LncRNA TINCR favors tumorigenesis via STAT3-TINCR-EGFR-feedback loop by recruiting DNMT1 and acting as a competing endogenous RNA in human breast ...
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Wang et al. Cell Death and Disease (2021)12:83
https://doi.org/10.1038/s41419-020-03188-0 Cell Death & Disease
ARTICLE Open Access
LncRNA TINCR favors tumorigenesis via
STAT3–TINCR–EGFR-feedback loop by recruiting
DNMT1 and acting as a competing endogenous
RNA in human breast cancer
Qin Wang1, Jiena Liu1, Zilong You1, Yanling Yin1, Lei Liu1, Yujuan Kang1, Siwei Li1, Shipeng Ning1, Hui Li1, Yajie Gong1,
Shouping Xu1 and Da Pang 1,2
Abstract
The long noncoding RNA (lncRNA) TINCR has recently been found to be associated with the progression of human
malignancies, but the molecular mechanism of TINCR action remains elusive, particularly in breast cancer. The
oncogenic role of TINCR was examined in vitro and in vivo in breast cancer. Next, the interaction between TINCR,
DNMT1, and miR-503-5p methylation was explored. Moreover, the mechanism by which TINCR enhances EGFR
expression and downstream signaling via an RNA–RNA interaction was comprehensively investigated. Furthermore,
upstream transcriptional regulation of TINCR expression by STAT3 was examined by performing chromatin
immunoprecipitation. Finally, feedback signaling in the STAT3–TINCR–EGFR downstream cascade was also
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investigated. TINCR is upregulated in human breast cancer tissues, and TINCR knockdown suppresses tumorigenesis
in vitro and in vivo. Mechanistically, TINCR recruits DNMT1 to the miR-503-5p locus promoter, which increases the
methylation and suppresses the transcriptional expression of miR-503-5p. Furthermore, TINCR also functions as a
competing endogenous RNA to upregulate EGFR expression by sponging miR-503-5p. In addition, TINCR stimulates
JAK2–STAT3 signaling downstream from EGFR, and STAT3 reciprocally enhances the transcriptional expression of
TINCR. Our findings broaden the current understanding of the diverse manners in which TINCR functions in cancer
biology. The newly identified STAT3–TINCR–EGFR-feedback loop could serve as a potential therapeutic target for
human cancer.
Introduction diagnostic biomarkers and potential targets for cancer
Long noncoding RNAs (lncRNAs) are >200-nt-long therapeutics3–5, and recent examination of increasing
RNA transcripts encoded by the genome that are mostly numbers of cancer transcriptomes by using next-
not translated into proteins, but play key roles in reg- generation sequencing has identified thousands of
ulating gene expression, chromatin dynamics, differ- lncRNAs whose aberrant expression is associated with
entiation, growth, and development1,2. Several lncRNAs different cancer types, including breast, pancreatic, lung,
are also aberrantly expressed in cancers and can serve as liver, gastric, head and neck, and colon cancers6–12.
Although lncRNAs have now been recognized as funda-
mental regulators of gene expression, most lncRNAs
Correspondence: Shouping Xu (Shoupingxu@hrbmu.edu.cn) or remain functionally uncharacterized in cancer.
Da Pang (pangda@ems.hrbmu.edu.cn)
1
Breast cancer (BC) is the most common female malig-
Department of Breast Surgery, Harbin Medical University Cancer Hospital,
nant tumor worldwide. A major feature is its challenging
Harbin, China
2
Heilongjiang Academy of Medical Sciences, Harbin, China heterogeneity at the clinical and molecular level13. Over
Edited by B. Rotblat
© The Author(s) 2021
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 2 of 16
the past few decades, BC has revealed that it is quite (Fig. 1f). Next, GO and KEGG pathway analyses were
complex and has no longer been considered a single performed to identify the potential functions of TINCR
disease, but rather a set of distinct subtypes14. BC sub- (Fig. 1g, h and Supplementary Table S2). The results
types have been classified according to immunohisto- indicated TINCR involvement in the biological processes
chemical markers, clinicopathologic features, genomic of mitotic nuclear division, cell division, regulation of the
alterations, and gene-expression profiling15,16. Aberrant cell cycle, DNA-replication initiation, DNA methylation,
expression of EGFR, a receptor tyrosine kinase of the chromosome segregation, cell proliferation, and
ERBB family17, can result in unregulated growth stimu- microtubule-based movement (Fig. 1g and Supplementary
lation and tumorigenesis in various types of cancer18–22. Table S2). Moreover, our results suggested potential
Moreover, inappropriate EGFR activation can occur TINCR participation in cell- cycle pathway, oocyte
through a range of complex mechanisms, including gene meiosis pathway, salivary-secretion pathway, p53 signal-
amplification, autocrine ligand–receptor stimulation, and ing pathway, progesterone-mediated oocyte-maturation
epigenetic modulation23–25. However, to determine how pathway, aldosterone synthesis, and secretion pathway,
intrinsic and acquired resistance to EGFR inhibitors in and other pathways related to tumorigenesis (Fig. 1h and
cancer treatments can be avoided, it is essential to com- Supplementary Table S2). The association of TINCR
prehensively elucidate the regulatory landscape of the overexpression with patient survival was analyzed in 125
ERBB family and the interaction and crosstalk between patients with breast cancer in the HMUCC cohort.
lncRNAs and ERBB-family members. Patients harboring tumors with upregulated TINCR
Here, we uncovered the diverse manners in which the expression were associated with poor overall survival (OS)
lncRNA TINCR functions in breast cancer biology. (Supplementary Fig. S1).
