Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is a common invasive malignancy worldwide with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been reported to be involved in cancer development. However, lncRNAs that are functional in ESCC and the underlying molecular mechanisms remain largely unknown.

Methods: Transcriptomic analysis was performed to identify dysregulated lncRNAs in ESCC tissue samples. The high expression of LINC00680 in ESCC was validated by RT-qPCR, and the oncogenic functions of LINC00680 was investigated by cell proliferation, colony formation, migration and invasion assays in ESCC cells in vitro and xenografts derived from ESCC cells in mice. RNA-seq, competitive endogenous RNA (ceRNA) network analysis, and luciferase reporter assays were carried out to identify LINC00680 target genes and the microRNAs (miRNAs) bound to LINC00680. Antisense oligonucleotides (ASOs) were used for in vivo treatment.

Results: Transcriptome profiling revealed that a large number of lncRNAs was dysregulated in ESCC tissues. Notably, LINC00680 was highly expressed, and upregulation of LINC00680 was associated with large tumor size, advanced tumor stage, and poor prognosis. Functionally, knockdown of LINC00680 restrained ESCC cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, LINC00680 was found to act as a ceRNA by sponging miR-423-5p to regulate PAK6 (p21-activated kinase 6) expression in ESCC cells. The cell viability and motility inhibition induced by LINC00680 knockdown was significantly reversed upon PAK6 restoration and miR-423-5p inhibition. Furthermore, ASO targeting LINC00680 substantially suppressed ESCC both in vitro and in vivo.

Conclusions: An oncogenic lncRNA, LINC00680, was identified in ESCC, which functions as a ceRNA by sponging miR-423-5p to promote PAK6 expression and ESCC. LINC00680/miR-423-5p/PAK6 axis may serve as promising diagnostic and prognostic biomarkers and therapeutic targets for ESCC.

Keywords: ASO; CeRNA; ESCC; LINC00680; LncRNA; PAK6; miR-423-5p.

Fig. 1

A large number of lncRNAs are dysregulated in ESCC. a Ten pairs of ESCC tumor tissues (T) and matched adjacent normal tissues (N) were collected and subjected to RNA-seq analysis followed by hierarchical cluster analysis. b Volcano plot shows the fold change against the q value for genes as described in (a). Blue and red dots represent genes with significant change (q < 0.05, FC > 1.5). c-d Pie chart shows the differentially expressed mRNAs (c) and lncRNAs (d) in ESCC tumor samples according to RNA-seq. e–f Heat map displays the expression of the differentially expressed mRNAs (e) and lncRNAs (f) as shown in (c) and (d), respectively. g Flowchart to search for clinical-relevant lncRNAs in ESCC. h–k The relative expression of LINC00680 (h), AC092910.3 (i), MIR4435-2HG (j), and GSEC (k) in ESCC tumor and normal tissues from lnCAR database. l-o The correlation between prognosis and the expression of LINC00680 (l), AC092910.3 (m), MIR4435-2HG (n), and GSEC (o) in ESCC patients from lnCAR database

Fig. 2

LINC00680 promotes cell proliferation, colony formation, migration, and invasion in vitro and tumor growth in vivo. a, b, g, i, k KYSE510 cells were transfected with negative control siRNA (si NC) or two independent siRNAs targeting LINC00680 (si LINC00680 #1 and si LINC00680 #2) followed by RT-qPCR analysis (a), cell proliferation assay (b), colony formation assay (g), wound healing assay (i), and transwell assay (k). Scale bar: 50 µm. h, j, l Quantification of the number of colonies (h), the percentage of wound area (j), and the number of invasive cells (l) as shown in (g), (i), and (k), respectively. c Schematic diagram of the chromosomal location of LINC00680 and the six isoforms confirmed by RACE and PCR assays. d cDNA from KYSE140 cells were subjected to 5’ and 3’ RACE assays to detect the full sequence of LINC00680. PCR products were separated by DNA agarose gel. The six isoforms were indicated by arrows. DNA marker was shown on the left. Sanger sequencing results of the 5’ and 3’ end from PCR products were shown at the bottom, detailed sequencing results could be found in Additional file 4: Table S3. m, n, o, q, s KYSE150 cells were transfected with control vector or vector expressing LINC00680 followed by RT-qPCR analysis (m), cell proliferation assay (n), colony formation assay (o), wound healing assay (q), and transwell assay (s). Scale bar: 50 µm. p, r, t Quantification of the number of colonies (p), the percentage of wound area (r), and the number of invasive cells (t) as shown in (o), (q), and (s), respectively. u KYSE510 cells stably transfected with negative control shRNA (sh NC) or two independent shRNAs targeting LINC00680 (sh LINC00680 #1 and sh LINC00680 #2) were subjected to RT-qPCR analysis. v Cells as described in (u) were subcutaneously injected into nude mice (n = 5 in each group), and images of excised tumors were shown. w The average of the weight of tumors as shown in (v). x The growth curves of tumors as shown in (v). The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Transcriptomic analysis reveals that PAK6 is regulated by LINC00680. a KYSE510 cells were subjected to polysome profiling and the resultant fractions were applied to RNA exaction and RT-qPCR analysis to examine the expression of LINC00680. Fractions 1 to 3: Free RNA (unbound with ribosome); Fraction 4: 40S (40S ribosomal subunit); Fractions 5 and 6: 60S (60S ribosomal subunit); Fractions 7 to 9: Monosome; Fractions 10 to 15: Polysome. b-c The subcellular distribution of LINC00680 in KYSE510 (b) and KYSE140 (c) cells was determined by subcellular fractionation followed by RT-qPCR analysis. d KYSE510 and KYSE140 cells transfected with negative control siRNA (si NC) and siRNA specifically targeting LINC00680 (si LINC00680) for three days were subjected to RNA-seq analysis, and gene expression patterns are presented by volcano plot. Blue and red dots represent genes with significant change (P < 0.001, FC > 1.5). e The expression of differentially expressed genes (P < 0.001, FC > 1.5) was represented by heat map. f CeRNA network constituting of LINC00680-miRNAs-mRNAs (genes positively-regulated by LINC00680, n = 68) was shown. Nodes in green, yellow, and light blue represent LINC00680, miRNAs, and mRNAs, respectively. g The flowchart to identify clinically relevant target genes of LINC00680 in ESCC. h The expression of PAK6 in a cohort of esophageal carcinoma (ESCA) samples (n = 182) and normal samples (n = 286) from GEPIA database (http://gepia.cancer-pku.cn/). i The correlation between the expression of PAK6 and prognosis of ESCA patients from Oncolnc database (http://www.oncolnc.org/). j-k KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or two independent siRNAs targeting LINC00680 (si LINC00680 #1 and si LINC00680 #2) followed by RT-qPCR (j) and immunoblotting analysis (k). l The expression of PAK6 in a cohort of ESCC tumor samples (n = 140) and normal samples (n = 140) in house. m The correlation between the expression of PAK6 and prognosis of ESCC patients in house (n = 60). n The correlation between the expression of PAK6 and LINC00680 in ESCC tumor samples in house (n = 140). The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

