The Cloning And Identification Of Differentially Expressed Genes In Malignant Transformation Cells Of Bronchial Epithelial Cells Induced By Mineral Powder In GeJiu

Objectives: It was known that GeJiu in Yunnan Province was a place where the incidence of lung cancer in miners was high. The occurrence of lung cancer in GeJiu miner was related with the abnormal expression of many kinds of genes. It was essential to clone,screen and identify differentially expressed genes for elucidating the molecular mechanism of lung cancer in GeJiu miner. Suppression subtractive hybridization(SSH) was the most effective technique to study the differentially expressed genes. SSH not only had a high screen efficiency and specificity, but also had a high sensitivity and can even isolate the differentially expressed genes in low abundance. To study the molecular mechanism of the malignant transformation in immortalized human bronchial epithelial cells (BEAS-2B) which were induced by mineral powder in GeJiu, and provided scientific proof for revealing the carcinogenesis of lung cancer in GeJiu miner, and also provided new research directions for seeking novel potential diagnostic and therapeutic targets of lung cancer in GeJiu miner.Methods: 1)The isolation of differentially expressed genes: BEAS-2B cell was used as driver, and its malignant transformation cell was considered as tester. Total RNA was respectively extracted from BEAS-2B and its malignant transformation cells. Then, mRNA was separated from total RNA. Switching mechanism at 5' end of the RNA transcript(SMART) cDNA synthesis technology was used to obtain sufficient double-stranded cDNA. Then SSH was used to isolate differentially expressed gene fragments between BEAS-2B cells and its malignant transformation cells. 2) The cloning of differentially expressed genes: The positive cDNA fragments were inserted into T/A plasmid vector, and then were transformed into E.coli to clone differentially expressed gene fragments between BEAS-2B cells and its malignant transformation cells. A cDNA subtractive library between BEAS-2B and its malignant transformation cells was constructed. 3) The screening of differentially expressed genes: All white clones were preliminarily screened by anabacteria PCR or digesting plasmids with EcoR I. 4)DNA sequencing and homology analysis: After preliminary screening, the positive gene fragments we obtained were sequenced. Then the results of DNA sequencing were analyzed in GenBank with Blast software.5) The identification of differentially expressed genes: After homology analysis, the positive cDNA fragments we obtained were identified by Dot blot or Northern blot.6) The analysis of differentially expressed genes: The analysis of mutation and function for known genes were carried out by the technology of bioinformatics.Results: 1) A cDNA subtractive library with high subtractive efficiency between BEAS-2B and its malignant transformation cells was constructed successfully by SSH and T/A clone. The library included 107 white clones. 2) All white clones were preliminarily screened by anabacteria PCR or digesting plasmids we extracted with EcoR I. Then 101 positive clones were obtained. 3) All positive gene fragments were sequenced and analyzed for homology in GenBank with Blast software. Among them, 44 gene fragments represented 20 kinds of known genes, and 36 gene fragments represented 14 kinds of known ESTs, and 11 gene fragments represented 6 kinds of new ESTs. 4) 19 kinds of known genes. 14 kinds of known ESTs and 6 kinds of new ESTs were confirmed to be differentially expressed gene fragments between BEAS-2B cells and its malignant transformation cells after the identification of Dot blot or Northern blot. 5 )The analysis of gene mutation was preformed on 19 kinds of known genes by the technology of bioinformatics. The change of C→T in the base of 338 in mRNA sequences of beta-2-microglobulin(B2M) led to the change of Thr→Ile in its amino acids of 93. And the change of T→C in the base of 391 in mRNA sequences of CCR4-N0T transcription complex, subunit 7(CNOT7) led to the change of Ile→Thr in its amino acids of 31. And the change of A→G in the base of 672 in mRNA sequences of CNOT7 led to the change of Lys→Glu in its amino acids of 125. 6) The functional analysis was preformed on 19 kinds of known genes by the technology of bioinformatics. The function of some genes were unknown among them, but that of others were involved in the growth, differentiation, metabolism, movement, apoptosis, transcription, translation, signal transduction, immunereaction of cells, the degradation of extracellular matrix, and so on.Conclusions: 1) A novel cDNA subtractive library with high subtractive efficiency between BEAS-2B and its malignant transformation cells induced by GeJiu mineral powder was successfully constructed by SSH. SSH was proved to be a powerful method for seeking differentially expressed genes. 2) Anabacteria PCR after reformed was a kind of method which could effectively screen DNA fragments ligated with vector. 3) Many differentially expressed genes participated in the pathway of the malignant transformation in BEAS-2B cells which were induced by GeJiu mineral powder. The up-regulation of expression and point mutation of these differentially expressed genes in the malignant transformation cells of BEAS-2B may play a considerable role in the pathway of the malignant transformation in the BEAS-2B cells induced by mineral powder in GeJiu. These findings provided scientific proof for revealing the carcinogenesis of lung cancer in GeJiu miner. 4) There were 6 kinds of new ESTs among the differentially expressed gene fragments we identified. These 6 kinds of new ESTs might represent new genes. They had been submitted to GenBank, and had possessed their accession numbers in GenBank. These new findings provided new research directions for studying etiopathogenesis of lung cancer in GeJiu miner further. 5 ) It had been reported formerly that6 kinds of genes had relations with lung cancer among the 19 kinds of known genes we identified. It reflected the resemblance in etiopathogenesis between other lung cancer and lung cancer in GeJiu miner. And it was first time found that the remaining 13 kinds of genes had relations with lung cancer. These findings indicated the specificity in etiopathogenesis between other lung cancer and lung cancer in GeJiu miner. It maybe have relations with special mechanism of inducing cancer by GeJiu mineral powder. 6) Compared with differentially expressed genes cloned and identified by suppression subtractive hybridization in lung cancer reported formerly, our study had some common results and different results. It was reported formerly that calmodulin 2(CALM2), eukaryotic translation elongation factor 1 alpha 1(EEF1A1) and histone deacetylase 6(HDAC6) expressed higher in lung cancer or lung cancer cells than that in normal lung tissue or normal bronchial epithelial cells. And it was first time found that the remaining 16 kinds of genes appeared in cDNA subtractive library of lung cancer constructed by SSH. These findings had considerable significance for constructing and optimizing gene library of lung cancer.