Suppression subtraction hybridization in screening of differentially expressed genes in radiation-induced malignant transformation cellular model / 第二军医大学学报

ABSTRACT

Objective: To screen for the differentially expressed genes during irradiation-induced malignant transformation of human bronchus epithelium cells (BEAS-2B). Methods: Suppression subtraction hybridization (SSH) was used to construct a subtracted cDNA library of differentially expressed genes during irradiation-induced malignant transformation of BEAS-2B cells. Then the subtracted library was screened by PCR and the differential fragments were sequenced and analyzed with BLAST. Fluorescent real-time quantitative PCR was used to investigate some of the differentially expressed genes. The new EST was registered in GenBank. Results: Then 40 clones were chosen to be sequenced from the library of increased expression and decreased expression respectively according to the length of insertion element. Totally 73 sequences were obtained from the 80 sequenced clones. Forty-one sequences were decreased in the transformed cells; BLAST analysis indicated that there were 6 known sequences, 20 unknown sequences, 7 void sequences and 8 repeated sequences. Thirty-two sequences were increased in the transformed cells; Blast analysis indicated that there were 14 known sequences, 9 unknown sequences, and 9 repeated sequences. Fluorescent quantitative PCR revealed that, compared with control group, the expression of MY06, HACE1, ZNF143, and HNRPH1 were significantly increased in the radiation transforming group, with their mRNAs increased by 3.49, 29.38, 12.99 and 5.00 folds, respectively. Compared with control group, the expression of PCBP2, RPL15, and TCERG1 in the radiation transforming group was significantly decreased, with their mRNAs decreased by 1.89,48.77 and 11.95 folds, respectively. The 29 unknown sequences were registered in the GenBank (ID EB643220-EB643248). Conclusion: The cDNA library has been successfully established for malignant transformation cellular model by suppression subtractive hybridization; the library includes a number of unknown genes. The increased gene ZNF143 is associated with cell proliferation and cell division. TCERG1, as an assistant transcription activation factor, plays an important role in the mRNA transcription and later modification. PCBP2, a Polyc connection protein, plays a modulating role in protein translation. These genes have not been reported in the radiation carcinogenicity.