br lentivirus transduced groups As
lentivirus transduced groups. As caspase 3/7 are the downstream cas-pases in the apoptosis pathway, those findings suggested that RKO or HCT 116 FilipinIII underwent increased apoptosis after transduction of shSNRPA1 lentivirus. Therefore, SNRPA1 knocking down can promote the apoptosis of RKO or HCT 116 cells.
3.1.5. Knocking down of SNRPA1 inhibited tumor formation in vivo From the above in vitro experiments, we came to know that
SNRPA1 knocking down contributed to the inhibition of cell pro-liferation, the dampened colony formation and increased cell apoptosis in RKO or HCT 116 CRC cells. To verify those findings in vivo, we implanted shRNPA1 or control lentivirus transduced RKO cells into nude mice and observed the tumor formation in mice. The results in Fig. 3A–D studied diﬀerent characteristics of formed tumors. The gen-eral situation after formation of implanted tumors was illustrated in Fig. 3A and the implanted tumors were then harvested and exhibited in
Fig. 2. Celigo cell proliferation assay. RKO (A, C) or HCT116 (B, D) cells transduced with anti-SNRPA1 or control shRNA lentivirus for 3 days were seed into 96-well plates (2 × 103 cells/well) and the cell growth status was monitored by taking fluorescent images (Top panel of Fig. 2A, B), calculating cell count number or cell count fold changes (bottom panel of Fig. 2A, B), MTT cell viability assay (OD490) (Fig. 2C, D), respectively for consecutive 5 days as of the beginning of this experiment. The colony forming abilities of selected CRC cells were analyzed at day 8 after 3 days of anti-SNRPA1 or control shRNA lentivirus transduction. CRO or HCT 116 cells (500 cells/ well) were seed into 6-well plates. Shown below are the visible images of colony forming experiment for CRO (Fig. 2E) and HCT116 (Fig. 2F) and the corresponding quantitative data, respectively. The caspase 3/7 activities in CRO (Fig. 2G) or HCT 116 (Fig. 2H) treated with anti-SNRPA1 or control shRNA lentivirus. Shown results are the average one from three independent experiments, error bars represent standard deviation (SD) and ** means a significant statistical diﬀerence between groups with p-value < 0.01.
Fig. 3B. other parameters such as tumor weight and tumor volume were summarized in Fig. 3C and3D, respectively. Those results clearly in-dicated that nude mice implanted with shSNRPA1 lentivirus transduced RKO cells formed smaller size and number of tumors compared to the control groups (Fig. 3A, C). In terms of tumor weight and tumor
volume, the data suggested that the averaged tumor volume at each of 5 measuring points or averaged tumor weight at the end of mice sacrifice were much less in SNRPA knocking down groups than in their re-spective controls (Fig. 3C, D). The whole-body imaging analysis and corresponding quantitative data were illustrated in Fig. 3E–H, which
Fig. 3. Tumor formation in SPF nude mice by implanting anti-SNRPA1 or control shRNA lentivirus transduced RKO cells. At the end of the experiment, 10 mice for control or shRNA knocking down groups were lined up and imaged with (C) or without dissection (A) of the implant tumors. The corresponding tumor volume (B) or tumor weight (D) were plotted on the right, respectively. Whole-body ima-ging of implanted tumors in nude mice implanted with control shRNA (E) or shSNRPA1 (F) lentivirus transduced RKO cells. The corresponding quantitative analysis of total radiant eﬃciency for control (G) or shSNPRA1 knocking down group (H) were summarized below, re-spectively. T-test of diﬀerence of total radiant eﬃciency between control and SNRPA1 knocking down groups in-dicated a p-value of 0.002301.
were consistent with the results shown in Fig. 3A–D. Those in vivo experimental results further corroborated that previous findings in vitro
that SNRPA1 knocking down inhibited the cell proliferation, promoted
cell apoptosis of CRC cells.
Fig. 4. (A) Protein interaction network of SNRPA1. Important oncogenic or tumor suppressor genes associated with SNRPA1 are shown. (B) Western blotting detection of protein expression in selected target genes regulated by SNRPA1. NC- control group, KD-SNRPA1 knocking down group. GAPDH was the internal control. (C) quantitative analysis of WB results from (B), *-indicates a statistically significant diﬀerence between groups.
3.2. Screening for downstream genes regulated by SNRPA1 via microarray profiling analysis
To further decipher the regulatory mechanism of SNRPA1 in CRC, we performed a microarray gene expression profiling analysis. 6 gen-echips (Aﬀymetrix 901838) were hybridized with RNA isolated from 3 pairs of RKO cells transduced with control or shSNRPA1 lentivirus. After the processing of microarray raw data, the diﬀerences of nor-malized expression data between each of 3 pairs of control or knocking down samples were compared. Genes who appeared 3 times in 3 comparisons were finally selected into the pool of diﬀerentially ex-pressed genes (DEGs). Further analysis of the pool of DEGs identified 602 upregulated and 765 downregulated genes in SNRPA1 silencing groups compared with control, which are of particular interest in this study.