Methylation Analysis On Self-samples As Triage Tool For HPV-positive Women
A methylation, sample technology, used in cancer prevention and medical diagnostics, to solve problems such as inability to perform
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Embodiment 1
[0063] Example 1. HPV-immortalized (immortalized) keratinocyte line (HPV-immortalized keratinocyte cell lines) and cervical cancer cell lines Hsa-miR124 methylation
[0064]Because the mature Hsa-miR124 sequence is encoded at three distinct genomic locations (hsa-miR124-1[8p23.1], hsa-miR124-2[8q12.3], and hsa-miR124-3[20q13.33]), The hsa-miR124 methylation status was therefore determined at all 3 loci by quantitative methylation-specific PCR (MSP). The following amplicons were detected: hsa-miR124-1: nt811 to 904; hsa-miR124-2: nt701 to 838; hsa-miR124-3: nt897 to 991, each involving figure 2 sequence shown in . The housekeeping gene B-actin was used as an internal reference (Harden et al., J Urology 2003; 169:1138-1142). Using these assays, we did not find hsa-miR124 methylation (EK) in primary keratinocytes isolated from three different donors, however, in three hrHPV-containing cervical cancer cell lines (SiHa, HeLa and CaSki ) methylates the corresponding CpG is...
Embodiment 2
[0065] Example 2: Overexpression of hsa-miR124 in SiHa cells has anti-proliferative and anti-migratory effects effect
[0066] To determine the possible functional relevance of reduced hsa-miR124 expression in cervical carcinogenesis, two cervical cancer cell lines, SiHa and CaSki, were transduced with a retroviral vector containing the genetic components leading to stable expression of hsa-miR124. Expression analysis by RT-PCR confirmed ectopic expression of hsa-miR124 in hsa-miR124 transductants and lack of expression in cells transduced with empty retroviral vector. After 6 days of culture, ectopic expression of hsa-miR124 in SiHa and CaSki transductants resulted in a significant decrease in cell proliferation compared to hsa-miR-ctrl transducers (p<0.05). Furthermore, using the wound healing assay, SiHa hsa-miR124 transductants were found to have reduced migratory ability compared to SiHa hsa-miR-ctrl cells.
Embodiment 3
[0067] Example 3: Hsa-miR124 promoter methylation in cervical tissue samples
[0068] To determine if and when DNA hypermethylation of all three hsa-miR124 regulatory regions occurs during the course of cervical carcinogenesis in vivo, we performed mild CIN lesions ( CIN1), severe CIN lesions (CIN3) and cervical cancers (SCCs and AdCas), quantitative MSP analysis was performed using primers and probes as defined in Table 2. In normal cervix, 6% (1 / 18) samples were positively marked for methylation on hsa-miR124-3, while in none of the normal samples, methylation was detected on the other two gene regulatory regions ( hsa-miR124-1 and hsa-miR124-2). In CIN1 lesions, 28% (10 / 36) showed methylation on hsa-miR124-1, 6% (2 / 36) on hsa-miR124-2 and 11% ( 4 / 36). Methylation positivity in CIN3 lesions ranged from 46% (19 / 41) in hsa-miR124-1, 20% (8 / 41) in hsa-miR124-2 to 10% (4 / 41) in hsa-miR124-3 different. The frequencies of hsa-miR124-1, hsa-miR124-2 and hsa-miR124-3 as detec...
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