Overexpression of long noncoding RNA LINC00882 is associated with poor prognosis in hepatocellular carcinoma

Figures & data

Table 1 Primer sequence information used in qRT-PCR

LINC00882	Forward	5′-GCCGATACTTGACCTACGCA-3′
	Reverse	5′-AGATGGCAGGTGCAATCACA-3′
Ki67	Forward	5′-ATAAACACCCCAACACACACAA-3′
	Reverse	5′-GCCACTTCTTCATTCCAGTTACA-3′
PCNA	Forward	5′-TCCTCCTTCCCGCCTGCCTGTAGC-3′
	Reverse	5′-CGCGTTATCTTCGGCCCTTAGTGTA-3′
MMP2	Forward	5′-ATAACCTGGATGCCGTCGT-3′
	Reverse	5′-AGGCACCCTTGAAGAAGTAGC-3′
MMP9	Forward	5′-GAACCAATCTCACCGACAGG-3′
	Reverse	5′-GCCACCCGAGTGTAACCATA-3′

Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; MMP, matrix metalloproteinase.

Table 2 Results of differential expression of LINC00882

Gene	Locus on chromosomes	Transcripts	GSE55191		GSE64631
			Log FC	p-value	Log FC	p-value
LINC00882	chr3:106828642.106959485	NR_028303	3.2	0.0019	2.6	0.0097

Notes: Log FC represents the fold difference in expression; p-value indicates the statistical analysis of significant difference expression. LINC00882 expression is upregulated in the GEO dataset.

Abbreviations: FC, fold change; GEO, Gene Expression Omnibus.

Figure 1 High expression of LINC00882 is correlated with poor overall survival of HCC patients.

Notes: (A) Relative mRNA levels of LINC00882 in two GEO datasets (GSE55191 and GSE64631), noncancerous liver tissues (Con), HCC cancer tissues (HCC). *p < 0.05 vs Con in each corresponding database. (B) The expression levels of LINC00882 were detected by in situ hybridization in two separate cohorts (adjacent and independent cohort). A scoring criterion was used for semi-quantification of in situ hybridization. The final scores ranged from 0 to 1, and scores of 2 and 3 were regarded as low expression and high expression, respectively. Representative pictures of different scores are displayed; the upper row is noncancerous tissue and the lower row is HCC tissue. The magnification was 400×. (C) High expression of LINC00882 is correlated with poor overall survival of HCC patients.
Abbreviations: HCC, hepatocellular carcinoma; GEO, Gene Expression Omnibus; Con, control.

Table 3 The expression information of clinical tissues

Tissue samples	Tissue types		Total	p-value
	Liver cancertissue	Adjacent tissue
The expression level
Low expression	31	64	95	<0.01
High expression	56	23	79
Total	87	87	174

Table 4 Association between LINC00882 expression and clinicopathological characteristics in HCC patient

Variables	LINC00882 expression		p-value
	Low	High
Sex			0.907
Male	54	17
Female	13	3
Tumor numbers			1.000
1	55	17
>1	12	3
Clinical stage			0.184
T1	43	16
T2	24	4
Recurrence status			0.04
Present	44	8
Absent	23	12
HBV infection			0.352
Present	56	19
Absent	11	1
Cirrhosis			0.043
Present	61	14
Absent	6	6
Tumor capsule status			0.652
Present	33	11
Absent	34	9
Age (y)			0.535
<60	49	16
≥60	18	4
Tumor size (cm)			0.636
<3	11	5
3–5	19	6
>5	37	9

Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; y, years.

Table 5 Mean and median for survival time

Group	Meana				Median
	Estimate	Std error	95% CI		Estimate	Std error	95% CI
			Lower bound	Upper bound			Lower bound	Upper bound
−	49.300	3.581	42.281	56.319	48.000	2.236	43.617	52.383
+	36.701	2.667	31.473	41.930	29.000	4.092	20.979	37.021
Overall	39.908	2.326	35.349	44.468	37.000	3.492	30.156	43.844

Note:

a Estimation is limited to the largest survival time if it is censored.

Table 6 Overall comparisons

Test method	Chi-square	df	Sig
Log rank (Mantel–Cox)	4.093	1	0.043

Note: Test of equality of survival distributions for the different levels of group.

Table 7 COX regression analysis

Variables	β	SE	Sig	HR (95% CI)
Expression level of LINC00881	1.353	0.378	0.000	3.868 (1.844–8.117)
Sex	0.440	0.531	0.408	1.553 (0.548–4.399)
Age	0.296	0.373	0.427	1.344 (0.647–2.791)
Tumor numbers	0.240	0.387	0.536	1.271 (0.595–2.713)
Tumor capsule status	0.234	0.318	0.462	1.264 (0.677–2.359)
Clinical stage	0.420	0.335	0.211	1.521 (0.789–2.934)
Recurrence status	1.930	0.443	0.000	6.893 (2.895–16.411)
Cirrhosis	−0.930	0.479	0.052	0.395 (0.154–1.010)
HBsAg	−0.727	1.302	0.576	0.483 (0.038–6.198)
HBcAb	0.721	1.322	0.585	2.057 (0.154–27.423)
Tumor size			0.044
Tumor size (1)	1.054	0.553	0.057	2.868 (0.970–8.478)
Tumor size (2)	1.292	0.516	0.012	3.638 (1.322–10.012)

Abbreviations: HR, hazard ratio; SE, standard error.

Figure 2 LINC00882 knockdown results in expression inhibition of proliferative markers and invasion-related proteins.

Notes: (A) Endogenous expression of LINC00882 in normal cells and hepatoma cells. (B) Silencing of LINC00882 in two HCC cell lines was confirmed by qRT-PCR. Silencing of LINC00882 resulted in expression inhibition of proliferative markers Ki67/PCNA and invasive related proteins MMP2/MMP9 at both mRNA (C) and protein levels (D). *p < 0.05 and **P < 0.01 vs negative control lentivirus-infected cell group (Lv-NC-shRNA).
Abbreviations: HCC, hepatocellular carcinoma; Lv, lentivirus; MMP, matrix metalloproteinase; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; Con, control.

Figure 3 LINC00882 knockdown reduces abilities of proliferation, migration and invasion of HCC cells.

Notes: (A) The cell proliferation was detected using EdU assay (the magnification was 400×), and the percentage of EdU positive cells were counted and compared (C, right). (B) Transwell assay was used to assess the invasion ability of negative control lentivirus-infected HCC cells (Lv-NC-shRNA) and LINC00882-silencing HCC cells (Lv-882-shRNA). Meanwhile, the invasive cells were counted and compared (C, left). The magnification was 200×. *p < 0.05 vs negative control lentivirus-infected cell group (Lv-NC-shRNA). (D) The wound healing assay was performed to detect the migration ability of HCC cells in negative control lentivirus-infected cell group (NC) and LINC00882-silencing (882−) HCC cells. The magnification was 200×.
Abbreviations: EDU, 5-ethynyl-2′-deoxyuridine; HCC, hepatocellular carcinoma; Lv, lentivirus; NC, negative control.