Prdx2Regulates The Growth Of Human Colon Cancer Cells Through Wnt/Beta-catenin Signaling

Part oneExpression of Prdx2in colorectal carcinoma and its clinicalsignificanceObjective: To examine the expression of Prdx2in colon cancer tissuesand cell lines, explore its relationship with clinical pathologicalfeatures and the correlation with the expression of β–catenin.Methods:1. The expression of Prdx2and β–catenin in thirty-fivecolorectal adenocarcinoma specimens and their corresponding adjacentnormal colonic mucosa tissues as well as the relationship between Prdx2expression and clinical pathological characteristics of patients with coloncancer were analyzed by immunohistochemistry. The correlation betweenPrdx2expression and β-catenin expression was assessed using a Spearman’s rank correlation.2. The mRNA expression levels of Prdx2and β–catenin in human coloncancer tissue and normal colon mucosa tissues as well as colon cancer celllines and normal colonic mucosa cell lines were analyzed by Real-TimePCR.3. The total protein expression of Prdx2and β–catenin in human coloncancer tissue and normal colon mucosa tissues as well as the expression ofPrdx2and β–catenin in the nuclear fractions of colon cancer cell lines andnormal colonic mucosa cell lines were analyzed by Western blotting.4. The co-expression of Prdx2and β–catenin in both colon cancer cell lineswas analyzed by immunofluorescence analysis.Results:1. Abnormal expression of Prdx2and β–catenin were detectedin colon cancer tissues, and the Spearman’s rank correlation analysisshowed a significant positive correlation between Prdx2expression andβ–catenin expression in the colon cancer tissue samples(P<0.05). Prdx2expression was significantly associated with tumor metastasis and the TNMstage (P<0.05).2. The mRNA expression levels of Prdx2and β–catenin in human coloncancer tissues and colon cancer cell lines SW480and SW620weresignificantly elevated compared with the normal colonic mucosa tissuesand normal colonic mucosa cell lines FHC. The mRNA expression level ofPrdx2was overexpressed by (7.05±0.26) times in colon cancer tissues compared to normal colonic mucosa tissues, and the mRNA expressionlevels of Prdx2in colon cancer cell lines SW480and SW620were(2.56±0.15),(4.03±0.22) times higher than in normal colonic mucosa celllines FHC respectively(P<0.05).3. The total protein expression of Prdx2and β–catenin in human coloncancer tissue was (5.11±0.3),(4.84±0.17) fold higher compared to normalcolon mucosa tissues, and The total protein expression of Prdx2andβ–catenin in colon cancer cell lines was significantly increased comparedto the normal colonic mucosa cell lines FHC (P<0.05). The expression ofPrdx2and β–catenin in the nuclear fractions of colon cancer cell lines wasalso significantly increased compared to the normal colonic mucosa celllines FHC respectively (P<0.05).4. Immunofluorescence analysis showed co-expression of Prdx2andβ–catenin was located in nucleus of both colon cancer cell lines.Conclusion: Prdx2and β–catenin is highly expressed in human coloncancer tissues and cells and a significant positive correlation betweenPrdx2expression and β–catenin expression in the colon cancer tissuesamples. And Prdx2expression was significantly associated with tumormetastasis and the TNM stage. Part twoEffect of Prdx2shRNAon the proliferation and apoptosis in coloncancer cellsObjective: To investigate the effect of Prdx2silencing by RNAi on cellproliferation and apoptosis ability in colon cancer cell lines.Methods:1. The effect of Prdx2gene silencing on the proliferation andapoptosis in colon cancer cell lines SW480and SW620were detected usingMTT assay and flow cytometry.2. A reactive oxygen species assay kit was used to measure intracellularROS in SW480and SW620cell lines after Prdx2silencing.3. The effect of antioxidant NAC on the proliferation and apoptosis inPrdx2silenced colon cancer cell lines SW480and SW620were detected byMTT and flow cytometry assays.5. To observe the effect of Prdx2shRNAon the growth of human coloncancer xenograft tumor, SW480and SW620cells transduced with Prdx2shRNA or negative control vector were implanted subcutaneously into theright flank of nude mice to form xenograft tumors.Results:1. MTT assay results showed that the proliferation of SW480and SW620cells transfected with Prdx2shRNA was significantly decreased compared to controls. It