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Cervical carcinoma is one of the most common malignancies in women worldwide. Persistent infection with high risk human papillomavirus is the most significant factor for carcinogenesis of cervical cancer. Therapeutic modalities of cervical carcinoma are not efficiently controlling the progression of cancer in one third of patients. Therefore, understanding of molecular mechanisms in cancer progression is important to prevent disease progression and improve treatment efficacy leading to the reduction of incidence and mortality rate. We investigated genes differentially expressed in therapeutic response and non–response cervical carcinoma stage IIIB using the human whole genome microarrays. The differential expression of genes in each group was constructed for the network by IPA software. Differentially expressed genes in non–responses compared with responses were identified, which were 167 up-regulated and 146 down-regulated genes. The up-regulated genes have biological functions involved in signal transduction (CXCL10, ERBB2, TRAF5, FPRL1, IL1B, FGFBP1 and SCTR), cell proliferation (REG1A, AREG, TGFBI, ERBB2, PIM2, EIF5A and ISG20), proteolysis (PLAU, CRADD, C1R, KLK14, TMEM27, ICEBERG, BF and ADAMTS8), and cell migration (ERBB2, IL8, IL1B, and FPRL1). The down-regulated genes have biological functions involved in transcription (GLIS1, ZNF337, SOX4, PITX1, LASS4, GRHL3, CRABP2, SUHW3, TLE2 and KLF8), cell adhesion (PCDHB6, TRO, MAGI1, PVRL4, SLURP1 and MUC4), and signal transduction (CD38, CXCL14, HTR2B, CEACAM6, DPYSL3, NCR1, CRABP2, CLIC3, TLE2 and PLXNA2). Our study indicates that multiple genes play distinct roles in response or non-response to therapy in cervical carcinoma.
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It was well established that HPV infection, especially type 16 and 18 is the major cause of cervical cancer. Immune evasion of infected cells or tumor cells is also known as one of contributing factors of tumor development caused by down-regulation of MHC class I expression. The aim of this study is to investigate the level and pattern of MHC class I expression in 182 cervical cancerous tissues and 57 normal cervix using immunoperoxidase staining technique. Of 182 cervical cancerous tissues, 50 cases were classified as pre-cancerous lesions whereas, 132 cases were invasive cervical carcinoma. No significant association in grading or intensity of MHC class I was shown between patient and control group. Interestingly, the distribution pattern of MHC class I was significantly shown as non-surface presenting molecules accumulating in cytoplasm of cancerous tissues compared to normal cervix tissues with p-value \< 0.001 χ² test .This result indicates that tumor cells have lost their immune surveillance by reducing their antigen presenting MHC class I molecules at cell surface. Moreover, this alteration can be used as an early marker for cancer prognosis in pre-cancerous group. Therefore, the mechanism of the impairment in antigen presenting of MHC class I molecules should be investigated in further study.
RESUMEN
Cervical cancer is the most common cancer among women worldwide. Even though HPV infection is known as the major risk factor of cervical carcinogenesis, immune evasion of infected cells or tumor cells is also well established as the major contributing factors. Down regulation of MHC class I as well as the presence of non-surface presenting MHC class I molecules have been previously reported. Our aim of this study is to investigate the expression level of tapasin protein which is one of the MHC class I antigen processing proteins in various stages of cancerous tissues. Seventy nine dysplasia and 49 cases of cervical cancer and 49 normal cervix from myoma patients as control were included in this study. The association between the expression level and disease progression was analyzed using χ2 for trend. The reduction of tapasin expression is significantly associated with respect to the progression of staging in both dysplasia and cervical cancer group (p=0.001; χ2 for trend). In conclusion, our study illustrates that the reduction in tapasin expression might exert as one of the mechanism leading to the impairment of antigen presenting of MHC class I in cancerous tissues. Therefore, tapasin staining might be useful as an early prognostic marker in dysplasia. More antigen processing proteins should be also further investigated.
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Infection with high-risk HPV has been implicated as one of the major risk factors of cervical cancer. Integration of viral DNA into host DNA is essential for cervical carcinogenesis. This study was aimed to develop a multiplex real-time PCR to quantify Early gene 2 (E2 gene) and Early gene 6 (E6 gene) of HPV16 using Taqman probe. The ratio of E2 and E6 copy number was calculated to determine physical status of HPV16. The pure episomal form was expected to have an equivalent copy number of E2 and E6 gene giving rise to E2/E6 ratio of 1 whereas, viral integration resulted in less E2 than E6. The detection limit as well as precision of this developed method were obtained at 103 copies with CV of less than 10%. Cut off value of E2/E6 ratio for complete episomal form was found more than 0.83, whereas complete integration was expected to 0. This method was further analyzed with DNA from 15 pre-invasive or dysplasia lesions and 15 invasive cervical carcinoma tissues. The percentage of total integration form in invasive cases showed significantly higher than pre-invasive cervical lesions which obtained about 93%(14/15) and 66%(10/15), respectively (p value \< 0.05). The method described here is sensitive to assess the physical state as well as viral copy numbers, which suggests as a potential marker for disease progression. Furthermore, followed-up cases should be studied in large scale sample sizes in order to evaluate its potential as prognostic marker in cervical cancer.
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RESUMEN
Promoter hypermethylation of tumor suppressor genes are associated with an epigenetically mediated gene silencing, which is a common feature in various human cancers including cholangiocarcinoma. Even though methylation-specific PCR (MSP) has been accepted for its sensitivity to detect methylation in cancerous tissues, this technique is time-consuming, labor-intensive and usage of carcinogenic chemicals. To overcome this limitation, DNA biosensor using quartz crystal microbalance (QCM) was developed in this study. QCM, a mass sensor, is rapidly becoming an important tool for biological analysis. p16 methylation in KKU-M213 CCA cell line was investigated as a model for protocol validation. In principle, specific biotinylated DNA probe were immobilized via avidin–biotin system on gold surface of a QCM device. The specific hybridization between immobilized probe with methylated and unmethylated p16 products were monitored by decreasing in frequency due to mass change. The results showed that the optimal concentrations of mercaptoproprionic acid (MPA), avidin, and 5’-biotinylated DNA probe were 10 mM, 1 mg/dL and 1 mM, respectively. The oscillation signal obtained from methylation or unmethylation positive samples were clearly discriminated from negative ones. The cut-off values were calculated from negative methylation and unmethylation at 30 Hz with preferable lower than 15 % precision. The QCM sensor can detect positive signal at 1,000 fold dilution compared to agarore gel electrophoresis. No cross-hybridization was observed when unmethylation product was performed on methylation sensor and vice versa. Moreover, QCM can be reused for at least 3 times with considerable signal reduction. In conclusion, our developed QCM sensor for p16 methylation illustrated an acceptable performance compared to conventional method. The detection process can be reduced within 5 minutes. However, this prototype sensor as well as mini-PCR integration device for real time analysis should be further developed.