Role Of Genomic Imprinting And Osteopontin In Human Lung Cancer

Ⅰ. Expression of Genomic Imprinting of IGF2,H19 , MESTin Lung Cancer Background and objective: Genomic imprinting is a parental origin-specificchromosomal modification that causes differental expression of maternal and paternalalleles of a gene. It is a non-Mendelian inherited epigenetic form of gene regulation thatresults in monoallelic expression. Insulin-like growth factor 2 (IGF2), H19 andMesoderm-specific transccript(MEST ,also known as paternally expressed gene 1)are all imprinted genes.IGF2 and MEST usually transcribed from paternal allele andH19 from maternal allele. Accumulating evidence suggests that deregulation ofimprinted genes, including loss of imprinting(LOI), plays a role in oncogenesis. LOI ofthe three genes are found in a number of tumors,suggesting that LOI of them may playan important role in tumorigenesis. In the present study, we investigated allelicexpression of IGF2 , H19 and MEST in human lung cancer as well as in matchednormal lung tissiue in order to study the imprinting status and expression level of thethree genes and provide a clue for the mechanism of carcinogenesis of lung cancer. Methods: We compared the imprinting status and expression level of IGF2 ,H19and MEST in both lung cancer samples and the matched normal tissues. Genomic DNAfrom 32 lung cancer patients was amplified by polymerase chain reaction (PCR) andscreened for heterozygosity at IGF2 ,H19 and MEST using ApaⅠ,RslI and AflⅢrespectively. If heterozygous, polyadenylated RNA was isolated andreversed-transcribed into cDNA. cDNA then was amplified by PCR and the productswere digested with the same restriction enzymes to evaluate lung cancer expressionof ,H19 and MEST. Results: Twelve of 32 (37.5%)/11of 32 (34.4%) patients studied were informativefor IGF2 and MEST respectively. Lung cancer tissue from 10 of the 12 informativepatients(83.3%)exhibit LOI of IGF2; furthermore, the matched normal tissues from 4 ofthese 10 patients also shows weak biallelic expression of IGF2. LOI of MEST wasnoted in 81.8% (9 of 11) of informative cases. Monoallelic expression was maintainedin all the matched normal tissues examined. LOI of IGF2 was seen more frequently inmoderately to poorly differentiated samples. In contrast, H19 exhibited consistentmonoallelic expression in all the 14 informative samples. Conclusion: The findings indicated that independent deregulation took place inimprinted genes and suggested that aberrant imprinting and overexpression of IGF2 andMEST play an important role in carcinogenesis of lung cancer. LOI of IGF2 and MESTmay be a prophase manifestation of lung cancer. Ⅱ .Establishment of human osteopontin RNA and β-actin RNAfor the standard curve of real-time PCRBackground and method Today,real-time instruments are comprised of athermal cycler and a fluorometer for the detection of fluorescence during the cyclingprocess. Fluorescence data is collected by a computer that communicates with thereal-time machine. This data is displayed in a graphical format through softwaredeveloped for real-time analysis. Fluorescent data is collected at least once during each cycle of amplificationallowing for real-time monitoring of amplification. A user is able to determine whichsamples are amplifying on a cycle-by-cyle basis. This instant data allows them to seehow individual samples amplify in relation to known standards, positive controls andnegative controls. Not only is the user able to monitor the whoke reaction during theamplification process, but they can truly optimize their protocols based on theinformation they receive. This leads to increased sensitivity, specificity, and efficiency. After raw data is collected, the analysis can begin. The software for the real-timeinstrument normalizes the data to account for differences in background fluorescence.Once normalization is complete, a threshold level can be set. This is the level at whichfluorescence data is analyzed with corresponding concentrations are run, a linearregression analysis produces a standard curve form which the concentration of unknown...