaction was observed between the TERT SNP rs2853669 and HPV status as well as other hotspot TERT promoter (C228T and C250T) mutations determined in our previous study. In addition, the overall meta-analysis revealed a significant association of the SNP rs2853669 with other cancer types in different ethnic populations (OR=1.09; 95% CI=1.03-1.16; P=0.004). The present results suggested that the TERT SNP rs2853669 could play an important role in the risk of cervical cancer in a South Indian population.

EMT signaling: potential contribution of CRISPR/Cas gene editing.

Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.

LncRNA OIP5-AS1 is overexpressed in undifferentiated oral tumors and integrated analysis identifies as a downstream effector of stemness-associated transcription factors.

Long non-coding RNAs (lncRNAs) play an important role in the regulation of key cellular processes in early development and cancer. LncRNA Oip5-as1 facilitates stem cell self-renewal in mouse by sponging mmu-miR-7 and modulating NANOG level, yet its role in cancer is less understood. We analyzed OIP5-AS1 expression in oral tumors and in TCGA datasets. We observed overexpression of OIP5-AS1 in oral tumors (P < 0.001) and in tumors of epithelial origin from TCGA. OIP5-AS1 expression was strongly associated with undifferentiated tumors (P = 0.0038). In silico analysis showed miR-7 binding site is conserved in mouse and human OIP5-AS1. However, human NANOG 3'-UTR lost the binding site for hsa-miR-7a-3. Therefore, we screened for other miRNAs that can be sponged by OIP5-AS1 and identified six potential miRNAs and their downstream target genes. Expression analysis showed downregulation of miRNAs and upregulation of downstream target genes, particularly in undifferentiated tumors with high-level of OIP5-AS1 suggesting OIP5-AS1 could post-transcriptionally modulate the downstream target genes. Further, systematic epigenomic analysis of OIP5-AS1 promoter revealed binding motifs for MYC, NANOG and KLF4 suggesting that OIP5-AS1 could be transactivated by stemness-associated transcription factors in cancer. OIP5-AS1 overexpression in undifferentiated oral tumors may be suggestive of enhanced cancer stemness, and consequently, poor clinical outcome.

Comprehensive analysis of aberrantly expressed lncRNAs and construction of ceRNA network in gastric cancer.

Gastric cancer remains fifth most common cancer often diagnosed at an advanced stage and is the second leading cause of cancer-related death worldwide. Long non-coding RNAs (lncRNAs) involved in various cellular pathways are essential for tumor occurrence and progression and they have high potential to promote or suppress the expression of many genes. In this study, we profiled 19 selected cancer-associated lncRNAs in thirty gastric adenocarcinomas and matching normal tissues by qRT-PCR. Our results showed that most of the lncRNAs were significantly upregulated (12/19). Further, we performed bioinformatic screening of miRNAs that share common miRNA response elements (MREs) with lncRNAs and their downstream mRNA targets. The prediction identified three microRNAs (miR-21, miR-145 and miR-148a) and five gastric cancer-specific target genes (EGFR, KLF4, DNMT1 and AGO4) which also showed strong correlation with lncRNAs in regression analysis. Finally, we constructed an integrated lncRNA-miRNA-mRNA interaction network of the candidate genes to understand the post-transcriptional gene regulation. The ceRNA network analysis revealed that the differentially regulated miR-21 and miR-148a were playing as central candidates coordinating sponging activity of the lncRNAs analyzed (H19, TUG1 and MALAT1) in this study and the overexpression of H19 and miR-21 could be a signature event of gastric tumorigenesis that could serve as prognostic indicators and therapeutic targets.

Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma.

MicroRNAs (miRNAs) are reported to function as a major component in the cellular signaling circuit, which regulates epithelial-mesenchymal transition (EMT). Dysregulation of the microRNA-200 (miR-200) family and EMT-associated genes enables tumor metastasis and resistance to therapy. The present study profiled miR-200 family members miR-200a, miR-200b, miR-200c, miR-141 and miR-429, and also several EMT-regulatory genes including zinc finger E-box-binding homeobox (ZEB)1, ZEB2, epithelial cadherin and vimentin in 40 oral primary tumors in order to understand their role(s) in oral squamous cell carcinoma (OSCC). The reverse transcription-quantitative polymerase chain reaction was used to analyze each sample. Results demonstrated a significant downregulation of miR-200 family members in tumors with a history of tobacco chewing/smoking (P<0.0006, P=0.0467, P=0.0014, P=0.0087 and P=0.0230, respectively) and undifferentiated pathology (miR-200a, P=0.0067; miR-200c, P=0.0248). EMT markers ZEB2 (P=0.0451) and vimentin (P=0.0071) were significantly upregulated in the oral tumors. Furthermore, ZEB2 antisense RNA1 was overexpressed in 50% of OSCC samples (P=0.0075). EMT-regulatory genes did not exhibit any association with clinical outcome. The present study also analyzed the expression of EMT-regulatory genes in 523 head and neck squamous cell carcinoma (HNSCC) samples from The Cancer Genome Atlas (TCGA) database, and the association with treatment outcome. Analysis of TCGA datasets also demonstrated no significant association in the expression of EMT markers with disease recurrence and treatment outcome. The results of the present study revealed dysregulation of miR-200 family miRNAs and EMT-regulatory genes in OSCC without any significant effect on treatment outcome.