TINCR was found to be upregulated and positively cor- Besides breast cancer, multiple other cancers exhibited
related with EGFR expression in human breast cancer. TINCR upregulation, including adrenocortical carcinoma
Mechanistic analyses revealed that TINCR recruits (ACC), bladder urothelial carcinoma (BLCA), cervical
DNMT1 to the miR-503-5p locus promoter and thereby and endocervical cancers (CESC), lymphoid neoplasm-
increases miR-503-5p methylation and suppresses its diffuse large B-cell lymphoma (DLBC), acute myeloid
transcriptional expression, and further that TINCR also leukemia (LAML), lung squamous-cell carcinoma
functions as a competing endogenous RNA (ceRNA) to (LUSC), pancreatic adenocarcinoma (PAAD), testicular
upregulate EGFR expression by sponging miR-503-5p. germ-cell tumors (TGCT), thyroid carcinoma (THCA),
Moreover, TINCR stimulates JAK2–STAT3 signaling and thymoma (THYM), according to an analysis of the
downstream from EGFR, and STAT3, in turn, increases GEPIA database (Fig. 2a). To investigate the relationship
the transcriptional expression of TINCR. Our findings between TINCR expression and the prognosis of cancer
enhance the current understanding of the diverse roles of patients, Kaplan–Meier survival analysis and log-rank test
TINCR in cancer biology, and the newly identified were performed to assess the effects of TINCR expression
STAT3–TINCR–EGFR-feedback loop might represent a and clinical outcomes on the survival of patients with
potential therapeutic target for human cancer. breast cancer in the GEO database (two TINCR probes
were used here, 244374-at/229385-at) (Supplementary
Results Table S3) and other cancers in TCGA database. High
TINCR expression is upregulated and correlated with poor TINCR expression indicated markedly poorer prognosis
prognosis in breast cancer than low TINCR expression did in BRCA patients (Fig.
By using the large-scale cancer-genome RNA-seq 2b–g) and BLCA, kidney renal papillary-cell carcinoma
expression data from TCGA database, we examined the (KIRP), ovarian serous cystadenocarcinoma (OV), uterine
potential association of carcinogenesis with the lncRNAs corpus endometrial carcinoma (UCEC), esophageal car-
differentially expressed between breast-cancer and adja- cinoma (ESCA), THCA, kidney renal clear-cell carcinoma
cent normal tissues (Fig. 1a, b). Here, we focused on (KIRC), and stomach adenocarcinoma (STAD) patients
TINCR, which was found to be expressed at higher levels (Fig. 2h–s). EGFR showed higher expression in the 4T1,
in human breast-cancer than in normal tissues (Fig. 1c, d). MDA-MB-231, and UACC-812 cell lines than the other
To validate the result, we analyzed 250 tissue specimens— tested cell lines (Supplementary Fig. S2). Thus, the fol-
125 breast-cancer and 125 adjacent normal tissues—from lowing experiments were performed with these three cell
Harbin Medical University Cancer Center (HMUCC), and lines to examine the regulatory effects on EGFR
we found that TINCR was upregulated in breast-cancer expression.
tissues relative to the expression in normal tissues (Fig. 1e
and Supplementary Table S1). Moreover, TINCR TINCR promotes tumor growth in vivo and in vitro
expression was higher in advanced tumor-node metastasis We next investigated the potential biological functions
(TNM) stages III and IV than in TNM stages I and II of TINCR: we used shRNAs specifically designed to target
Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 3 of 16
A Normal Cancer B Volcano Plot
60
RP11−1100L3.8
FGF14−AS2
MIR497HG
AC016995.3
AC005682.5 48
RP11−161M6.2
AC074289.1
-log2(P-value)
RP11−295M3.4
AC097724.3
RP4−575N6.4 36
RP13−1016M1.2
RP11−70D24.2
MIR99AHG
AP000473.5
WDR86−AS1 24
RP11−394O4.5
ACTA2−AS1
RP11−474O21.5
LINC00961
RP11−536O18.1 12
RP11−104L21.2
RP11−855A2.5
AC093110.3
RP11−968O1.5
RP1−302G2.5 0
RP11−736K20.5 -3.07 -1.93 -0.78 0.36 1.51 2.65 3.8
MAGI2−AS3
RP11−1024P17.1 log2(FoldChange)
RP11−158M2.3
RP11−182J1.17
PP14571 C
RP11−459E5.1
LINC01605 20
LINC01614
RP11−417E7.2
TONSL−AS1
AC068580.5
HOTAIR ****
TINCR expression
RP11−465B22.8
RP11−510N19.5 16
RP1−86C11.7 10
TINCR
CTD−2510F5.4
CTA−384D8.35
RP11−353N14.2
TFAP2A−AS1 5
FAM83H−AS1 12
RP11−429J17.7
U47924.27
RP11−22C11.2 0
RP11−1136G4.2
RP6−65G23.3
AC104667.3 8
RP11−92K15.3 −5
C6orf99
AC009005.2 Tumor Normal
RP3−395M20.12 BRCA
RP5−963E22.6
ATP2A1−AS1 −10 (Num(T)=1104;Num(N)=113)
TYMSOS
D
* E F *
20
6
6 ****
Relative TINCR expression
Relative TINCR expression
TINCR expression
5
16
4
4
3
2 12
2
1
0
8
0
Tumor Normal Tumor Normal Stage Stage
BRCA (N=125) I&II III&IV
(Num(T)=1085; Num(N)=291)
G GO:0007067~mitotic nuclear division
H
hsa04110:Cell cycle
GO:0051301~cell division hsa04114:Oocyte meiosis
GO:0051726~regulation of cell cycle hsa04970:Salivary secretion
GO:0007062~sister chromatid cohesion hsa04530:Tight junction
GO:0000281~mitotic cytokinesis
hsa04115:p53 signaling pathway
GO:0000082~G1/S transition of mitotic cell cycle
hsa04670:Leukocyte transendothelial migration
GO:0007052~mitotic spindle organization
hsa04914:Progesterone-mediated oocyte maturation
GO:0042267~natural killer cell mediated cytotoxicity
hsa04540:Gap junction
GO:0006270~DNA replication initiation
hsa03460:Fanconi anemia pathway
GO:0007059~chromosome segregation
hsa04925:Aldosterone synthesis and secretion
GO:0007018~microtubule-based movement
hsa00750:Vitamin B6 metabolism
GO:0000086~G2/M transition of mitotic cell cycle
GO:0051256~mitotic spindle midzone assembly hsa04520:Adherens junction
GO:0030855~epithelial cell differentiation hsa01230:Biosynthesis of amino acids
hsa04911:Insulin secretion
GO:0006306~DNA methylation
GO:0007017~microtubule-based process hsa00601:Glycosphingolipid biosynthesis
GO:0008283~cell proliferation hsa05130:Pathogenic Escherichia coli infection
GO:0016055~Wnt signaling pathway hsa00310:Lysine degradation
GO:0007049~cell cycle hsa04261:Adrenergic signaling in cardiomyocytes
GO:1902749~regulation of cell cycle G2/M phase transition hsa04710:Circadian rhythm
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7
-lg(P value) -lg(P value)
Fig. 1 (See legend on next page.)
Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 4 of 16
(see figure on previous page)
Fig. 1 TINCR is highly expressed in human breast cancer. a Hierarchical-clustering heatmap of differentially expressed lncRNAs in breast-cancer
and adjacent normal tissues, generated from RNA-seq data from TCGA. Colors: expression levels indicated by log2-transformed scale bar below the
matrix; red and blue: max and min expression levels, respectively. b Volcano plot of lncRNAs differentially expressed between breast-cancer tissues
normal tissues. P < 0.001, fold change >1.2 or fold change < –1.2. c–e TINCR expression in cancer and normal tissues from 1104 breast cancer patients
in TCGA cohort (c), 1,085 breast-cancer patients in GEPIA cohort (d), and 125 breast-cancer patients in HMUCC cohort (e). f TINCR expression in breast
cancers of different TNM stages in TCGA database. (g, h) GO and KEGG analyses of TINCR. P < 0.05 indicated statistical significance. Vertical axis:
biological process or pathway category; horizontal axis: −log10 (P value) of significant biological process or pathway. *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001.
the mouse homolog of the gene, Tincr, and injected Balb/ Considering the aforementioned results, we sought to
c mice with 4T1 cells in vivo. Tumor growth was mark- ascertain whether TINCR knockdown and EGFR-
edly inhibited in mice injected with TINCR-knockdown inhibitor treatment used together would produce a
cells as compared with the growth in mice injected with synergistic anticancer effect. When TINCR knockdown
control cells (Fig. 3a, b), and the median tumor weight was combined with treatment with the EGFR inhibitor
was also lower in the knockdown than in the control gefitinib, cell-inhibition activity was higher than in the
group (Fig. 3c). Furthermore, the results of in vitro assays gefitinib group (IC50 = 13.29 and 10.02 μmol/L vs.
showed that proliferation and colony formation were 26.3 μmol/L) (Fig. 4g). Furthermore, this combination
lower in TINCR-knockdown cells relative to control (Fig. treatment more effectively inhibited tumor growth than
3d–g), and TINCR knockdown also decreased the inva- the single treatments in vivo (Fig. 4h, i), and the median
siveness of these cells (Fig. 3h, i). tumor weight was lower in the combination- treatment
group than in the gefitinib or control group (Fig. 4j).
TINCR upregulates EGFR expression and downstream
signaling in human breast cancer TINCR upregulates EGFR expression by acting as a ceRNA
The aforementioned guilt-by-association analysis and our to sponge miR-503-5p
previous study26 suggested that TINCR could perform its Next, we investigated the potential mechanism by which
potential oncogenic function through EGFR and the EGFR TINCR regulates EGFR expression. First, the subcellular
downstream signaling pathway. Here, TINCR knockdown localization of TINCR was examined, which revealed
decreased EGFR mRNA and protein expression relative to TINCR localization in both the cytoplasm and the nucleus
control in UACC-812 and MDA-MB-231 cells (Fig. 4a, b). in breast-cancer cells (Fig. 5a and Supplementary Fig. S6).
We also examined the relationship between TINCR and LncRNAs localized in the cytoplasm typically act as
EGFR protein expression in 120 breast-cancer samples from ceRNAs to influence the expression of their target
HMUCC, and the results showed that the EGFR level was genes27. To identify miRNA candidates that might be
higher in TINCR high-expression tissues than in TINCR affected in TINCR-knockdown and scrambled-control
low-expression tissues (Fig. 4c, Supplementary Fig.S3, and groups, transcriptome miRNA sequencing was performed
Supplementary Table S4). Next, we tested how TINCR to identify microRNAs that potentially bind TINCR
knockdown affects EGFR downstream signaling. As per our transcript on the HMUCC cohort samples. We identified
expectation, TINCR knockdown led to a decrease in p- 138 potential microRNA candidates in TINCR-
JAK2, STAT3, and p-STAT3 relative to their levels in knockdown compared to the control groups (Supple-
control cells (Fig. 4d). To gain insights into the role of mentary Table S5). Furthermore, the public databases
TINCR in EGFR downstream signaling in a clinical context, TargetScan, RNA22, and StarBase were used to predict
we performed immunohistochemical analysis on samples potential miRNAs that bind to EGFR 3ʹ-UTR (Supple-
from the HMUCC cohort, which revealed that JAK2 and mentary Table S5), and overlapping miRNAs were
STAT3 were more abundant in TINCR high-expression examined among the transcriptome-sequencing data from
tissues than TINCR low-expression tissues (Fig. 4e, Sup- the HMUCC cohort and the TargetScan, RNA22, and
plementary Fig. S4, and Supplementary Table S4). Fur- StarBase databases (Fig. 5b and Supplementary Table S5).
thermore, in Balb/c mice injected with 4T1 cells in vivo, Ultimately, miR-503-5p was selected for the following
EGFR and its downstream-signaling molecules were experiments. The knockdown of TINCR increased the
downregulated in the TINCR-knockdown group relative to expression of miR-503-5p (Fig. 5c). Moreover, the levels
the control group (Fig. 4f). To further validate the result, we of TINCR, EGFR mRNA, and EGFR protein were
analyzed tissue specimens from HMUCC, and we found decreased and increased, respectively, after transfection of
that EGFR, JAK2, and STAT3 were upregulated in breast- miR-503-5p mimic and its corresponding inhibitor (Fig.
