[Screening of aryl hydrocarbon receptor gene specific RNA interference fragment by quantitative competitive RT-PCR].

OBJECTIVE: To evaluate and screen the specific RNAi fragments which can effectively inhibit Aryl hydrocarbon receptor(AHR) gene mRNA expression in human bronchial epithelial cell line (16HBE). METHODS: AHR mRNA of 16HBE cells transfected 4 different AHR gene interfere sites were determined quantitatively with the quantitative competitive RT-PCR by using self-prepared internal standard as competitive templates, and the RNA interfere effect wasevaluated. RESULTS: AHR mRNA average expression per 40ng total RNA of 16HBE cells transfected 4 different AHR gene interfere fragments were 5.65fg, 14.78fg, 3.14fg and 0.68fg respectively, the average rates of inhibition were 61.6%, -0.5%, 78.6% and 95.4% respectively. CONCLUSION: AHR gene specific effective RNA interfere sequence ware screened by quantitative competitive RT-PCR which could accurately quantify gene mRNA level, and offered condition for studying the gene function of AHR.

[Study on E-cadherin, Cyclin D1 and Cyclin E expression in anti-malignant transformation by chlorophyllin].

OBJECTIVE: To investigate the effects of chlorophyllin on the regulations of proteins related to cell cycle in vitro in human bronchial epithelial cell line 16HBE transformed by trans-benzo(a) pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (trans-BPDE). METHODS: RT-PCR and fluoroimmunocytochemistry methods were used to detect the expression of E-cadherin, in mRNA and protein levels, among untreated control cells, malignant transformed cells induced by trans-BPDE and anti-transformed cells treated with chlorophyllin. The expression of cyclins such as Cyclin D1 and Cyclin E was also detected by fluoroimmunocytochemistry method. RESULTS: The loss of E-cadherin expression was found in mRNA and protein levels after being transformed by trans-BPDE, while its expression existed normally after being anti-transformed by chlorophyllin. Cyclin D1 and Cyclin E had no expression in control 16HBE, but expressions of the proteins were enhanced obviously in malignant transformed cell line while those were inhibited significantly in the anti-transformed cells treated with 100pmol/L chlorophyllin. CONCLUSION: The ability of chlorophyllin to anti-malignant transformants 16HBE cells exposed to trans-BPDE is correlated with arrest the loss of E-cadherin expression. Chlorophyllin has a significant effect on expressions of Cyclin Dl and Cyclin E during the course of anti-malignant transformation.

[Expressions of 8-OH-dG, k-ras, and p53 genes in pleural effusion cells and their clinical significances].

BACKGROUND & OBJECTIVE: Oxidative DNA damage plays an important role in carcinogens-induced carcinogenesis. 8-hydoxy-2deoxy-guanosine (8-OH-dG), a biomarker of oxidative DNA damage, plays important roles in initiation, progression, and prognosis of lung cancer, and closely relates with mutations of k-ras and p53 genes in carcinogenesis of lung tissue. This study was to detect protein expressions of 8-OH-dG, k-ras, and p53 genes in lung cancer tissues, and to analyze their values in distinguished diagnosis of lung cancer. METHODS: Protein levels of 8-OH-dG, k-ras, and p53 in pleural effusion cells from 53 patients with lung cancer, and 53 patients with other benign lung diseases were detected by immunocytochemistry. RESULTS: Positive rates of 8-OH-dG, k-ras, and p53 protein in cancer group were significantly higher than those in benign disease group [75.5% (40/53) vs. 15.1% (8/53), P < 0.01; 64.2% (34/53) vs. 3.8% (2/53), P < 0.01; and 69.8% (37/53) vs. 18.9% (10/53), P < 0.01; respectively]. Protein levels of 8-OH-dG, k-ras, and p53 protein in cancer group were 1.68+/-1.21, 1.32+/-1.06, and 1.57+/-1.15,respectively. Rank correlation analysis showed that protein expression of 8-OH-dG positively correlated with those of k-ras (RS=0.643, P < 0.01), and p53 (RS=0.827, P < 0.01)u protein expression of k-ras positively correlated with that of p53 (RS=0.897, P < 0.01). CONCLUSIONS: Protein expressions of 8-OH-dG, k-ras, and p53 are up-regulated in pleural effusion cells of lung cancer, and have mutual relations. They may be used as reference markers in diagnosing and screening for lung cancer.

[A study on the role of DNA repair gene O6-methylguanine-DNA methyltransferase in the development of human lung cancer].

OBJECTIVE: To study the role of O(6)-methylguanine-DNA methyltransferase (hMGMT) in the development of human lung cancer. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) method was applied to measure hMGMT mRNA expression in 150 lung cancer specimens, 40 normal lung tissues, and in the peripheral mononuclear blood cells from 50 lung cancer cases and 50 normal controls. The protein expressions of p53, C-MYC and K-RAS were assessed by immuno-histochemistry. The effects of some exposure factors on the expression of hMGMT gene were analyzed. The relationships between hMGMT gene and cancer related genes p53, C-MYC and K-RAS were investigated. RESULTS: The mRNA of hMGMT was low or absent in 49 of 150 (32.7%) lung cancer specimens, whereas 2 of 40 (5%) normal lung tissues had reduced the levels of hMGMT mRNA. The low expression of hMGMT seemed to be a risk factor of lung cancer, with a OR of 9.22 (2.05-57.65). Reduced expression levels of hMGMT mRNA were observed in 10 of 50 (20%) lung cancer patients' peripheral mononuclear blood cells, and 2 of 50 (4%) blood cells among normal controls. When investigating the exposure factors which affecting the expression of hMGMT gene, we noticed that smoking was suppressing the expression of hMGMT gene. Interestingly, over-expression of K-RAS oncogene was significantly correlated with low expression of hMGMT (P < 0.05). However, the expressions of p53 and C-myc were not correlated with the status of hMGMT gene. CONCLUSION: hMGMT might play an important role in the development of human lung cancer. Low expression of hMGMT gene seemed to be a risk factor for lung cancer which could be used as a valuable biomarker on susceptibility of human lung cancers.