Genotyping of CYP2C9 and VKORC1 polymorphisms predicts south Indian patients with deep vein thrombosis as fast metabolizers of warfarin/acenocoumarin.

Deep vein thrombosis (DVT) is a life-threatening disease. Warfarin and acenocoumarol are anticoagulants used to treat DVT and vary among individuals in terms of treatment response/toxicity. Single nucleotide polymorphisms (SNPs) in CYP2C9 and VKORC1 play a role in the pharmacokinetics and dynamics of warfarin and acenocoumarol and they determine the efficacy of treatment by controlling drug clearance in treated individuals. The aim of the current study was to genotype the critical SNPs of CYP2C9 and VKORC1 genes in a south Indian population in order to understand the metabolizer phenotype of patients with DVT. CYP2C9 (rs1799853, rs1057910, rs1057909, rs28371686) and VKORC1 (rs9923231) SNPs were genotyped in 124 cases of DVT. Genomic regions of these SNPs from genomic DNA were amplified with PCR and directly sequenced using Sanger sequencing except for the SNP rs1799853, which was detected using Sau96I restriction endonuclease-based digestion of variant alleles. Among south Indian patients with DVT, 6.5% (8/124) had the rs1799853 SNP of CYP2C9 and 11% (14/124) had the rs1057910 SNP while 16% (20/124) had the rs9923231 SNP of VKORC1 which were associated with the response to warfarin treatment. None of the patients tested positive for poor drug metabolizing genotypes of the CYP2C9 gene and only 1.6% of the south Indian population was sensitive to warfarin treatment. Genotyping results suggest that a relatively greater amount of the therapeutic drug is required to achieve/maintain the international normalized ratio (INR) in south Indian patients with DVT.

Expression profiling of long non-coding RNA identifies linc-RoR as a prognostic biomarker in oral cancer.

Oral squamous cell carcinoma is the most aggressive cancer that is associated with high recurrence, metastasis, and poor treatment outcome. Dysregulation of long non-coding RNAs has been shown to promote tumor growth and metastasis in several cancers. In this study, we investigated the expression of 11 selected long non-coding RNAs that are associated with cell proliferation, metastasis, and tumor suppression in oral squamous cell carcinomas and normal tissues by quantitative real-time polymerase chain reaction. Out of the 11 long non-coding RNAs profiled, 9 were significantly overexpressed in tumors with tobacco chewing history. Moreover, the long non-coding RNA profile was similar to the head and neck cancer datasets of The Cancer Genome Atlas database. Linc-RoR, a regulator of reprogramming, implicated in tumorigenesis was found to be overexpressed in undifferentiated tumors and showed strong association with tumor recurrence and poor therapeutic response. In oral squamous cell carcinomas, for the first time, we observed linc-RoR overexpression, downregulation of miR-145-5p, and overexpression of c-Myc, Klf4, Oct4, and Sox2, suggesting the existence of linc-RoR-mediated competing endogenous RNA network in undifferentiated tumors. Taken together, this study demonstrated the association of linc-RoR overexpression in undifferentiated oral tumors and its prognostic value to predict the therapeutic response.

Long non-coding RNA CCAT1 is overexpressed in oral squamous cell carcinomas and predicts poor prognosis.