cancer tissues relative to the expression in normal tissues 5d, e). Notably, combining TINCR knockdown with miR-
(Supplementary Fig. S5). 503-5p-inhibitor treatment attenuated the decrease in
Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 5 of 16
A B C
CA
YM
CA
SC
BC
L
CA
AD
SC
CT
C
M
TINCR 244374_at TINCR 244374_at
AC
BR
CE
TG
BL
DL
LA
LU
TH
TH
PA
Transcripts Per Million (TPM)
90
100
100
80
Disease free survival(%)
Overall survival(%)
70
80
80
60
60
60
50
40
TINCR High (n=588)
40
40
30 TINCR Low (n=698) TINCR High (n=1503)
20 Log-rank p=0.008 TINCR Low (n=1663)
HR=1.52 Log-rank p=0.048
20
20
10 HR=1.17
0
0
0
(n= 7)
n= )
N 404)
n= )
(n= )
T ( 291)
N 06)
T ( 3)
(n= )
T ( 337)
N 173)
T ( 70)
(n= )
n= )
(n= )
n= )
(n= )
T ( 65)
(n= )
T ( 337)
(n= )
9)
T ( 128
T ( =28
N 085
N =47
N 486
T ( 338
N 179
T ( 171
N 137
N 512
N =118
N n=7
33
1
3
(n=
(n=
1
0 50 100 150 0 50 100 150 200 250
1
n=
n=
n=
n=
(n
n
T(
n
Time (months) Time (months)
D E F G
TINCR 244374_at TINCR 229385_s_at TINCR 229385_s_at TINCR 229385_s_at
Distant metastasis free survival(%)
Distant metastasis free survival(%)
100
100
100
100
Disease free survival(%)
Overall survival(%)
80
80
80
80
60
60
60
60
TINCR High (n=615)
TINCR High (n=613) TINCR Low (n=671) TINCR High (n=618)
TINCR Low (n=697)
40
40
40
40
Log-rank p=0.049 TINCR Low (n=692)
Log-rank p=0.047 HR=1.35 TINCR High (n=1535)
TINCR Low (n=1631) Log-rank p=0.014
HR=1.25
HR=1.5
20
20
20
Log-rank p=0.024
20
HR=1.23
0
0
0
0
0 50 100 150 0 50 100 150 0 50 100 150 200 250 0 50 100 150
Time (months) Time (months) Time (months) Time (months)
H BLCA
I BLCA
J KIRP
K KIRP
100
100
100
100
Disease free survival(%)
Disease free survival(%)
TINCR High (n=122)
TINCR Low (n=363)
Overall survival(%)
Overall survival(%)
80
80
80
80
Log-rank p=0.048
HR=1.32
60
60
60
60
40
40
40
40
TINCR High (n=133) TINCR High (n=125)
TINCR Low (n=105) TINCR High (n=103) TINCR Low (n=280)
20
20
20
20
Log-rank p=0.047 TINCR Low (n=270) Log-rank p=0.004
HR=2.09 Log-rank p=9.7e-06 HR=2.97
HR=3.55
0
0
0
0
0 50 100 150 0 50 100 150 0 50 100 150 200 0 20 40 60 80 100 120
Time (months) Time (months) Time (months) Time (months)
L M N O
OV OV UCEC UCEC
100
100
100
100
Disease free survival(%)
Disease free survival(%)
TINCR High (n=193)
Overall survival(%)
Overall survival(%)
TINCR Low (n=310) TINCR High (n=149)
80
80
80
80
Log-rank p=0.028 TINCR Low (n=62)
HR=1.34 Log-rank p=0.049
60
60
60
60
HR=1.43
TINCR High (n=351)
40
40
40
40
TINCR Low (n=205)
TINCR High (n=517) Log-rank p=0.045
HR=1.79
20
20
20
20
TINCR Low (n=196)
Log-rank p=0.001
HR=2.8
0
0
0
0
0 50 100 150 0 50 100 150 0 50 100 150 200 0 50 100 150 200
Time (months) Time (months) Time (months) Time (months)
P ESCA Q THCA R KIRC S STAD
100
100
100
100
TINCR High (n=30) TINCR High (n=284)
Disease free survival(%)
Disease free survival(%)
TINCR Low (n=493)
TINCR Low (n=96)
Overall survival(%)
Overall survival(%)
Log-rank p=7.5e-12
80
80
80
80
Log-rank p=0.019 HR=2.79
HR=2.78
60
60
60
60
TINCR High (n=279)
TINCR Low (n=192)
40
40
Log-rank p=0.045 TINCR High (n=146)
40
40
HR=2.36 TINCR Low (n=201)
Log-rank p=0.001
20
20
20
20
HR=2.96
0
0
0
0
0 20 40 60 80 0 50 100 150 0 50 100 150 0 20 40 60 80 100 120
Time (months) Time (months) Time (months) Time (months)
Fig. 2 High expression of TINCR is correlated with poor prognosis in human cancer. a Scatter diagram depicting TINCR expression in multiple
cancer and adjacent normal tissues, generated from GEPIA database. b–g Kaplan–Meier analysis of OS (b, e), DFS (c, f), and DMFS (d, g) of breast-
cancer patients according to TINCR expression, generated from GEO database. h–s Kaplan–Meier analysis of OS and DFS in BLCA (h, i), KIRP (j, k), OV
(l, m), UCEC (n, o), ESCA (p), THCA (q), KIRC (r), and STAD (s) patients according to TINCR expression, generated from TCGA database. Data are shown
as means ± standard deviation (SD); Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 6 of 16
A B C
Balb/C 2000
Scramble *
Sh Tincr #1 1.0
Tumor volume(mm3)
1500
Weight of tumor(g)
Scramble *** 0.8
***
1000 *** 0.6
* 0.4
Sh Tincr#1
500 *
* 0.2
0 0.0
6 8 10 12 14 16 18 20 (days) Scramble Sh Tincr #1
D E F
4T1 UACC812 4T1
0.8 0.6 ****
Number of colonies
Scramble NC ***
250
Sh Tincr #1 SiTINCR #1 Scramble Sh Tincr #1 Sh Tincr #2
Cell viability(OD450)
Cell viability(OD450)
0.6 0.5
**
200
** **
Sh Tincr #2 SiTINCR #2
150
0.4 0.4 ** 100
50
0.2 0.3 0
e
#1
#2
bl
cr
cr
m
ra
n
n
Ti
Ti
0.0 0.2
Sc
Sh
Sh
