Upregulated long noncoding RNA PANDAR predicts an unfavorable prognosis and promotes tumorigenesis in cholangiocarcinoma

Figures & data

Figure 1 Relative expression of PANDAR in CCA and its clinical significance.

Notes: (A) Relative expression of PANDAR in CCA tissues and adjacent nontumor tissues were determined by qRT-PCR; (B) Relative expression of PANDAR in HIBEC and CCA cell lines were determined by qRT-PCR; (C) Kaplan–Meier survival curves showed that overexpression of PANDAR decreased overall survival in patients with CCA. *P<0.05, **P<0.01, ***P<0.001.
Abbreviations: CCA, cholangiocarcinoma; qRT-PCR, quantitative real-time PCR.

Table 1 Association between PANDAR expression and clinicopathological characteristics of CCA

Clinicopathological features	No of patients (n)	PANDAR expression		P-value
		High (n)	Low (n)
Gender				0.457
Male	29	19	10
Female	38	21	17
Age in years				0.307
<60	41	22	19
≥60	26	18	8
Tumor site				0.382
Intrahepatic	13	7	6
Perihilar	30	16	14
Distal	24	17	7
Tumor stage				0.799
T1–2	24	15	9
T3–4	43	25	18
Lymph node invasion				0.004
Present	44	32	12
Absent	23	8	15
Vascular invasion				0.794
Positive	21	12	9
Negative	46	28	18
TNM stage				0.034
I–II	15	5	10
III–IV	52	35	17
Histology type				0.142
Papillary	9	3	6
Nonpapillary	58	37	21
Differentiation grade				0.602
Well/moderately	22	12	10
Poorly/undifferentiated	45	28	17
Postoperative recurrence				0.006
Present	52	36	16
Absent	15	4	11
Serum CEA level				0.193
>5 ng/mL	44	29	15
≤5 ng/mL	23	11	12
Serum CA19-9 level				0.221
>37 U/mL	53	34	19
≤37 U/mL	14	6	8
HBV infection				0.788
Positive	19	12	7
Negative	48	28	20

Abbreviations: CEA, carcinoembryonic antigen; CA19-9, carbohydrate antigen 19-9; HBV, hepatitis B virus.

Table 2 Univariate and multivariate analysis of prognostic factors for overall survival in patients with CCA

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P-value	HR	95% CI	P-value
Overall survival
Gender (male vs female)	1.085	0.643–1.832	0.759
Age (≥60 vs <60 years)	1.063	0.626–1.805	0.822
Tumor site (extrahepatic vs intrahepatic)	1.034	0.535–2.000	0.920
Tumor stage (T3–4 vs T1–2)	1.433	0.824–2.490	0.202
Lymph node invasion (positive vs negative)	1.953	1.096–3.480	0.023			0.189
Vascular invasion (positive vs negative)	1.341	0.773–2.328	0.296
TNM stage (III–IV vs I–II)	2.36	1.147–4.859	0.020			0.307
Histology type (papillary vs nonpapillary)	0.616	0.264–1.440	0.264
Differentiation grade (poorly/undifferentiated vs well/moderately)	1.748	0.987–3.098	0.056			0.145
Postoperative recurrence (present vs absent)	2.903	1.409–5.982	0.004	2.433	1.157–5.118	0.019
Serum CEA level (>5 ng/mL vs ≤5 ng/mL)	1.368	0.781–2.396	0.273
Serum CA19-9 level (>37 U/mL vs ≤37 U/mL)	1.475	0.761–2.859	0.250
HBV infection (positive vs negative)	1.426	0.806–2.521	0.223
PANDAR expression (high vs low)	2.228	1.269–3.913	0.005	1.830	1.026–3.264	0.041

Note: Data in bold indicates statistical significance at P<0.05.

Abbreviations: CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; HBV, hepatitis B virus.

Figure 2 Transfection and knockdown efficiency of PANDAR-specific siRNAs and PANDAR depletion retarded cell proliferation and colony formation in CCA cells.

Notes: (A) Transfection efficiency detected by flow cytometry in QBC939 and RBE cells; (B) Relative expression of PANDAR in QBC939 and RBE cells was significantly decreased by three siRNAs specifically targeting PANDAR compared with si-NC; (C) CCK-8 assay showed that silenced PANDAR inhibited cell proliferation of QBC939 and RBE cells; (D) Clonogenic assay showed that silenced PANDAR dramatically impaired the colony formation ability of QBC939 and RBE cells. *P<0.05, **P<0.01.
Abbreviation: CCA, cholangiocarcinoma.

Figure 3 PANDAR depletion increased apoptosis in CCA cells.

Notes: (A) Flow cytometry for apoptosis analysis showed that silenced PANDAR significantly promoted apoptosis of QBC939 and RBE cells compared to negative control siRNA; (B) AO/EB double fluorescence staining assay showed that silenced PANDAR significantly promoted apoptosis of QBC939 and RBE cells compared to negative control siRNA (×200); (C) Relative expression of caspase-3 and caspase-9 in QBC939 and RBE cells were activated by si-PANDAR-2; (D) Western blot analysis showed that knockdown of PANDAR restrained the expression of Bcl-2 and increased Bax expression in QBC939 and RBE cells. *P<0.05, **P<0.01.
Abbreviations: CCA, cholangiocarcinoma; AO/EB, acridine orange/ethidium bromide.

Figure 4 Knockdown of PANDAR impaired migration and invasion capacity in vitro and reversed EMT of CCA cells.

Notes: (A) Wound healing assay showed that silenced PANDAR inhibited migration ability of QBC939 and RBE cells (×50); (B) Transwell migration assay showed that silenced PANDAR inhibited migration capacity of QBC939 and RBE cells (×100); (C) Transwell invasion assay showed that silenced PANDAR inhibited invasion potential of QBC939 and RBE cells (×100); (D) Western blot analysis showed that knockdown of PANDAR restrained the expression of MMP-9 in QBC939 and RBE cells; (E) Knockdown of PANDAR reversed EMT in QBC939 and RBE cells. *P<0.05, **P<0.01.
Abbreviations: CCA, cholangiocarcinoma; EMT, epithelial-to-mesenchymal transition.