Effects of RNA interference of COX-2 gene expression on malignant proliferation of A549 cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the inhibition of COX-2 gene expression and its effects on malignant proliferation of human lung adenocarcinoma A549 cells after interfering at different target sites in vitro.</p><p><b>METHODS</b>The 3rd, 7th and 10th exon of COX-2 were selected as the targets and three COX-2 siRNA expression vectors with human U6 promoter were constructed. Three siRNA expression vectors and two vacant vectors were transfected into A549 cells expressing COX-2 with lipofectamine, respectively. The transfected cell strains were constructed and the change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A549 cells after interfering at different target sites were studied by cell growth curve and colony formation assay in vitro.</p><p><b>RESULTS</b>The three siRNAs and U6 promoter were validated by PCR, restriction endonuclease digestion, DNA sequencing and BLAST alignment, and cloned into the pEGFP vector. The cell strains transfected were named as A549-3, A549-7, A549-10, A549-p and A549-pU6, respectively. A549-p cells showed expression of GFP and A549-3, A549-7, A549-10, A549-p and A549-pU6 cells did not show at 24, 48 and 72 hours after transfection. The results of RT-PCR and Western blot showed an inhibition of COX-2 expression after interfering at three target sites (3rd, 7th and 10th exons). In contrast to A549 cells, the levels of COX-2 mRNA of A549-3, A549-7 and A549-10 cells were reduced by 10.6%, 33.4% and 61.2%, respectively. The levels of COX-2 protein of A549-3, A549-7 and A549-10 cells were reduced by 26.7%, 44.7% and 56.2%, respectively. The results of cell growth curve and colony formation assay showed a slowing down of the growth of A549-10 cells and reduction of their colony formation rate. The other two targets had no apparent effect on the growth of A549 cells.</p><p><b>CONCLUSION</b>There is a significant inhibiting effect of RNA interference on the malignant proliferation of A549 cells in vitro, and the most striking effect can be seen when the 10th exon of COX-2 is taken as the interference target.</p>

The relationship between genetic polymorphism of metabolizing enzymes and the genetic susceptibility to lung cancer / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the gene polymorphism of metabolizing enzymes and the genetic susceptibility to lung cancer as well as to study the synergistic effects between smoking and the genes.</p><p><b>METHODS</b>A case-control study (case = 217, control = 200) was carried out to compare the frequent distribution of CYP1A1, 2E1, 2D6 and GSTM1 genotypes between the lung cancer group and the control group with a polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) method and to analyze the relationship between these genes and smoking.</p><p><b>RESULTS</b>GSTM1-null genotype frequency was 58.5% in the lung cancer group and 47.5% in the control group with significant difference (P = 0.02). The frequent distribution of CYP1A1, 2E1, 2D6 genotypes was not significantly different in the two groups (P > 0.05). Synergistic effects were found between smoking and GSTM1 but not between smoking and CYP1A1, 2E1, 2D6.</p><p><b>CONCLUSION</b>Smoking and GSTM1-null genotype seemed to be the risk factors of lung cancer. Those who carrying GSTM1-null genotype and smoking cigarettes were prone to suffer from lung cancer to become the high-risk population of the disease.</p>

Growth inhibitory effect of adriamycin conjugated to single-chain antibody on human lung adenocarcinoma in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the growth inhibitory effect of adriamycin (ADM) conjugated to an anti-lung cancer single-chain antibody (ScFv) 2A7-1 on lung adenocarcinoma cell line A2 in vitro.</p><p><b>METHODS</b>2A7-1 cell culture medium was concentrated by ultra-filtration (with Amicon P10Z filter), and soluble ScFv was purified using RPAS purification kit. ADM was conjugated to 2A7-1 by glutaraldehyde. A(280) and A(490) of the conjugate 2A7-1-ADM were determined by spectrophotometry and the molar ratio of 2A7-1 to ADM was calculated. Immunoreactivity of the conjugate was detected by immunohistochemistry. Its growth inhibitory effect on lung adenocarcinoma cell line A2 was determined by colony formation assay in vitro.</p><p><b>RESULTS</b>The molar ratio of 2A7-1 to ADM was 1:3.2. The conjugate strongly reacted with A2 cell. Its growth inhibitory effect on A2 cells was 4 times as potent as ADM.</p><p><b>CONCLUSION</b>Adriamycin conjugated to anti-lung cancer single-chain antibody 2A7-1 has much higher cytotoxic activity than unconjugated adriamycin against human lung adenocarcinoma.</p>

Inhibitory effect of p53 with deletion of c-terminal 356 - 393 amino acids on malignant phenotype of human lung cancer cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of extraneous p53 gene with deletion of c-terminal 356 - 393 amino acids on inhibition of malignant phenotype of human lung cancer cell line.</p><p><b>METHODS</b>Recombinant plasmid pEGFP-p53 (del) with codon deletion of c-terminal 37 amino acids from 393 to 356 region and pEGFP-p53 (wild type) were constructed. The human lung cancer cell line 801D served as a receipt cell had p53 deletion and mutation at 248 codon. 801D cells, having been transfected by pEGFP-p53 (wild type), pEGFP-p53 (del) or pEGFP, were selected by G418. Growing transfected cells were cloned respectively by method of dilution. Presence of extraneous gene was detected by PCR, their expression in cells was examined by fluorescence microscopy. Cloning efficiency was in vitro tested to examine the cellular proliferating ability. The xenograft in nude mice was performed and xenograft tumors were weighed one month later. Expression of GFP in tumor and transplanted cellular mass were detected by blot slices.</p><p><b>RESULTS</b>pEGFP-p53 (del)-801D, pEGFP-p53-801D and pEGFP-801D were established. Extraneous p53 gene and expression of GFP were found in pEGFP-p53 (del)-801D and pEGFP-p53-801D. Inhibitory rate of colony was 99.6% for pEGFP-p53 (del)-801D and 81.0% for pEGFP-p53-801D. Inhibition of malignant proliferation of extraneous p53 (del) was higher than that of p53 (wild type) (P < 0.01). Even when inhibition of malignant proliferation extraneous pEGFP-p53 (del) was obvious, 0.2% colonies were formed, extraneous p53 and expression of GFP were observed. Animal test showed that tumor on the nude mice was positive (4/4, 4/4) in the control group (801D and pEGFP-801D), but negative (0/4, 0/4) in the experiment group [pEGFP-p53 (del) 801D and pEGFP-p53 (wild type) 801D]. Expression of GFP in the cells of cellular mass transplanted by pEGFP-p53 (del) 801D or pEGFP-p53 (wild type) 801D was observed.</p><p><b>CONCLUSION</b>In vitro inhibitory effect of extraneous p53 gene with deletion of C-terminal 356 - 393 amino acids on malignant growth of lung cancer cell with p53 mutation or deletion at 248 codon is marked. Inhibitory action of p53 on malignant proliferation of cancer cells is heterogeneous.</p>