24h 48h 72h 24h 48h 72h 96h
G UACC812 I 4T1 UACC 812 MDA-MB-231
**
Number of colonies
NC Si TINCR #1 Si TINCR #2 300 **
0h 0h
200
Scramble
NC
100
0
C
#1
#2
24h 24h
N
R
R
C
C
N
N
TI
TI
Si
Si
H 4T1 UACC 812 MDA-MB-231
0h 0h
Scramble
Si TINCR #1
Sh Tincr #1
NC
NC
24h 24h
Si TINCR #1
Si TINCR #1
Sh Tincr #1
0h 0h
Si TINCR #2
Si TINCR #2
Si TINCR #2
Sh Tincr #2
Sh Tincr #2
24h 24h
Cell numbers/well
Cell numbers/well
Cell numbers/well
*** 800
*** 200
**
400 *** *** **
600
150
300
400
** ***
percentage%(24h/0h)
percentage%(24h/0h)
percentage%(24h/0h)
100
200
150 80 ** 100 ****
200
**
Wound healing
Wound healing
Wound healing
100 50
0
*** 60
80
0 0 100
60
e
1
#2
C
#1
#2
C
#1
#2
40
bl
r#
N
N
40
cr
R
R
R
R
m
nc
50
C
C
C
C
ra
n
20
N
N
IN
IN
Ti
Ti
20
Sc
TI
TI
T
T
Sh
Sh
Si
Si
Si
Si
0 0 0
e
#1
#2
C
#1
#2
C
#1
#2
bl
N
N
cr
cr
R
R
R
R
m
C
C
C
C
ra
n
n
N
N
N
N
Ti
Ti
Sc
TI
TI
TI
TI
Sh
Sh
Si
Si
Si
Si
Fig. 3 TINCR promotes tumor growth in vivo and in vitro. a Tumorigenesis and tumor tissue of each group of Balb/c mice (n = 6/group).
b, c Tumor volume and weight measured in Balb/c mice after injection of 4T1 cells transfected with Tincr-specific or scrambled-control shRNA. Tumor
tissues were measured at the indicated time points and dissected at the endpoint. d–i Knockdown of Tincr/TINCR expression inhibits proliferation
(d, e), colony formation (f, g), and migration (h, i) of 4T1, UACC 812, and MDA-MB-231 cells. Scale bar, 100 μm. Data are presented as means from
three independent experiments ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 7 of 16 Fig. 4 TINCR regulates EGFR expression and downstream signaling in human cancer. a, b TINCR knockdown decreases expression of EGFR mRNA (a) and protein (b) in UACC 812 and MBA-MB-231 cells. c EGFR protein expression is higher in TINCR-high breast-cancer tissues than in TINCR-low tissues and vice versa in 122 patients in the HMUCC cohort. Scale bar, 400 μm. d TINCR knockdown decreases the levels of p-JAK2, STAT3, and p- STAT3 in UACC 812 cells. e JAK2 and STAT3 protein expression is higher in TINCR-high breast-cancer tissues than in TINCR-low tissues and vice versa in 125 patients in the HMUCC cohort. Scale bar, 400 μm. f TINCR knockdown decreases the levels of EGFR, JAK2, p-JAK2, STAT3, and p-STAT3 in vivo (n = 6/group). g Gefitinib IC50 analysis after Tincr knockdown. h Tumorigenesis and tumor tissue of each group of Balb/c mice (n = 6/group). i, j Tumor growth and weight measured in Balb/c mice in each group. Tumors were measured at the indicated time points and dissected at the endpoint. Data are presented as means from three independent experiments ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Official journal of the Cell Death Differentiation Association
Wang et al. Cell Death and Disease (2021)12:83 Page 8 of 16
Nucleus
A Cytoplasm B StarBase TargetScan
1.0
**
Relative TINCR expression
** ** **** 27 591
0.8 **** *
0.6 22 102 1
0.4 630 26
59 0
0.2
23
0.0 684 2
1.0 **** **** **** **** **** 52 5
*** RNA22 HMUCC
29
Relative U1 expression
0.8
0.6 C
6
NC Si TINCR #1 Si TINCR #2
0.4
*
0.2
Relative expression
4
0.0
1.0
**** **** **** *** **** **** **
* *****
Relative GAPDH expression
** ***
0.8
****
**
2
0.6 ****
0.4
0
0.2
3p
p
p
p
p
p
p
p
p
p
3p
5p
-3
-5
-3
-3
-5
-5
-3
-5
-3
1-
2-
a-
-2
49
12
4b
4c
24
03
03
0b
b-
b-
99
29
-1
-2
-3
-4
-5
-5
-3
-1
25
25
-1
-1
iR
iR
iR
iR
iR
iR
iR
iR
0.0
iR
-1
-1
iR
m
m
m
m
m
m
m
m
iR
iR
m
m
m
m
2
7D
F7
68
31
53
UACC812
81
-4
-2
-4
MC
T4
CC
MB
MB
MB
UA
A-
A-
A-
D E
MD
MD
MD
NC _ _ _ _
+ + +
Si TINCR _ _ _ _ _ + +
miR-503-5p mimic
_ + _ _ _ _ _
MDA-MB-231 UACC812 MDA-MB-231 UACC812 _ _ _ _ _
miR-503-5p inhibitor + +
4
** **
Relative EGFR expression
Relative TINCR expression
2.5
*** 2.0
p-EGFR 170kDa
3
UACC812
1.0 0.5 1.0 2.3 1.0 0.5 1.3
*** 1.5 EGFR 170kDa
2 *** *** 1.0 0.3 1.0 2.8 1.0 0.5 1.3
*** * 1.0
GAPDH 38kDa
1
0.5
0 0.0 p-EGFR 170kDa
MDA-MB-231
-5 m C
hi c
-5 m C
hi c
-5 m C
hi c
-5 m C
hi c
r
r
r
r
to
to
to
to
1.0 0.4 1.0 2.9 1.0 0.7 1.1
in imi
in imi
in imi
in imi
03 5p N
03 5p N
03 5p N
03 5p N
bi
bi
bi
bi
EGFR 170kDa
p
p
p
p
-5 3-
-5 3-
-5 3-
-5 3-
iR 0
iR 0
iR 50
iR 50
m iR-5
m R-5
1.0 0.3 1.0 3.0 1.0 0.5 1.2
m iR-
m iR-
i
m
m
m
m
GAPDH 38kDa
F Position chr19:5567847-5567864 H I
pmirGLO pmirGLO pmirGLO pmirGLO
Relative luciferase activity
Relative luciferase activity
TINCR WT 5’ CGGACGAGGCGCUGCUGCTACCGC 3’ 1.5 TINCR TINCR-mut 1.5 EGFR EGFR-mut
hsa-miR-503-5p 3’ UUGACAAGGGCGACGAGCTACCGC 5’
.....