was showed that the apoptosis in thePrdx2silenced SW480and SW620cells was increased significantlycompared with the control groups by flow cytometry analysis (P<0.05).2. It was showed that the content of endogenous ROS in both cell linestransduced with Prdx2shRNA was significantly increased compared withcontrols (P<0.05).3. After treatment of Prdx2silenced cells with different concentrations ofNAC for24h, the results indicated significantly increased proliferation anddecreased apoptosis in NAC-treated silenced cells compared to untreatedsilenced cells (P<0.05).4. It was showed that the average tumor volume was significantly smallerin the Prdx2shRNA transduction group compared to the control groupthroughout the study period (P<0.05).Conclusion: Knockdown of Prdx2suppressed the proliferation andactivated the apoptosis SW480and SW620cells, may be due to an increasein ROS. Part threeThe Study of Prdx2Silencing on Modulating Wnt/β–catenin PathwayObjective: To investigate the effect of Prdx2knockdown on expressionof Wnt/β–catenin pathway related genes, to reveal the effeetive mechanismof Prdx2on colon cancer growth.Methods:1. Western blot was used to detect the expression of nuclearβ–catenin and cytoplasmic β–catenin, a critical factor of Wnt/β–cateninpathway, other proteins associated with the Wnt signaling pathway such asp-β-cateninser33/37, GSK-3β, p-GSK-3βser7and two Wnt target gene c-Myc,Survivin in Prdx2silenced cells and controls. The mRNA expression levelsof c-Myc, surviving were also analyzed by Real-Time PCR.2. An alteration of intracellular distribution of β–catenin in Prdx2silencedcells and control cells were detected by immunofluorescence staining.3. The effect of Wnt/β–catenin signaling pathway inhibitor XAV-939onthe biological characteristics of colon cancer cells transduced with Prdx2shRNA. Two kinds of cells were divided into the following4groups:①solvent control group: DMSO;②Blank control group: XAV-939(–) andPrdx2shRNA (–);③Interference group: XAV-939(–) and Prdx2shRNA(+);④Inhibitor group: XAV-939(+) and Prdx2shRNA (–);⑤ the combination of interference and inhibitor group: XAV-939(+) andPrdx2shRNA (+). The proliferation and apoptosis of colon cancer cellsin each group were detected by MTT assay and flow cytometry.4. Western blot assay was used to detect the effects of Wnt/β-cateninpathway inhibitor XAV-939on the expression of endogenous Prdx2proteinin colon cancer cells.Results:1. Following Prdx2silencing, the levels of intranuclearβ–catenin were significantly decreased in SW480and SW620cells,whereas the levels of cytoplasmic β–catenin were significantly increased inSW480and SW620cells (P<0.05). In addition, the levels ofphosphorylated β–catenin (Ser33/37), which generates the degradationsignal, were significantly increased following Prdx2silencing (P<0.05).However, the total expression levels of β–catenin were largely unchanged.The levels of GSK-3β were significantly increased in Prdx2-silenced cells,whereas the levels of phosphorylated GSK-3βSer9were significantlydecreased in Prdx2-silenced cells (P<0.05). Furthermore, thedown-regulation of Survivin and c-Myc mRNA and protein expressionlevels in cells following transduction with Prdx2shRNAwere determinatedby Real-Time PCR and Western blot assays.2. It was showed that β-catenin was observed to translocate from itslocation in the nucleus in negative control-transduced cells to thecytoplasm in silenced cells. 3. XAV-939alone and Prdx2shRNA alone significantly reduced coloncancer cells proliferation compared with the blank control group (P<0.05).However, treatment with XAV-939in colon cancer cells with Prdx2knockdown did not further reduced the proliferation compared to coloncancer cells with Prdx2knockdown alone. Moreover, XAV-939alone andPrdx2shRNA alone significantly increased apoptosis rate in colon cancercells, compared with the blank control group (P<0.05). However, treatmentwith XAV-939in colon cancer cells with Prdx2knockdown did not furtherincreased apoptosis rate compared to colon cancer cells with Prdx2knockdown alone.4. The protein expression of Prdx2was obviously downregulated in theSW480and SW620cells that treated with XAV-939.Conclusion: Prdx2silencing inhibited the expression of Wnt/β-cateninpathway related genes. Prdx2plays a critical role in the growth of humancolon cancer possibly through modulating the Wnt/β-catenin pathway.