PAK6 promotes the malignant behaviors in ESCC cells. a, b, c, d, e, g, i, k, m, o KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or siRNA targeting PAK6 (si PAK6 #1 and si PAK6 #2) followed by RT-qPCR analysis (a), immunoblotting analysis (b), cell proliferation assay (c-d), colony formation assay (e, g), wound healing assay (i, k), and transwell assay (m, o). Scale bar: 50 µm. f, h, j, l, n, p Quantification of the number of colonies (f, h), the percentage of wound area (j, l), and the number of invasive cells (n, p). q, r, s, t, v, x KYSE150 cells were transfected with control vector or vector expressing PAK6 followed by RT-qPCR analysis (q), immunoblotting analysis (r), cell proliferation assay (s), colony formation assay (t), wound healing assay (v), and transwell assay (x). Scale bar: 50 µm. u, w, y Quantification of the number of colonies (u), the percentage of wound area (w), and the number of invasive cells (y) as shown in (t), (v), and (x), respectively. The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

PAK6 restoration attenuates the inhibitory effects of LINC00680 knockdown on the malignant behaviors in ESCC cells. a, b, c, d, f, h, j, l, n KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or siRNA targeting LINC00680 (si LINC00680) in the presence or absence of vector expressing PAK6 followed by immunoblotting analysis (a), cell proliferation assay (b-c), colony formation assay (d, f), wound healing assay (h, j), and transwell assay (l, n). Scale bar: 50 µm. e, g, j, k, m, o Quantification of the number of colonies (e, g), the percentage of wound area (i, k), and the number of invasive cells (m, o). The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

LINC00680 acts as a miRNA sponge for miR-423-5p to regulate the expression of PAK6 and the malignant behaviors in ESCC cells. a-b Sequence match between miR-423-5p and wild-type (WT) LINC00680 (a) or the 3’ UTR of PAK6 (b) as well as the corresponding mutant form (MT) with the predicted miR-423-5p binding site mutated is shown. c-d KYSE510 (c) and KYSE140 (d) cells were transfected with luciferase reporter vectors containing wild-type (WT-luc) or mutated (MT-luc) LINC00680 in the presence or absence of negative control miRNA (miR-NC) or miR-423-5p mimic (miR-423-5p) followed by dual-luciferase reporter assay. e–f KYSE510 (e) and KYSE140 (f) cells were transfected with luciferase reporter vectors containing wild-type (WT-luc) or mutated (MT-luc) 3’ UTR of PAK6 in the presence or absence of negative control miRNA (miR-NC) or miR-423-5p mimic (miR-423-5p) followed by dual-luciferase reporter assay. g, h, i, j, k, l, n, p, r, t, v KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or siRNA targeting LINC00680 (si LINC00680) in the presence or absence of miR-423-5p inhibitor followed by RT-qPCR analysis (g, h), immunoblotting analysis (i), cell proliferation assay (j, k), colony formation assay (l, n), wound healing assay (p, r), and transwell assay (t, v). Scale bar: 50 µm. m, o, q, s, u, w Quantification of the number of colonies (m, o), the percentage of wound area (q, s), and the number of invasive cells (u, w). The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

LINC00680 is a potential therapeutic target in ESCC. a, b, c, d, e, g, i, k, m, o KYSE510 and KYSE140 cells were transfected with negative control ASO (ASO NC) or ASO specifically targeting LINC00680 (ASO LINC00680) followed by RT-qPCR analysis (a, b), cell proliferation assay (c, d), colony formation assay (e, g), wound healing assay (i, k), and transwell assay (m, o). Scale bar: 50 µm. f, h, j, l, n, p Quantification of the number of colonies (f, h), the percentage of wound area (j, l), and the number of invasive cells (n, p). q Graphic illustration of ASO NC or ASO LINC00680 injection in nude mice. r Images of excised tumors as described in (q) are shown. s The average of the weight of tumors as shown in (r). (t) The growth curves of tumors as shown in (r). u, v The expression of LINC00680 (u) and PAK6 (v) in tumors as described in (r) was examined by RT-qPCR analysis. w A proposed model of LINC00680 function in ESCC. The highly expressed LINC00680 in ESCC cells functions as a miRNA sponge to sponge miR-423-5p to release its repression on PAK6, leading to the aberrant expression of PAK6 and ESCC tumorigenesis. The experiments were repeated for three times, and representative data is shown with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001