.....
.....
.....
.....
.....
.....
1.0 1.0
TINCR mut 5’ CGGACGAGGAT AGT AGGCTACCGC 3’
***
0.5 0.5
G Position chr7:55274319-55274324 *
EGFR WT 5’ AGACUACAAAAAUGAAGCUGCUC 3’ 0.0 0.0
NC ic NC ic NC ic NC ic
GACGUCUUGACAAGGGCGACGAU iR im iR im iR im iR im
hsa-miR-503-5p 3’ 5’ M m M m M m M m
p p p p
.....
.....
.....
.....
.....
.....
5 5 5 5
03- 0 3- 03- 0 3-
EGFR mut 5’ AGACUACAAAAAUGAAT AGT AGC 3’ -5 -5 -5 -5
iR iR iR iR
M M M M
Fig. 5 (See legend on next page.)
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 9 of 16
(see figure on previous page)
Fig. 5 TINCR promotes EGFR expression by acting as a ceRNA. a Subcellular localization of TINCR in breast-cancer cell lines, assessed using
nuclear/cytoplasmic extract isolation assay. b Overlapping miRNAs in transcriptome miRNA sequencing data from HMUCC cohort and TargetScan,
RNA22, and starBase databases. Venn diagrams were generated using Venny online software (http://bioinfogp.cnb.csic.es/tools/ venny/). c qRT-PCR
examination of the expression of miRNAs in UACC 812 cells transfected with a TINCR-specific siRNA or scrambled siRNA. d, e Expression of TINCR,
EGFR mRNA (d), and EGFR protein (e) in UACC 812 and MDA-MB-231 cells treated with miR-503-5p mimic or inhibitor. f, g Complementarity between
miR-503-5p seed sequence and 3ʹ-UTRs of TINCR (f) and EGFR (g) predicted through a computational and bioinformatics-based approach by using
TargetScan and StarBase online databases. Watson–Crick complementarity is connected by “|.” Nucleotide-replacement mutations made to the genes
are underlined. h, i Luciferase-reporter assay for assessing interactions between miR-503-5p and its binding sites or mutated binding sites in 3ʹ-UTRs
of TINCR (h) and EGFR (i) in HEK293T cells. Data are presented as means from three independent experiments ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001.
EGFR protein levels (Fig. 5e). Last, the results of 503-5p was upregulated when DNMT1 expression was
luciferase-reporter assays to investigate physical interac- knocked down (Fig. 6e), and the results of ChIP-PCR
tions showed that both EGFR and TINCR with their wild- assays indicated DNMT1 enrichment at the miR-503-5p
type 3ʹ-UTRs were regulated by miR-503-5p, and that this promoter (Fig. 6f). Notably, RIP-assay results indicated
effect could be abolished by mutating their miRNA- markedly higher TINCR enrichment in the DNMT1
binding sites (Fig. 5f–i). These results indicate that EGFR group than the IgG group (Fig. 6g), and DNMT1
and TINCR are bona fide direct targets of miR-503-5p. enrichment at the miR-503-5p promoter was decreased
relative to control after knockdown of TINCR (Fig. 6h).
