The Study On The Correlation Of GOLPH3 Gene And Wnt Signaling Pathway In Human Colon Cancer Cells

Objective: The overexpression of GOLPH3 was found in colorectal cancer tissue, and it may promote cell proliferation through activating Akt/m TOR signaling pathway. The subject researched the correlation between GOLPH3 gene and Wnt signaling pathway, to explore the mechanism of the overexpression of GOLPH3 gene promote proliferation in human colon cancer cells.Methods: 1. Measured the expression of GOLPH3 m RNA in the human colon cancer cell lines of HCT116, HT29, SW480 and SW620 by RT-PCR, the cell lines of the highest expression were selected and divided into groups to experiment follow-up. 2. Dividing the human colon cancer cells of SW620 into four groups: the negative control group, the group of GOLPH3 si RNA were transfected(si RNA-GOLPH3), the group of added inhibitor of Akt(Tricinbine), and the group of appended inhibitor of GSK-3β(TWS119). 3. After the si RNA-GOLPH3 were transfected into human colon cancer cells, to examined the silencing effect of GOLPH3 gene in way of RT-PCR. 4. Assessed the activity of Wnt signaling pathway in all groups with the method of Topflash. 5. By the methods of MTT and colony formation assay, detected the proliferation of cancer cells in all groups. 6. Inspected the apoptosis of cancer cells in all groups with flow cytometry method. 7. The expression of proteins with GOLPH3、β-catenin、GSK-3β and p S9-GSK-3β in cancer cells of all groups were checked by Western Blot. 8. The experimental result of all groups were analyzed statistically, to investigated the correlation of GOLPH3 gene overexpression promoted proliferation through activatingthe Wnt signaling pathway in human colon cancer cells.Results: 1. The GOLPH3 m RNA expression levels in the human colon cancer cells of HCT116, HT29, SW480 and SW620 by RT-PCR were 1.00±0.097, 2.872±0.064, 1.957±0.133 and 3.958±0.169. Obviously, the level of SW620 was highest. 2. GOLPH3 m RNA expression levels were 0.236±0.076 in si RNA-GOLPH3 group and 1.000±0.092 in negative control group,and the expression level of GOLPH3 m RNA was significantly lower than the negative control group(P<0.001). 3. The result of Topflash(the relative value of RLU): 1.000±0.164 in negative control group, 0.342±0.061 in si RNA-GOLPH3 group, 0.359±0.125 in Tricinbine group, and 0.365±0.155 in TWS119 group, the relative value of RLU in the experimental groups were significantly lower than the negative control group(P<0.001). Meanwhile, no significant difference of the value of RLU between the experimental groups, no statistical significance(P> 0.05). 4. The growth inhibition ratio of cancer cells in four groups by MTT: 0 in negative control group, 59.9% in si RNA-GOLPH3 group, 56.6% in Tricinbine group, and 51.3% in TWS119 group, the growth inhibition ratio of cancer cells in experimental groups were higher than the negative control group(P<0.05). Meanwhile, the growth inhibition of cancer cells between the experimental groups without statistical significance(P> 0.05). 5. Cancer cell colony numbers: 207.333±17.786 in negative control group, 82.333±15.144 in si RNA-GOLPH3 group, 93.333±12.342 in Tricinbine group, and 95.667±16.563 in TWS119 group, the cell colony numbers in experimental groups were significantly lower than the negative control group(P<0.001). In addition, no significant difference in cell colony numbers between the experimental groups, no statistical significance(P> 0.05). 6. The apoptosis rate of cancer cells: 1.680±0.576% in negative control group, 52.467±4.373% in si RNA-GOLPH3 group, 46.167±5.293% in Tricinbine group, and 50.167±6.506% in TWS119 group, the percentage of apoptosis in cancer cells ofexperimental groups were visiblely more than the negative control group(P<0.001). Moreover, no significant difference in percentage of apoptosis of cancer cells between the experimental groups, no statistical significance(P> 0.05). 7. Compared with the negative control group: ① In si RNA-GOLPH3 group, the expression levels of GOLPH3, β-catenin and p S9-GSK3β of cancer cells appeared to be lower(P<0.05). While no significant difference in the expression levels of GSK-3β(P>0.05). ② In Tricinbine group, the expression levels of GOLPH3 and GSK-3β of cancer cells showed no difference(P>0.05), however the expression levels of β-catenin and p S9-GSK3β significantly lower(P <0.001). ③ In TWS119 group, the expression levels of GOLPH3 and GSK-3β of cancer cells showed no difference(P>0.05), however the expression levels of β-catenin and p S9-GSK3β significantly lower(P <0.001). Comparing in the experimental groups: ① The expression levels of GOLPH3 of cancer cells, the si RNA-GOLPH3 group lower than the Tricinbine group, also the TWS119 group(P <0.05), but there were no difference between the latter two group(P>0.05). ② The expression levels of β-catenin of cancer cells between si RNA-GOLPH3 group and Tricinbine group seemed no difference to each other(P>0.05), but either lower than the TWS119 group(P <0.05). ③ The expression levels of GSK-3β and p S9-GSK3β of cancer cells in experimental groups were no obvious difference(P>0.05).Conclusions: 1. The overexpression of GOLPH3 gene could activate the Wnt signaling pathway, as well as increase the expression of β-catenin, promote proliferation and restrain apoptosis in human colon cancer cells. 2. The Akt, GSK-3β with activation could irritate the Wnt signaling pathway, raise the expression of β-catenin similarity, promote proliferation and restrain apoptosis in human colon cancer cells.3. The overexpression of GOLPH3 gene could activate the Wnt signaling pathway, the mechanism of action as that the overexpression of GOLPH3 gene activated Akt, which may also further activate the Wnt signaling pathway by the mediation of GSK-3β, and promoted proliferation in human colon cancer cells as a result.