br To investigate the e ects
To investigate the eﬀects of SNRPA1 knocking down on the cells’ proliferation abilities, we applied the Celigo cell proliferation assay to RKO and HCT116 Filipin III transduced with anti-SNRPA1 shRNA or control Biomedicine & Pharmacotherapy 117 (2019) 109076
lentivirus 3 days before the experiment. The GFP labelled cells were imaged for consecutive 5 days after the beginning of this experiment. The representative fluorescent images and the cell count number or fold changes of cell growth for RKO and HCT 116 cells were shown in Fig. 2A, B, respectively. For MTT assay, the cell growth curves were shown in Fig. 3C, D. Notably, judging from the cell count number (Fig. 2C, D), the rate of cell proliferation in the anti-SNRPA1 lentivirus treatment groups are significant slower than that in the control groups, regardless of RKO or HCT 116 cells, indicating the downregulation of SNRPA1 can eﬀectively inhibit the cell proliferation. The cell count number or fold changes of cell count diﬀer dramatically between the two groups, the number of RKO cells in anti-SNRPA1 shRNA lentivirus treated group even decreased after 5 days of growth in contrast with the robust growth in the control groups. Compared to that in RKO cells, the cell count number or fold changes of cell count in HCT116 cells re-mained largely unchanged over the 5-day experiment, which seems in accordance with the results in Fig. 1 that anti-SNRPA1 shRNA lentivirus works better for RKO cells. The cell growth curves in Fig. 2C, D gen-erally concur with the cell count results in Fig. 2A, B, with a much faster growth seen in the control groups in RKO or HCT116 cells. These results suggest that knocking down SNRPA1 impaired greatly the cell pro-liferation in RKO and HCT 116 cells, which highlights the potential of inhibiting SNRPA1 in restricting the cell proliferation of CRC cells.
3.1.3. Knocking down of SNRPA1 inhibited colony formation of RKO and HCT116 cells
To further investigate how SNRPA1 dampens the cell proliferation in RKO and HCT 116 cells, we looked at the abilities of those cells to form colonies after 3 days of anti-SNRPA1 or control shRNA lentivirus transduction. Lentivirus treated RKO or HCT116 cells were then seed into 6-well plates (500 cells/ well) and the visible colonies formed in each group were checked after 8 days of culture. The results shown in Fig. 2E–F are representative images of those treated cells. Strikingly, we noticed that the number of formed visible colonies in anti-SNRPA1 shRNA lentivirus treated RKO or HCT116 cells are significantly less than that in the control groups (Fig. 2E). The quantitative data in Fig. 2F corroborated our observation in Fig. 2E. However, there is a little discrepancy regarding the number of cells in the control RKO and HCT116 cells, with RKO group showing about averaged 275 cells versus around 140 cells in HCT116 control groups. In addition, it seems that the inhibition of colony forming abilities by anti-SNRPA1 shRNA len-tivirus in RKO cells was a little less prominent compared to that in HCT116 cells. Overall, the results clearly demonstrated that knocking down SNRPA1 will damage the colony forming abilities of CRC cells, which thus makes sense of the inhibited cell proliferation aforemen-tioned.
3.1.4. Knocking down of SNRPA1 promoted the apoptosis of RKO and HCT116 cells
The inhibited cell proliferation and dampened colony formation in RKO or HCT116 cells conferred by shSNRPA1 lentivirus indicated the probability of apoptosis in those two cells. During the occurring of apoptosis, it’s well-known that certain caspases (caspase-8, 9, 10 in humans) play upstream initiator roles in apoptosis by coupling cell death stimuli to the downstream eﬀector caspases (caspase-3, 6, 7) . Due to the crucial roles of caspase 3,7 in apoptosis and as caspase 3 and caspase 7 exhibited almost indistinguishable activity toward certain synthetic peptide substrates , measurement of activated caspase 3/7 activities simultaneously has become a simple and reliable method of detecting cell apoptosis. To verify that, we studied the ac-tivated caspase 3/7 activities in them after shRNA lentivirus trans-duction. The results shown in Fig. 2G–H stated that the caspase 3/7 activities are significantly higher in shSNRPA1 lentivirus transduced RKO (left) or HCT116(right) cells compared to the control shRNA len-tivirus treated groups. Both cell lines implicated a statistically sig-nificant diﬀerence (p < 0.01) between control and shSNRPA1
Fig. 1. (A) qRT-PCR analysis of transcriptional expression of SNRPA1 in four CRC cell lines (SW480, RKO, HT-29 and HCT 116). GAPDH was used as an internal control, Ct (SNRPA1-GAPDH) value was shown on the y-axis, x-axis states each cell line used. SD are calculated from three repeating experiments. SNRPA1 knocking down by shRNA lentivirus. qRT-PCR detection of SNRPA1 mRNA transcripts in RKO (B) and HCT116(C) cells. Western blotting analysis of SNRPA1 (D) in RKO and HCT cells. Lane 1-RKO controls cells; Lane 2-RKO cells transduced with anti-SNRPA1 shRNA lentivirus; Lane 3-HCT116 control cells; Lane 4-HCT 116 cells transduced with anti-SNRPA1 shRNA lentivirus. GAPDH was the internal control for both qPCR and Western blotting experiments. **Indicates a statistical sig-nificance with p-value < 0.01.