The impact of C-MYC gene expression on gastric cancer cell

Abstract

The upregulation or mutation of C-MYC has been observed in gastric, colon, breast, and lung tumors and in Burkitt's lymphoma. However, little is known about the role C-MYC plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of C-MYC on the growth, proliferation, apoptosis, invasion, and cell cycle of the gastric cancer cell line SGC7901 and the gastric cell line HFE145. C-MYC cDNA was subcloned into a constitutive vector PCDNA3.1 followed by transfection in normal gastric cell line HFE145 by using liposome. Then stable transfectants were selected and appraised. Specific inhibition of C-MYC was achieved using a vector-based siRNA system which was transfected in gastric cancer cell line SGC7901. The apoptosis and cell cycles of these clones were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay, respectively. The invasion of these clones was analyzed by using cell migration assay. The C-MYC stable expression clones (HFE-Myc) and C-MYC RNAi cells (SGC-MR) were detected and compared with their control groups, respectively. HFE-Myc grew faster than HFE145 and HFE-PC (HFE145 transfected with PCDNA3.1 vector). SGC-MR1, 2 grew slower than SGC7901 and SGC-MS1, 2 (SGC7901 transfected with scrambled control duplexes). The cell counts of HFE-Myc in the third, fourth, fifth, sixth, and seventh days were significantly more than those of control groups (P < 0.05). Those of SGC-MR1, 2 in the fourth, fifth, sixth, and seventh days were significantly fewer than those of control groups (P < 0.05). Cell cycle analysis showed that proportions of HFE-Myc and SGC-MR cells in G0-G1 and G2-M were different significantly with their control groups, respectively (P < 0.05). The apoptosis rate of HFE-Myc was significantly higher than those of control groups (P < 0.05). Results of colony-forming assay showed that the colony formation rate of HFE-Myc was higher than those of control groups; otherwise, the rate of SGC-MR was lower than those of their control groups (P < 0.05). The results of cell migration assay showed that there were no significant differences between experimental groups and control groups (P > 0.05). In conclusion, C-MYC can promote the growth and proliferation of normal gastric cells, and knockdown of C-MYC can restrain the growth and proliferation of gastric cancer cells. It can induce cell apoptosis and help tumor cell maintain malignant phenotype. But it can have not a detectable influence on the ability of invasion of gastric cancer cells.

Fig. 1

The results of RT-PCR for C-MYC in SGC7901 cell and HFE145 cell. S1, S2 were the results of RT-PCR for C-MYC, N-MYC, and β-actin control in SGC7901 cell. H1 and H2 were the results of RT-PCR for C-MYC, N-MYC, and β-actin control in HFE145 cell. The results showed that there was C-MYC expression in SGC7901 cell but no detectable expression of C-MYC gene in HFE145 cell. On the other hand, there was no detectable expression of N-MYC gene in SGC7901 or HFE145 cell

Fig. 2

The immunocytochemistry result of C-MYC in HFE145 and SGC7901 cell lines (×200). a There was no positive signal in HFE145 cell. The results showed that there was no detectable expression of C-MYC gene in HFE145 cell. Bar 20 μm. b The brown positive signals were mainly distributed in cytoplasm and nucleus. The results showed that there was expression of C-MYC gene in SGC7901 cell line. Bar 25 μm). (Color figure online)

Fig. 3

The RT-PCR results of C-MYC in HFE-Myc and HFE-PC cells. H_M1 and H_M2 were the results of RT-PCR for C-MYC and β-actin control in HFE-Myc cell. H_P1 and H_P2 were the results of RT-PCR for C-MYC and β-actin control in HFE-PC cell. The results showed that there were expressions of C-MYC gene in HFE-Myc cell but no expression in HFE-PC cell

Fig. 4

The immunocytochemistry results of C-MYC in HFE-PC and HFE-Myc cells (×400). a There was no positive signal in HFE -PC cell. Bar 20 μm. b The brown positive signals were mainly distributed in cytoplasm of HFE-Myc cells. The results showed that there was expression of C-MYC gene in HFE-Myc cell line. Bar 20 μm. c The brown positive signals were mainly distributed in nucleus and cytoplasm of positive control cells. Bar 20 μm. d There was no positive signal in HFE-Myc cells which were immunostained using PBS instead of C-MYC antibody as negative control. Bar 20 μm. (Color figure online)

Fig. 5

The western blot results of C-MYC in HFE-Myc, HFE-PC, and HFE145 cells. H_M was the result of western blot for C-MYC and β-actin control in HFE-Myc cell, H_P was the result of western blot for C-MYC and β-actin control in HFE-PC cell, and H was that in HFE145 cell. The results showed that there was expression of C-MYC gene in HFE-Myc cell but no expression in HFE-PC and HFE145 cell

Fig. 6

The RT-PCR results of C-MYC in SGC-MR1, SGC-MR2, SGC-MS1, SGC-MS2, and untreated SGC7901 cells. S_S1 and S_S2 were the results of RT-PCR for C-MYC and β-actin control in SGC-MS1 and SGC-MS2 cells, respectively; S_R1 and S_R2 were the results of RT-PCR for C-MYC and β-actin control in SGC-MR1 and SGC-MR2 cells, respectively. S_G was that in untreated SGC7901 cell. The results showed that the expression level of C-MYC gene in SGC-MR1 and SGC-MR2 cells was decreased when compared with the control groups

Fig. 7

The western blot results of C-MYC in SGC-MR1, SGC-MR2, SGC-MS1, SGC-MS2, and untreated SGC7901 cells. S_R1 and S_R2 were the results of western blot for C-MYC and β-actin control in SGC-MR1 and SGC-MR2 cells, respectively; S_S1 and S_S2 were the results of western blot for C-MYC and β-actin control in SGC-MS1 and SGC-MS2 cells, respectively. S was that in untreated SGC7901 cell. The results showed that the expression level of C-MYC gene in SGC-MR1 and SGC-MR2 cells was decreased when compared with the control groups

Fig. 8

The growth curves of C-MYC-transfected and C-MYC-RNAi cell lines. a The growth curves of HFE-Myc, HFE-PC, and HFE145 groups. b The growth curves of SGC-MR1, 2, SGC-MS1, 2, and untreated SGC7901 groups. The unit of vertical axis was ×10⁵; horizontal axis was the number of days

Fig. 9

The results of colony formation assay for C-MYC-transfected and C-MYC-RNAi cell lines. a The results of colony formation assay of HFE-Myc, HFE-PC and HFE145 cells. 1 the colony formation rate of HFE-Myc cell, 2 that of HFE-PC cell, 3 that of HFE145 cell line. b The results of colony formation assay of SGC-MR1, SGC-MR2, SGC-MS1, SGC-MS2, and untreated SGC7901 cell lines. 1, 2 the colony formation rate of SGC-MR1, SGC-MR2 cells, respectively; 3, 4 that of SGC-MS1, SGC-MS2 cells, respectively; 5 that of SGC7901 cell line

Fig. 10

The results of cell migration assay for C-MYC-transfected and C-MYC-RNAi cell lines. a The results of cell migration assay of HFE-Myc, HFE-PC, and untreated HFE145 cell lines. 1 the cell migration rate of HFE-Myc cell line, 2 that of HFE-PC cell line, 3 that of untreated HFE145 cell line. b The results of cell migration assay of SGC-MR1, SGC-MR2, SGC-MS1, SGC-MS2, and untreated SGC7901 cell lines. 1, 2 the cell migration rate of SGC-MR1, SGC-MR2 cells, respectively; 3, 4 that of SGC-MS1, SGC-MS2 cells, respectively; 5 that of SGC7901 cell line