TINCR recruits DNMT1 to miR-503-5p locus and suppresses
its expression via DNA methylation Identification of positive-feedback loop in
As mentioned in the preceding section, TINCR was also STAT3–TINCR–EGFR axis
localized in the nucleus (Fig. 5a and Supplementary Fig. To investigate the transcriptional regulation upstream
S6), which suggested that TINCR could exert its reg- of TINCR, we used the online database JASPAR (http://
ulatory effect through epigenetic modification of target jaspar.genereg.net/) to search for transcription factors
genes, and knockdown of TINCR expression was found to that might potentially be enriched at the promoter of the
upregulate miR-503-5p (Fig. 5c). First, TINCR knock- TINCR locus. The analysis yielded putative STAT3-
down increased pri-mir-503-5p and pre-mir-503-5p level binding sites in the region upstream of the TSS of
relative to control in UACC 812 cells (Fig. 6a). Because TINCR (Fig. 7a). Moreover, there were obvious enrich-
DNA methylation is a major epigenetic-modification ment peaks of STAT3 in the promotor region (chr19-
mechanism that results in the downregulation of gene 5572112–5573399) of TINCR in HCC70 cells in
expression11, we tested whether methylation of the miR- ENCODE database (Fig. 7b). Next, from the CCLE data-
503 locus was regulated through TINCR. Next, we base, TINCR expression was found to be positively cor-
obtained information on the region around the tran- related with STAT3 expression in human cancer cells
scriptional start site (TSS, −2000 to +200 bases; “−”: (Fig. 7c). Last, STAT3 knockdown decreased the expres-
upstream of TSS; “+”: downstream of TSS) by using the sion of TINCR (Fig. 7d), and the results of ChIP-PCR
University of California at Santa Cruz (UCSC) database assays indicated that STAT3 was highly enriched at the
(http://genome.ucsc.edu/cgi-bin/hgGateway), and we TINCR promoter relative to mock control (Fig. 7e). Col-
then located a CpG island in the promoter region of miR- lectively, our results indicate that TINCR stimulates the
503 locus from the public data of the Li Lab (http://www. activation of EGFR and its key downstream-signaling
urogene.org/methprimer/index.html) (Fig. 6b). Bisulfite effectors, including STAT3, which, reciprocally, promotes
sequencing was performed to investigate the role of the transcriptional expression of TINCR.
TINCR in CpG-island methylation in the miR-503 locus.
As expected, TINCR knockdown led to a decrease in miR- Discussion
503-5p CpG-island methylation at positions LncRNAs have been found to be associated with
ChrX.133680280 and ChrX.133680391 (Supplementary tumorigenesis and drug resistance in various types of
Table S6), and pri-miR-503-5p, pre-miR-503-5p, and cancer29–33. Identification of novel onco-lncRNAs and
miR-503-5p expression was increased following treatment their regulatory mechanisms, and development of novel
with the DNA methyltransferase inhibitor decitabine lncRNA-based targeted strategies for combating cancer,
(1 μM) (Fig. 6c, d). Because DNMT1 is a maintenance appears promising and may introduce a new paradigm in
methyltransferase that preserves the methylation state cancer research. Moreover, EGFR activity has been
across mitotic divisions28, we tested whether miR-503-5p reported to be modulated by diverse signaling path-
expression is regulated by DNMT1. Accordingly, miR- ways34–40. However, the comprehensive regulation of
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 10 of 16
A UACC812 B miR-503-5p
(TSS,+1)
C UACC812 MDA-MB-231
** chrX:
4
CTL
6
**** **** NC (-310,+92)
****
**** *** Decitabine
Relative gene level
Si TINCR #1 3
Relative gene level
CpG island
4
**** Si TINCR #2
2
**
2
1
0 0
Pri-miR-503-5p Pre-miR-503-5p
p
p
p
p
-5
-5
-5
-5
03
03
03
03
-5
-5
-5
-5
iR
iR
iR
iR
i-m
m
i-m
m
e-
e-
Pr
Pr
UACC812 MDA-MB-231
Pr
Pr
D E F
** * **
Relative miR-503-5p expression
Relative miR-503-5p expression
4 2.0
Input IgG DNMT1
**** **
3 1.5
miR-503-5p 247bp UACC812
**
2 1.0
miR-503-5p 247bp MDA-MB-231
1 0.5
0 0.0
CTL Decitabine CTL Decitabine
C
#1
#2
C
#1
#2
N
N
T1
T1
T1
T1
MDA-MB-231
M
M
M
M
UACC812
N
N
N
N
D
D
D
D
G H
Si
Si
Si
Si
0.020
** 15
0.015 ###
***
% Input
% Input
10
0.010
5
0.005
0.000 0
)
T1
)
T1
)
T1
)
T1
G
G
G
G
Ig
Ig
Ig
Ig
M
M
M
M
k(
N
k(
N
k(
N
k(
N
D
D
D
D
oc
oc
oc
oc
M
M
M
M
UACC812 MDA-MB-231 NC Si TINCR
Fig. 6 TINCR recruits DNMT1 to miR-503-5p promoter to regulate miR-503-5p gene methylation. a TINCR knockdown increases Pri-MiR-503-
5p and Pre-MiR-503-5p expression in UACC 812 cells. b Schematic representation of predicted CpG island in miR-503-5p locus. CpG island located in
the promoter region of miR-503-5p locus around the transcriptional start site (TSS, −310 to +92 bases; “−”: upstream of TSS; “+”: downstream of TSS).
c Pri-MiR-503-5p and Pre-MiR-503-5p expression is upregulated in UACC 812 and MDA-MB-231 cells treated with decitabine (1 μM) for 10 h. d MiR-
503-5p expression is upregulated in UACC-812 and MDA-MB-231 cells treated with decitabine (1 μM) for 10 h. e miR-503-5p expression is
upregulated in UACC 812 and MDA-MB-231 cells following DNMT1 knockdown. f ChIP assay results indicating that DNMT1 is enriched at miR-503-5p
locus CpG islands in UACC 812 and MDA-MB-231 cells. g RIP-assay results showing higher enrichment of TINCR in DNMT1 group than IgG group in
UACC 812 and MDA-MB-231 cells. IgG: negative control. h ChIP assay results indicating diminished enrichment of DNMT1 at the promoter of miR-
503-5p locus after TINCR knockdown. Data are presented as means from three independent experiments ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001, ###P < 0.001.
EGFR by lncRNAs remains poorly understood. Our study model of the molecular mechanism underlying the dual
revealed that TINCR upregulated EGFR expression regulation by TINCR of EGFR and its downstream genes
through a ceRNA interaction to trigger enhanced through miR-503-5p.
