The Roles Of Wnt/β-catenin Signaling Pathway In Hormesis Of Cell Proliferation Induced By Cadmium Chloride In HEK293Cells

Objective:To study the roles of Wnt/β-catenin signaling pathway in hormesis of cell proliferationinduced by Cadmium Chloride in HEK293cells, to explore the mechanisms of cadmiumchloride induced cell proliferation and provide a reference for further understanding and safetyof hormesis.Methods:1. Three pairs of shRNA were designed and synthesized for β-catenin gene mRNA sequences,shRNA plasmid was constructed to interfere with β-catenin gene and blocked Wnt/β-cateninsignaling pathway in HEK293cells.2. The CCK-8test was used to detect the hormesis of cell proliferation induced by the serialdose of Cadmium Chloride in24h and48h exposure, at the same time, to determine the doserange of hormesis (Hormetic Zone).3. The CCK-8test was used to detect the cell proliferation of β-catenin interference HEK293cells induced by the serial dose of Cadmium Chloride in24h and48h exposure and toexplore the roles of Wnt/β-catenin signaling pathway in hormesis of cell proliferationinduced by Cadmium Chloride.4. The real time quantitative RCR test was used to detect the mRNA expression of Cyclin D1and c-myc in HEK293cells and β-catenin interference HEK293cells induced by the serialof Cadmium Chloride in24h.5. The Flow Cytometry (FCM) was used to analysis the cell cycle in HEK293and β-catenininterference HEK293cells induced by the serial of Cadmium Chloride in24h.Results:1. Three shRNA interference plasmid vectors were constructed. RT-PCR and Western Blotwere used to detect the interference efficiency of shRNA plasmid vector, in which the β-catenin mRNA expression inhibition rate was67.68%and the protein expression inhibitionrate was61.27%.2. The CCK-8test revealed that low dose Cadmium Chloride could induce HEK293cells tooccur cell proliferation hormesis, and the dose range of hormesis were0.1μmol/L~1μmol/Lin24h and0.001μmol/L~0.5μmol/L in48h respectively.3. The CCK-8test revealed that only0.5μmol/L Cadmium Chloride could induce β-catenininterference HEK293cells to occur cell proliferation hormesis in24h, and all the serial dosecould not induce β-catenin interference HEK293cells to occur cell proliferation hormesis in 48h.Thehormesis of cell proliferation was inhibited by blocking Wnt/β-catenin signalingpathway.4. Quantitative PCR results showed that the dose range of Cadmium Chloride at0.01μmol/L~1μmol/L elevated the expression of Cyclin D1, which was statistically significant (P<0.05)that compared with the control group. The expression levels of Cyclin D1presented firstincreased and then decreased trend in the dose range from0.01μmol/L to10μmol/L. Theexpression level of Cyclin D1reached the maximum at1μmol/L group. While the serial doseof Cadmium Chloride effected on β-catenin gene interference HEK293cells in24h, theexpression levels of Cyclin D1at all the dose groups showed no significant difference(P>0.05) comparing with control group.5. The dose range of Cadmium Chloride at0.1μmol/L~10μmol/L elevated the expression of c-myc, which was statistically significant (P<0.05) that compared with the control group. Thedose of Cadmium Chloride and the expression of c-myc showed a linear dose-responserelationship. The expression level of c-myc reached the maximum at10μmol/L group. Whilethe serial dose of Cadmium Chloride effected on β-catenin gene interference HEK293cellsin24h, the expression level of c-myc at10μmol/L dose group was higher than control group,there was significant difference (P>0.05).6. Cadmium Chloride acted on HEK293cells for24h, the cell phase proportion of G1phasewas reduced by0.1μmol/L and1μmol/L Cadmium Chloride, and cell phase proportion in Sphase was elevated by1μmol/L Cadmium Chloride, which were statistically significant(P<0.05) similarly that compared with the control group. Cadmium Chloride acted on β-catenin interference HEK293cells for24h, the cell proportion in G1phase and S phase wereno difference with the control group(P>0.05)Conclusion:1. Low doses of Cadmium Chloride can induce cell proliferation hormesis in HEK293cells,The Hormetic Zone were0.1~1μmol/L(24h),0.001~0.5μmol/L(48h) respectively.2. Wnt/β-catenin signaling pathway plays an important role in hormesis of cell proliferationthat induced by low dose Cadmium Chloride in HEK293cells. Cadmium Chloride in lowdose may activate the Wnt/β-catenin signaling pathway to increase the expression of CyclinD1to induce the change of cell cycle, then induce cell proliferation.