Oral squamous cell carcinoma (SCC) is the most common malignant tumor in India with 5-year survival rates totaling <50%. Recently, dysregulation of non-coding RNA was reported as a potential hallmark of carcinogenesis. Colon Cancer Associated Transcript 1 (CCAT1), an lncRNA located in chromosome 8q24, close to the c-Myc gene, has been reported to be overexpressed in many human cancers. In the present study, the authors analyzed the expression of CCAT1, c-Myc and the miRNAs miR155-5p, let7b-5p, miR490-3p and miR218-5p sponged by CCAT1 in 60 oral tumor and 8 normal tissue samples by reverse transcription-quantitative polymerase chain reaction. CCAT1 was significantly overexpressed in 27% (16/60) of oral tumors. Interestingly, a high level of c-Myc expression was observed in all CCAT1 overexpressing cases (P=0.0473). Furthermore, CCAT1 overexpression significantly downregulated miR155-5p (P=0.03) and let7b-5p (P<0.0001). Oral cancer cases expressing high level of CCAT1 (P=0.01) presented poor therapeutic outcome. To the best of the authors' knowledge, this is the first study to report the overexpression of the CCAT1 in oral SCCs, and the results suggested that CCAT1 overexpression may sponge miR155-5p and let7b-5p, and may account for poor treatment response.

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.

Oral squamous cell carcinoma: microRNA expression profiling and integrative analyses for elucidation of tumourigenesis mechanism.

BACKGROUND: The advantages and utility of microRNAs (miRNAs) as diagnostic and prognostic cancer markers is at the vanguard in recent years. In this study, we attempted to identify and validate the differential expression of miRNAs in oral squamous cell carcinoma (OSCC), to correlate their expression with the clinico-pathological profile of tumours and to identify the signaling pathways through which the aberrantly expressed miRNAs effect tumourigenesis. METHODS: miRCURY LNA™ array with probes specific to 1168 miRNAs and TaqMan assays specific for 10 miRNAs was employed to evaluate and validate miRNA expression in a discovery cohort (n = 29) and validation cohort (n = 61) of primary OSCC tissue specimens, respectively. A computational pipeline with sequential integration of data from miRTarBase, CytoScape, UniProtKB and DIANA-miRPath was utilized to map the target genes of deregulated miRNAs and associated molecular pathways. RESULTS: Microarray profiling identified 46 miRNAs that were differentially expressed in OSCC. Unsupervised clustering demonstrated a high degree of molecular heterogeneity across the tumour samples as the clusters did not represent any of their clinico-pathological characteristics. The differential expression of 10 miRNAs were validated by RT-qPCR (let-7a, let-7d, let-7f and miR-16 were downregulated while miR-29b, miR-142-3p, miR-144, miR-203, and miR-223 were upregulated in OSCC; the expression of miR-1275 was variable in tumours, with high levels associated to regional lymph node invasion; additionally, miR-223 exhibited an association with advanced tumour stage/size). In silico analyses of the experimentally confirmed target genes of miRNAs revamp the relationship of upregulated miRNAs with tumour suppressor genes and of downregulated miRNAs with oncogenes. Further, the differentially expressed miRNAs may play a role by simultaneously activating genes of PI3K/Akt signaling on one hand and by repressing genes of p53 signaling pathway on the other. CONCLUSIONS: The identified differentially expressed miRNAs and signaling pathways deregulated in OSCC have implications for the development of novel therapeutic strategies. To the best of our knowledge, this is the first report to show the association of miR-1275 with nodal invasion and the upregulation of miR-144 in OSCC.

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.

Down Regulation of miR-34a and miR-143 May Indirectly Inhibit p53 in Oral Squamous Cell Carcinoma: a Pilot Study.

BACKGROUND: Aberrant microRNA expression has been associated with the pathogenesis of a variety of human malignancies including oral squamous cell carcinoma (SCC). In this study, we examined primary oral SCCs for the expression of 6 candidate miRNAs, of which five (miR-34a, miR-143, miR-373, miR-380-5p, and miR- 504) regulate the tumor suppressor TP53 and one (miR-99a) is involved in AKT/mTOR signaling. MATERIALS AND METHODS: Tumor tissues (punch biopsies) were collected from 52 oral cancer patients and as a control, 8 independent adjacent normal tissue samples were also obtained. After RNA isolation, we assessed the mature miRNA levels of the 6 selected candidates against RNU44 and RNU48 as endogenous controls, using specific TaqMan miRNA assays. RESULTS: miR-34a, miR-99a, miR-143 and miR-380-5p were significantly down-regulated in tumors compared to controls. Moreover, high levels of miR-34a were associated with alcohol consumption while those of miR-99a and miR-143 were associated with advanced tumor size. No significant difference was observed in the levels of miR-504 between the tumors and controls whereas miR-373 was below the detection level in all but two tumor samples. CONCLUSIONS: Low levels of miR-380-5p and miR-504 that directly target the 3'UTR of TP53 suggest that p53 may not be repressed by these two miRNAs in OSCC. On the other hand, low levels of miR-34a or miR-143 may relieve MDM4 and SIRT1 or MDM2 respectively, which will sequester p53 indicating an indirect mode of p53 suppression in oral tumors.