Functional Study Of GPC5 Gene In Lung Adenocarcinoma Cell Lines.

AbstractObjectiveLung cancer is one of the most common malignant tumor in the world. Recently, a multicenter genome-wide association studies(GWAS) study reported that genetic variations of glypican-5(GPC5) may significantly contribute to an increased risk of lung cancer in never smokers. GPC5 belongs to the phosphatidylinositol proteoglycans(HSPGs),which are linked to the exocytoplasmic surface of the plasma membrane via a glycosylphosphatidylinositol anchor, playing an important role in the cell proliferation and differentiation. The role of GPC5 gene in lung cancer cell lines has been markerd by arguments and controversies. In our present study, we constructed overexpressing and interference vector to regulate the expression of GPC5 and studied the biological effects of GPC5 in lung adenocarcinoma cell lines.Method1. Reverse transcription PCR(RT-PCR) and real-time quantitative PCR(q RT-PCR) have been done to determine the m RNA expression level of GPC5 in the lung adenocarcinoma cell lines.Western blot technology has been done to determine the protein expression level of GPC5 in the lung adenocarcinoma cell lines.2. GPC5 overexpression vector and interference vector have been build.GPC5 overexpression and GPC5 silence cell model have been constructed by liposome transfection.3. CCK-8 has been done to explore the cell proliferation in the GPC5 overexpression and GPC5 silence cell model.4. Flow cytometry analysis has been done to explore the cell cycle and apoptosis in the GPC5 overexpression and GPC5 silence cell model.5. Transwell has been done to explore the cell invasion and migration ability in the GPC5 overexpression and GPC5 silence cell model.Result1. GPC5 m RNA expression level was detected in four lung adenocarcinoma cell lines(A549, H1299, H358 and H1975) and normal lung tissue. We found that, GPC5 m RNA expression level in lung adenocarcinoma cell lines were lower than human normal lung tissue. In terms of cell lines, the expression of GPC5 was relatively low in A549 cell line but was high in H1299 cell line. The protein detection results were consistent with the m RNA level.2. We used the A549 cells to construct GPC5 overexpression cell line and used the H1299 cells to construct GPC5 down-expression cell line.3. We examined the growth inhibition effect by ectopic GPC5 expression in A549 by the CCK8 assay. Ectopic expression of GPC5 in A549 cell line caused a significant decrease in cell viability(p<0.05). On the other hand, we knocked down GPC5 expression in H1299 cell line by si RNA vector transfection. Knockdown of GPC5 expression significantly increased the growth of H1299 cell line(p<0.05).4. We examined the effect of GPC5 expression on the cell cycle distribution. We found that the number of GPC5-transfected A549 cells with G1 DNA content was significantly increased(p < 0.05) compared to that of control vector-transfected cells, whereas S DNA content was decreased in these cells(p < 0.05). In addition, knockdown of GPC5 expression led to a significant decrease in the number of H1299 cells in G1 phase and an increase of those in S phase.5. We further examined the effect of GPC5 re-expression in lung cancer cells on apoptosis. Compared to the negative control, GPC5-transfected A549 cells total apoptosis rate decreased from 10.03% to 8.99%, but the difference was not statistically significant(P>0.1). In addition, knockdown of GPC5 expression in H1299 cells did not induce apoptosis.6. We investigate the effects of GPC5 expression on lung cancer cell migration by Transwell assays. We found that migration of A549 cells was reduced following upregulation of GPC5 expression(P<0.05). In addition, knockdown of GPC5 expression increased H1299 cells migration significantly(P<0.05)7. We then used Matrigel to evaluate lung cancer cell invasion. We found that invasion of A549 cells was reduced following upregulation of GPC5 expression(P<0.05). In addition, knockdown of GPC5 expression increased H1299 cells invasion significantly(P<0.05)ConclusionGPC5 could inhibit cell proliferation and cell cycle in lung cancer cell lines, GPC5 could suppress tumor probably by inhibiting the lung cancer cell migration and invasion, suggesting that GPC5 is a tumor suppressor gene...