br Tumor sections were further
Tumor sections were further under IHC staining using antibody against CPA4 and the proteins associated with downstream pathways.
2.10. Flow cytometric analysis of cell apoptosis and cell cycle
The extent of apoptosis was measured with an Annexin V-APC Apo-ptosis Detection kit (BD Biosciences) according to the manufacturer's instructions. Cells were collected, washed with ice-cold PBS twice,
gently resuspended in 100 μL of 1× binding buffer containing 2.5 μL APC-conjugated annexin-V, then incubated at room temperature in the dark for 15 min. For cell cycle analysis, the cells were fixed using 75% pre-cooled ethanol at 4 °C overnight. Cells were then stained with 50 μg/mL propidium iodide (PI; Kaiji, China) containing RNaseI (Kaiji, China). The stained cells were analyzed by flow cytometry (Guava easyCyte). Three independent replicates of this experiment were performed.
Fig. 3. Overexpression of CPA4 promoted the proliferation and tumorigenesis of human CRC cells in vivo and in vitro. (A) and (B) The efficiency of CPA4 overexpression in SW620 and RKO cells was determined by western blot, with GAPDH as a loading control. (C–F) Overexpression of CPA4 promoted cell proliferation as determined by CCK-8 assays and colony formation assays. (G–H) Overexpression of CPA4 promoted the migratory abilities and invasiveness of SW620 and RKO cells. (I) Tumorigenesis assay by subcutaneous injection of RKO/Vector and RKO/CPA4 cells in nude mice (n = 6/group). (J) Tumor volumes were measured every 5 days, and tumor weights were measured at the end of the experiment. (K) The sections of tumor processed by IHC staining using 701977-08-4 against CPA4, STAT3, and ERK. The results are presented as the mean ± SD. ( p b 0.05, p b 0.01, p b 0.001).
2.11. Statistical analysis
Experiments were conducted in triplicate, and the data are pre-sented as mean values ± standard deviation (SD). The expression level of CPA4 in paired non-tumor and tumor tissues was compared using a paired Student's t-test. An independent Student's t-test was used to compare the mean values of any two preselected groups. Sur-vival data were analyzed with the Kaplan-Meier method and were com-pared using the log-rank test. The correlations between IHC results and clinicopathologic parameters were determined using the chi-square test. p b 0.05 was considered statistically significant. All data were ana-lyzed using SPSS version 21.0 for Windows (IBM, Armonk, NY, USA). Designations for levels of statistical significance are as follows: * repre-sents p b 0.05, ** represents p b 0.01, and *** represents p b 0.001.
3.1. CPA4 expression in CRC cell lines and tumor tissues
CPA4 mRNA expression levels were investigated in 42 paired CRC and adjacent non-tumor tissue samples by qRT-PCR (Fig. 1A). CPA4 ex-pression was significantly upregulated in 76.2% (32/42) of CRC tissues compared with that in adjacent normal tissues (Fig. 1B). We also mea-sured CPA4 mRNA and protein expression levels in CRC cell lines and colon cell lines by qRT-PCR and western blot. Among five CRC cell lines, CPA4 mRNA expression was significantly increased in the HCT116 and LS123 lines and decreased in SW620 and RKO lines. CPA4 protein expression levels were determined using western blot and were consistent with relative levels of CPA4 mRNA (Fig. 1C, D).
3.2. Prognostic and clinicopathological significance of CPA4 in CRC
To explore whether CPA4 expression was associated with overall survival and clinicopathological features of CRC cases, IHC staining was performed in 120 human CRC samples, and high CPA4 protein ex-pression was detected in 35.0% (42/120) of those CRC samples (Fig. 1E). Kaplan-Meier analysis revealed that patients with low CPA4 expression levels had better overall survival rates than patients with high CPA4 expression (Fig. 1F). Furthermore, high CPA4 expression pos-itively correlated with TNM stage (p = 0.013) and histologic
differentiation (p = 0.009) (Table 1). In order to verify our findings, the expression level of CPA4 was analyzed using The Cancer Genome Atlas (TCGA). CPA4 was significantly up-regulated in both Colon Adeno-carcinoma (COAD) and Rectal Adenocarcinoma (READ) tissues com-pared with normal tissues (Fig. 1G).
3.3. Knockdown of CPA4 suppressed the proliferation and tumorigenesis of CRC cells
CPA4 expression was knocked down in HCT116 and LS123 lines by two different shRNAs (#1 and #2). The knockdown efficiency of each shRNA was confirmed by western blot (Fig. 2A, B). CCK-8 and colony formation assays indicated that knocking down CPA4 greatly inhibited the cell growth of HCT116 and LS123 cells compared to growth of con-trol cells (Fig. 2C–F). Transwell assays showed that knocking down CPA4 significantly attenuated the migratory and invasive abilities of HCT116 and LS123 cells (Fig. 2G, H). Tumorigenesis assays by subcuta-neous injection with shCPA4-stably transfected HCT116 cells were per-formed in nude mice, and tumor growth was monitored. Tumor volume and weight were significantly lower in the CPA4 knockdown group than in the negative control group (Fig. 2I, J). Moreover, much lower expres-sion of CPA4, STAT3 and ERK was found in CPA4 knockdown group than that in the control group, as detected by IHC analysis (Fig. 2K).