Differential promoter usages of PTCH1 and down regulation of HHIP are associated with HNSCC progression

Chakraborty, Balarko; Basu, Mukta; Mukhopadhyay, Debnath; Alam, Neyaz; Ghosh, Supratim; Dutta, Sankhadeep; Panda, Chinmay Kumar

doi:10.1016/j.prp.2022.153827

Cited by 6 publications

(8 citation statements)

References 37 publications

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“…In addition, the different layers (basal-parabasal/spinous layers) of serial cryo-sections (5µM) of normal cervical epithelium were also separated by the LCM technique and collected into a microcentrifuge tube [Fig S1]. DNA from the micro-dissected samples, PBL, plasma (isolated from PBL) and the CACX cell lines (SiHa and HeLa) were isolated by Proteinase-K digestion followed by the phenolchloroform method [Chakraborty et al, 2022].…”

Section: Dna Extraction From Samplesmentioning

confidence: 99%

“…The primer sequences are listed in Table S2B. The dCt value was obtained using the formula: dCt= [(Ct value) target gene-(Ct value) B2M, [Chakraborty et al, 2022] B. Immunohistochemical characterization of proteins:…”

Section: Expression Analysismentioning

confidence: 99%

“…In deletion mapping, a standard PCR was carried out using a γ[p32] ATP labeled forward primer and T4polynucleotide kinase (Promega), followed by amplifying the genomic DNA and nally performed electrophoresis on 8% denaturing polyacrylamide gel containing 8M urea. Loss of heterozygosity (LOH) was scored by densitometric scanning (Bio-Rad GS-800, USA) to determine the loss of one allele in the tumour compared to the same allele in normal (PBL) [Chakraborty et al, 2022]. Microsatellite size alteration (MA) was detected as a shift in one (MA-I) allele compared with their normal alleles.…”

Section: Promoter Methylation Analysismentioning

confidence: 99%

“…The value of fold change (ddCt) ≤0.66 and (ddCt) ≥1.5 was considered as under-expression and overexpression respectively [Chakraborty et al, 2022].…”

Section: Treatment Of Chemotherapeutic Drug (Cisplatin) On Cacx Cell ...mentioning

confidence: 99%

“…The promoter methylation frequency of the genes was also quanti ed by real-time PCR using a power SYBR Green assay (Applied Biosystems, USA). The percentage of hypomethylation was calculated using the following formula: (Ct [digested]/Ct [undigested] ×100) -100), [Chakraborty et al, 2022].…”

Section: Promoter Methylation Analysismentioning

confidence: 99%

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Down-regulation of FA-BRCA pathway in the molecular pathogenesis of uterine cervical carcinoma of Indian population: clinical and prognostic implications

Dutta

Pal

Sultana

et al. 2022

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Objective: Our study was aimed to understand the importance of FA-BRCA pathway genes in cervical carcinogenesis and their association with the prognosis of the disease. Material and methods: we analysed the molecular profiles of the key regulatory genes of FA-BRCA pathway (BRCA1, BRCA2, FANCC, FANCD2) in 109 cervical lesions at different clinical stages and validated in different bioinformatical analysis as well. The results were next correlated with different clinicopathological parameters. Furthermore, the drug tolerance mechanism of the genes was characterized by treating two CACX cell lines (SiHa and HeLa) in presence of the chemotherapeutic drug cisplatin. Result: Our data showed that the expression pattern (mRNA/Protein) of the genes of FA-BRCA pathway was gradually decreased from normal cervical epithelium to the development of carcinogenesis, also validated in different GEO datasets. Further, in-depth look into the results revealed that genetic (deletion) and epigenetic alterations (promoter methylation) [30 to 55 %] of the genes was strongly correlated with their reduced expression and development of cervical cancer among the patients, resulting in worst 5-year overall survival trend. Incidentally, the prevalence of promoter methylation in both plasma and respective tumour DNA of invasive cervical carcinoma patients implicated its prognostic importance and association with disease recurrence in this study. In continuation of that, our in-vitro study revealed that cisplatin could upregulate the FA-BRCA pathway genes gradually with increasing drug concentrations in the CACX cell lines through promoter hypomethylation due to reduced expression of DNMT1, indicating the intrinsic mechanism of drug tolerance of residual tumour cells of the disease.Conclusion: Our data showed that the inactivation of FA-BRCA pathway was associated with the development and prognosis of CACX. Up-regulation of the pathway genes in presence of cisplatin in the CACX cell lines suggested a plausible mechanism of non-responsiveness to the therapy.

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Section: Dna Extraction From Samplesmentioning

confidence: 99%

Section: Expression Analysismentioning

confidence: 99%

Section: Promoter Methylation Analysismentioning

confidence: 99%

“…The value of fold change (ddCt) ≤0.66 and (ddCt) ≥1.5 was considered as under-expression and overexpression respectively [Chakraborty et al, 2022].…”

Section: Treatment Of Chemotherapeutic Drug (Cisplatin) On Cacx Cell ...mentioning

confidence: 99%

Section: Promoter Methylation Analysismentioning

confidence: 99%

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