Silence of long noncoding RNA PANDAR switches low-dose curcumin-induced senescence to apoptosis in colorectal cancer cells

Figures & data

Table 1 Primers for real-time polymerase chain reaction analysis

Gene name	Forward	Reverse
GAPDH	5′-AGCCACATCGCTCAGACAC-3′	5′-GCCCAATACGACCAAATCC-3′
PANDAR	5′-TGCACACATTTAACCCGAAG-3′	5′-CCCCAAAGCTACATCTATGACA-3′
FAS	5′-GCTGGGCATCTGGACCCTCCTACCT-3′	5′-CAGTCACTTGGGCATTAACACTT-3′
NOXA	5′-AGCAGAGCTGGAAGTCGAGTGTG-3′	5′-TGATGCAGTCAGGTTCCTGAGC-3′
PUMA	5′-ACGACCTCAACGCACAGTACGAG-3′	5′-TAATTGGGCTCCATCTCGGG-3′
CCNB1	5′-AATAAGGCGAAGATCAACATGGC-3′	5′-TTTGTTACCAATGTCCCCAAGAG-3′
CDC25C	5′-TCTACGGAACTCTTCTCATCCAC-3′	5′-TCCAGGAGCAGGTTTAACATTTT-3′
CDK1	5′-AAATGTGTGTAGGTCTCAC-3′	5′-ATGATTTAAGCCAACTCAAA-3′

Figure 1 LncRNA PANDAR is undifferentiated in human colorectal cancer (CRC) tissues and does not affect cancer cell growth in vitro.

Notes: (A) PANDAR expression was undifferentiated in CRC tissues in 23 CRC patients by qRT-PCR. (B) The relative expression level of PANDAR in CRC cell lines was detected by qRT-PCR. (C) DLD-1 cells were transfected with PANDAR siRNA and control siRNA. (D) CCK8 assay was performed to determine the proliferation of DLD-1 cells. (E) Colony-forming growth assay was performed to determine the proliferation of DLD-1 cells. The colonies were counted and captured. The experiments were repeated at least three times. *P<0.05, **P<0.01.
Abbreviations: CCK8, Cell Counting Kit-8; LncRNA, long noncoding RNAs; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; NS, nonsignificant; si-NC, small interference-negative control; OD, optical density.

Figure 2 Low-dose curcumin triggers senescence but not apoptosis response in colorectal cancer cells.

Notes: (A) Curcumin was given to DLD-1 cells in vitro at a dose of 1.25–5 μM for 24 h. Then, cell viability was measured using CCK8 assay. (B) Colony-forming growth assay was used to investigate the proliferation of DLD-1 cells. (C) DLD-1 cells were treated with curcumin at a dose of 5 μM for 24 h, and the effect of curcumin on cellular senescence was observed by senescence-associated β-galactosidase staining. (D) The percentage of apoptotic cells was determined by flow-cytometric analysis. The experiments were repeated at least three times. *P<0.05, **P<0.01.
Abbreviations: CCK8, Cell Counting Kit-8; DMSO, dimethylsulfoxide; SA-β-Gal, senescence-associated β-galactosidase; NS, nonsignificant; OD, optical density; FL, fluorescence.

Figure 3 Downregulation of PANDAR in curcumin-treated CRC cells increases apoptosis but attenuates senescence.

Notes: (A) Expression of PANDAR was increased in DLD-1 cells treated with curcumin (5 μM) for 24 h by qRT-PCR. (B) CCK8 assay was used to investigate the proliferation of DLD-1 cells treated with knockdown of PANDAR combined with curcumin. (C) Colony-forming growth assay was used to investigate the proliferation of DLD-1 cells treated with knockdown of PANDAR combined with curcumin. (D) Flow-cytometric analysis was performed to determine the apoptotic percentage of curcumin-treated cells following PANDAR siRNA. (E) SA-β-Gal staining was performed to determine the cellular senescence of curcumin-treated cells following PANDAR siRNA. The experiments were repeated at least three times. *P<0.05, **P<0.01.
Abbreviations: CCK8, Cell Counting Kit-8; CRC, colorectal cancer; DMSO, dimethylsulfoxide; qRT-PCR, quantitative real-time polymerase chain reaction; SA-β-Gal, senescence-associated β-galactosidase; siRNA, small interfering RNA; NS, nonsignificant; FL, fluorescence; OD, optical density; si-NC, small interference-negative control.

Figure 4 qRT-PCR and Western blotting screening genes associated with the switch from senescence to apoptosis.

Notes: (A) qRT-PCR was used to detect the mRNA levels of FAS, NOXA, PUMA, CCNB1, CDC25C, and CDK1 in the DLD-1 cells treated with negative control, si-PANDAR combined with curcumin, curcumin alone, and si-PANDAR alone. (B) Western blotting analysis showed that the protein levels of PUMA were detected in DLD-1 negative control, si-PANDAR combined with curcumin, curcumin alone, and si-PANDAR cells. The experiments were repeated at least three times. *P<0.05, **P<0.01.
Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; si-NC, small interference-negative control.