The Relationship Between WTX Gene And P Glycoprotein And The Effects Of WTX On MDR Gastric Cancer Cells

Objective1. To study the expression of the tumor suppressor gene WTX, β-catenin and P-gp in the tumor tissue of different survival phases of patients with gastric carcinoma, as well as study the correlation of these three proteins; to set up a Logistic regression model, and analyze the factors influencing prognosis of gastric carcinoma.2. To build a over expression vector of lentivirus for WTX, and to research the effects of WTX on the expression of P-gp taking multi-drug resistance(MDR) gastric cancer cells SGC7901/VCR which with high expression of P-gp for example.3. To study the WTX expression mechanism on P-gp controlling, and study the effects of WTX on multidrug-resistance gastric carcinoma cells.Methods1. According to the survival time, we classify patients into two groups, as in group of good prognosis (survival time>5 years) and in group of poor prognosis (dead within 2 years because of the tumor), and immunohistochemical-stain the section of the carcinoma tissue and the paraffin section of the normal tissue. We examine the expression of WTX, β-catenin and P-gp in the tumor tissue of different survival phases of patients with gastric carcinoma, as well as the correlation of these three proteins. Set up a Logistic regression model, and analyze the correlated factors influencing prognosis of gastric carcinoma.2. Build a over expression vector of lentivirus for WTX, observe the expression of the green fluorescent protein, examine the mRNA expression level of WTX with quantitative-PCR(qPCR). Analyze the expression of WTX, mdr-1, MRP-1 and BCRP in SGC7901 and SGC7901/VCR cells with qPCR. Classify the SGC7901/VCR into three groups:control(CON), negative control(NC), and over expression (OE) group. Infect the SGC7901/VCR cells with lentivirus, and then examine the expression of mRNA and protein of P-gp in SGC7901/VCR after the over-expression of WTX with qPCR and Western blot.3. Infect SGC7901/VCR with lentivirus after the classification, and examine the changes of β-catenin, TCF4, CBP, p300, TP53, MDM2, NF-κB, E-cadherin, c-myc, and cyclinD1 before and after the over expression of WTX. Discuss the WTX controlling mechanism on P-gp. Study the influence of WTX on the growth of gastric carcinoma cells. Examine the cell apoptosis-rate by staining Annexin V/PI with flow cytometry. Examine the cell proliferative activity with CCK8 and then draft the cell growth curve. Examine the cell circle time with flow cytometry.Results:1. The expression of WTX in the good prognosis group is higher than in the poor prognosis group, while the expression of β-catenin and P-gp is lower than in the poor prognosis group. It has been showed in the related analysis that the expression of WTX is negatively correlated with that of β-catenin and P-gp, while the expression of β-catenin is positively correlated to that of P-gp. By setting up a Logistic regression model, we can land at the following variable quantities and the ultimate formulation, that is Age, TNM staging, WTX, β-catenin, and P-gp scores. The results are as followed:(1) age:with the the Youth Group (<40 years old) as a reference, we can get age Ⅰ as a result when we compare the mid-age group (≥40 years to< 60 years), and age Ⅱ as a result when we compare the senior-age group (≥60 years), and we can get the conclusion that B-value is a negative figure, and Exp (B)< 1, and we have learned that age is a factor that is negatively correlated with prognosis, that means young patients has a worse prognosis than the old ones. (2) TNM staging:B-value is a positive number, and Exp (B)> 1, which shows that TNM staging is positively correlated to the disease prognosis, that means when TNM staging increases, the prognosis will be worsen. OR=12.76, and 95% confident interval is between 1.119 and 145.435. (3) WTX scores:when B-value is a negative number, and Exp(B)< 1, then WTX scores is negatively correlated with prognosis, which means the higher the grading is, the better the prognosis will be. (4) β-catenin scores:when B-value is a positive number, and Exp (B)> 1,β-catenin scores is positively correlated with prognosis, which means the higher the scores is, the worse the prognosis will be. (5) P-gp scores:when B-value is a positive number, and Exp (B)> 1, then P-gp scores is positively correlated to prognosis, which means the higher the grading is, and the worse the prognosis will be.2. After we successfully build the over expression vector of lentivirus for WTX, we found that the WTX is both low expression in the SGC7901 and in SGC7901/VCR; MRP-1 and BCRP both have high expression in the SGC7901 and SGC7901/VCR; and the mdr-1 is low expression in the SGC7901 and high expression in SGC7901/VCR. After the examine of SGC7901/VCR which has infected the lentivirus with qPCR, the mRNA of WTX has risen as high as 108.9 times, and has successfully over expressed WTX. After the examine of over expression of WTX with qPCR and Western blot test, the expression of mdr-1 mRNA and P-gp protein have both decreased.3. After the examination of over expression of WTX with qPCR, the differences of the expression of TCF4、p300 and NF-κB is statistically meaningless (P>0.05). When the expression of c-myc decreases(P<0.05), the β-catenin、NCBP、TP53、 MDM2、E-cadherin and cyclinD1 increases with the differences statistically meaningful (P<0.05).And thus, we can conclude that with P53, WTX may control P-gp, restrain the expression of c-myc, and have influence on the cell adhesion and circle time.Obvious cells apoptosis has not been found with the examination of flow cytometry. After examing SGC7901/VCR growth curve with CCK8, we found that the SGC7901/VCR proliferation has been restrained after the over expression of WTX. And after examing with flow cytometry, we found that the cell circle time was restrained in S phase.Conclusions1. The expression of WTX,β-catenin and P-gp are correlated with the prognosis of gastric carcinoma. The expression of WTX is negatively correlated with the prognosis of gastric carcinoma, while the expression of β-catenin and P-gp are positively correlated with the prognosis of gastric carcinoma. Age, TMN staging, WTX scores,β-catenin scores and P-gp scores are factors that infected the prognosis of gastric carcinoma.2. SGC7901/VCR with high expression is a good model to study MDR mediated by P-gp. The over expression of WTX can lower the mRNA and protein expression of P-gp in SGC7901/VCR.3. With p53 pathway, WTX may control P-gp, restrain the expression of c-myc, and have influence on the cell adhesion and cycle. No obvious apoptosis of SGC7901/VCR has been found after the over expression of WTX. And the cells proliferation has been restrained because that cell cycle has been stopped in S phase after the overexpression of WTX.