| Objective:To investigate the effects of overexpressing hnRNPAB subtypes on human colorectal cancer stem cells,and conduct a preliminary study of its molecular mechanism.Methods:SW480 and HT29 cells were transfected with the lentiviral vector hnRNPA2/B1-GFP-LV and the negative control virus NC-GFP-LV,which were called experimental groups(SW480/hnRNPA2/B1,HT29-hnRNPA2/B1)and control group(SW480-NC,HT29-NC)respectively.The two groups of cells were tested as follows:(1)Determining the effect of overexpression:(1)The relative expression of hnRNPA2/B1 mRNA was detected by RT-qPCR;(2)The relative expression of hnRNPA2/B1 protein was detected by Western blot;(2)Detection of stemness on tumor cell:(1)Detection of tumor clone formation ability:plate clone formation experiment.(2)Detection of cancer stem cells markers:CD133 and CD44 were detected by flow direct-labeled antibody analysis;(3)Tumor tumorigenicity experiment in vivo:Detected by nude mice tumorigenicity experiment;(3)Detection of other malignant biological properties:(1)Detection of tumor invasion ability by Transwell;(2)Detection of tumor migration ability by Scratch test;(3)The tumor proliferation ability was detected by CCK8;(4)Detection of indicators of WNT/?-catenin signaling pathway:WNT3A,WNT5A,?-cantenin protein expression was detected by Western blot.Results:(1)The overexpression of effect determination:(1)The relative expression of hnRNPA2/B1 mRNA detected by RT-qPCR:The relative expression of hnRNPA2/B1 mRNA in the SW480 cell experimental group were(3.53±0.31)and that were(0.97±0.32)in the control group respectively.The relative expression of hnRNPA2/B1 in the experimental group was about 3.5 times that of the control group.(P<0.01);at the same time,the relative expression values of hnRNPA2/B1 mRNA in the HT29 cell experimental group were(3.32±0.33)and that were(0.98±0.32)in the control group(P<0.01);(2)The protein expression levels of hnRNPA2/B1 was detected by Western blot:The protein expression of hnRNPA2/B1 were(0.72±0.03)and(0.52±0.02)in the SW480 cell experimental group and control group,and(1.10±0.04)and.0.78±0.03)were in the HT29 cell experimental group and control group respectively.The protein expression of hnRNPA2/B1 in the experimental group increased significantly(P<0.01),indicating that overexpression of hnRNPA2/B1 succeeded,and cell model established successfully;(2)Detection of stemness on tumor cell:(1)Detection of tumor colony forming ability:The colony forming rates in the SW480 experimental group were(18.8±0.42)%and that were(5.6±0.21)%in the control group,respectively(P<0.01);at the same time,the colony forming rates in the HT29 experimental group were(16.1±0.53)%and that were(5.7±0.31)%in the control group,respectively(P<0.01);(2)Detection of cancer stem cell markers:The positive rates of CD133 were(63.2±4.5)%and(47.3±3.3)%in the SW480 experimental group and the control group respectively and that were(67.1±4.6)%and(49.5±5.2)%about CD44;At the same time,the positive rates of CD133 in the HT29 experimental group and the control group were(81.4±4.5)%and(64.5±3.4)%respectively and that were(79.4±4.6)%and(61.7±6.1)%about CD44.After overexpression of hnRNPA2/B1,the expression of two cancer stem cell markers increased significantly;(P<0.01)(3)Tumorigenicity experimet in vivo:The tumor volume in the SW480 cell experimental group and the control group were(1.575±0.38)cm~3 and(0.865±0.30)cm~3 respectively and that were(1.49±0.25)cm~3 and(0.81±0.23)cm~3 in the HT29 cell experimental group and control group.Compared with the control group,the tumor volume in experimental group was significantly higher.;(P<0.01)(3)Detection of other malignant biological properties:(1)Detection of tumor cell invasion ability:The number of cells passing through the transwell chamber were(315±6.7)and(62±4.3)in the SW480 cell experimental group and the control group,and that were(289±7.2)and(54±5.2)in the HT29 cell experimental group and the control group.Compared with the control group,the number of cells passing through the transwell chamber in the experimental group increased significantly;(P<0.01)(2)Detection of tumor cell migration ability:The healing rates of the SW480 cell experimental group were(19.6±0.9)%and that were(2.23±0.8)%in the control group,respectively,and that were(40.3±0.7)%and(6.4±0.6)%in the HT29 cell experimental group and the control group.Compared with the control group,the migration ability of tumor cells was significantly improved after overexpressing hnRNPA2/B1;(P<0.01)(3)Detection of tumor cell proliferation ability:The proliferation inhibition rates of the those two groups of cells were measured at three time points:24h,48h,and 72h.The OD value in SW480 cell experimental group and the control group were:24h(0.47±0.017)and(0.33±0.024);48h(0.62±0.027)and(0.40±0.034);and 72h(0.65±0.043)and(0.42±0.037);And that were:24h(0.49±0.032)and(0.31±0.033);48h(0.65±0.34)and(0.43±0.29);and 72h(0.67±0.042)and(0.47±0.035)in HT29 cell experimental group and the control group;The results suggest that compared with the control group,the OD value of the cells in the experimental group at three time points decreased significantly;(P<0.01)(4)Western blot detection of WNT3A protein expression were(0.87±0.04)and(0.27±0.03),and WNT5A protein expression were(0.92±0.05)and(0.37±0.04),then?-cantenin protein expression were(0.54±0.03)and(0.32±0.02)in SW480 cells in the experimental group and control group,while WNT3A protein expression in HT29 cell experimental group and control group were(0.91±0.03)and(0.52±0.04),and that were(1.01±0.06)and(0.28±0.05)and on WNT5A protein expression,then that were(0.64±0.04)and(0.39±0.03)on?-cantenin protein expression.Compared with the control group,the expression of the three proteins in the experimental group was significantly increased;(P<0.01)Conclusion:Overexpression of hnRNPA2/B1 could promote the acquisition of stemness in colorectal cancer cells SW480 and HT29,and its mechanism might be related to the high expression of hnRNPA2/B1 activating WNT/?-catenin signaling pathway. |
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