Analysis Of Annexin â…¡ Gene Expression In Human Lung Cancer And Study On Biological Effects Of Annexin â…¡ Antisense Vector In Lung Cancer Cells

Annexin â…¡ plays a very important role in DNA synthesis and cell proliferation. Expression of annexin â…¡ is significantly elevated in several human cancers, which is correlated with malignant degree, prognosis, invasion and metastasis. Thereby, selective inhibition of annexin â…¡ gene expression may inhibit the proliferation of tumor cells and reduce invasion and metastasis. This will provide new target in gene therapy of lung cancer. In this study, we analyzed expression of annexin â…¡ mRNA in primary human lung cancer cases by RT-PCR and electrophoresis band opacity density(OD) method, and the antisense expression vector of annexin â…¡ gene was constructed and transfected to human lung adenocarcinoma cell line SPC-A-1 in order to inhibit annexin â…¡ expression. At last we observed the biological behaviors of SPC-A-1 before and after transfection in vitro and vivo. Methods: 1. Semi-quantification expression analysis of annexin â…¡ mRNA in human lung cancer and corresponding normal lung tissues were performed by RT-PCR and electrophoresis band opacity density comparison analysis. 2. The total RNA was isolated from human lung cancer cell line SPC-A-1 and reversed into cDNA. Primers were designed according to human annexin â…¡ mRNA from Genebank database and the target DNA fragments were amplified by RT-PCR. The antisense expression vector was constructed by double restriction endonucleases cleavage directional clone method. 3. Annexin â…¡ antisense expression vector was introduced into SPC-A-1 cells by liposome transfection reagent. Positive clones were screened with G418. PCR was used to confirm whether the recombinant vector DNA integrated with the genomic DNA of SPC-A-1 cells. Semi-quantification expression analysis of annexin â…¡ mRNA was performed in SPC-A-1 cells before and after transfection. 4. The cell growth curves, clonogenicity efficiency in plating, 3H-TdR incorporaionand the cell cycle were measured to observe the changes in cell proliferation. Tumor cell apoptosis was observed by in situ apoptosis assessment. Transplantation tumor experiment in nude mouse was carried out to investigate the effects on tumor cells before and after transfection in vivo. Results: 1. The mRNA expression of annexin â…¡ in human lung cancer tissues was significantly higher than that in normal lung tissues. 2. The construction of the antisense vector was confirmed with plate screening, double endonucleases cleavage and DNA sequencing. The homogeneity between the insertion fragment and annexin â…¡ gene is 99.6%. 3. Positive clones were obtained after G418 screening. PCR verified the successful integration of antisense vector with the genomic DNA of SPC-A-1 cells. Semi-quantification RT-PCR showed that the annexin â…¡ mRNA expression reduced two thirds in transfected cells compared with that in untransfected cells. 4. Compared with the untransfected cells, transfected cells decreased significantly in cell growth, clonogenicity efficiency in plating and DNA synthesis. Cell cycle was blocked in G0-G1 phase. There was no marked difference in apoptosis rate between two groups. Compared with the group transplanted with SPC-A-1, tumor volume was smaller and tumor weight was lighter in antisense group. There was no invasion in antisense group whereas there were two cases of invasion to neighbouring tissues in SPC-A-1 group. Conclusions: 1. The mRNA expression of annexin â…¡ gene increased significantly in human lung cancer tissue compared with that in normal lung tissue. 2. The antisense expression vector encoding for human lung cancer annexin â…¡ gene was constructed successfully. 3. The antisense vector was transfected successfully to SPC-A-1 cells with liposome, which integrated with SPC-A-1 cell genome and markedly inhibited the expression of annexin â…¡ mRNA in SPC-A-1 cells. 4. The antisense vector inhibited cell proliferation of SPC-A-1 and invasion to neighbouring tissues of allografts.