JAK2–STAT3 downstream signaling, and that STAT3, in Generally, lncRNAs interact with chromatin DNA,
turn, increased TINCR transcriptional expression. Our RNA, or protein, thereby regulating chromatin accessi-
study uncovers a distinctive facet of tumorigenesis by bility, RNA stability, and protein activity or stability in cis-
identifying a previously unknown positive-feedback loop or trans-manner41,42. Unlike the classical theory, the
in the STAT3–TINCR–EGFR signaling pathway; this identification of TINCR ceRNA and RNA–protein-
suggests that the feedback loop could represent a new mediated dual regulation in this study reflects the
therapeutic target in pharmacological strategies developed lncRNA-mediated complex regulation of its target genes.
for human cancers. In Fig. 7f, we present a schematic In terms of their regulatory mechanism, lncRNAs
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 11 of 16
A C
Relative STAT3 expression
10
R=0.1067
P=0.0006
5
N=1019
0
Matrix ID Name Score Relative score Start End Predicted sequence -5
MA0144.2 STAT3 7.56 0.89 297 307 CCTCTGGGAAA
-10
MA0144.2 STAT3 5.28 0.86 52 62 ATGCCTGTAAT
-15
MA0144.2 STAT3 4.33 0.85 2259 2269 GCTCCTGGAAG -5 0 5 10
MA0144.2 STAT3 4.12 0.85 404 414 GTACCTAGAAG Relative TINCR expression
MA0144.2 STAT3 4.01 0.85 1431 1441 GTGCTGGGATT D
****
Relative TINCR expression
MA0144.2 STAT3 2.91 0.83 1749 1759 GTCCTTAAAAA
1.4
MA0144.2 STAT3 2.36 0.83 363 373 GTCATGAGAAA 1.2 ****
MA0144.2 STAT3 2.06 0.82 1439 1449 ATTCCAGGCAT 1.0
0.8
MA0144.2 STAT3 1.89 0.82 13 23 CTACTAAAAAT
0.05
MA0144.2 STAT3 0.92 0.81 69 79 TACTCGGGAAG 0.04
0.03
0.02
0.01
0.00
B refGene TINCR NC Si STAT3 #1 Si STAT3 #2
Ruler p13.3
chr19 5570K 5580K E 8
*
0.7
chr19:5572112-5573399
** Primer #1
Primer #2
6
*
** * **
Primer #3
84620_HCC70_STAT3
% Input
Primer #4
4
0.0
0.27 2
NS
NS NS
84621_HCC70_STAT3 NS
0
STAT3
STAT3
STAT3
STAT3
STAT3
STAT3
STAT3
STAT3
Mock(IgG)
Mock(IgG)
0.00
NC Si STAT3 #1
F
P P Cytoplasm
P
P
JAK2 EGFR mRNA
P
miR-503-5p
STAT3 TINCR
P
1 Activation
Inhibition
P Phosphorylation
STAT3 TINCR DNMT1
P
2 TINCR miR-503 EGFR
TINCR
Pri-miRNA
Pre-miRNA
Nucleus
Fig. 7 (See legend on next page.)
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 12 of 16
(see figure on previous page)
Fig. 7 Identification of the positive-feedback loop in STAT3–TINCR–EGFR signaling axis. a Upper corner of picture: STAT3-binding motif; lower
table: prediction of STAT3-binding sites within TINCR promoter region, from JASPAR database. b Enrichment peak of STAT3 in the promoter of TINCR
in ChIP-seq of HCC70 cells in ENCODE database. c TINCR expression is positively correlated with STAT3 mRNA expression in 1019 cancer cell lines in
CCLE cohort. d STAT3 knockdown decreases TINCR expression in MBA-MB-231 cells. e ChIP assay results indicating STAT3 enrichment at TINCR
promoter and diminished STAT3 enrichment at the promoter after STAT3 knockdown. f Molecular mechanism of dual regulation by TINCR of EGFR
and its downstream genes. Data are presented as means from three independent experiments ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
localized in the cytoplasm have been widely reported to broadinstitute.org/ccle/about), and Gene Expression
potentially compete for miRNA-response elements Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). All
(MREs) with target genes closely related to cancer transcripts were normalized by the log2 method. Wil-
occurrence and development by acting as ceRNAs; this coxon rank-sum statistical analysis was used to detect
would weaken the inhibitory effect of miRNAs on target significant differences among tumors or between tumor
genes and indirectly regulate the expression levels of the and normal samples. The correlations between genes were
target genes43–46. Our study suggests that TINCR func- assessed by Spearman’s correlation coefficients. To cal-
tions as a ceRNA to upregulate EGFR expression by acting culate Spearman’s correlation coefficient, we rank and
as a sponge for miR-503-5p. Conversely, lncRNAs loca- compare data sets to find Σd2, then plug the value into the
lized in the nucleus can regulate gene expression by acting standard version of Spearman’s rank- correlation coeffi-
as epigenetic modulators47,48. However, no previous study cient formula according to previous study49 Guilt-by-
has reported lncRNA regulation of miRNAs through the association analysis was performed to identify coding
modification of methylation. We have shown here for the genes that were positively or negatively correlated with
first time that TINCR can recruit DNMT1 to the miR- TINCR expression. JASPAR (http://jaspar.genereg.net/)
503-5p promoter to regulate miR-503-5p locus methyla- was used to examine the transcription factors that might
tion and thus transcription. Thus, this study fills in the potentially be enriched at the TINCR locus promoter.
theoretical gap in the lncRNA- regulation model and Correlations between TINCR and EGFR/JAK2 mRNA
provides a comprehensive understanding regarding the levels were assessed using Pearson correlation coeffi-
above-mentioned dual-regulatory network in human cients. Unpaired Student’s t tests were used to detect
breast cancer. significant differences among tumors or between tumor
In conclusion, this study has revealed that TINCR and normal samples. Survival was calculated by the
promotes tumorigenesis through a STAT3–TINCR– Kaplan–Meier method, with the log-rank test applied for
EGFR-feedback loop by recruiting DNMT1 and acting as comparison. Overall survival (OS) was calculated as the
a ceRNA in human breast cancer. We have demonstrated time from surgery until death; disease-free survival (DFS)
for the first time that TINCR upregulates EGFR expres- was measured as the time for which patients were disease-
sion by means of a dual molecular mechanism through free, and distant metastasis-free survival (DMFS) was
the aforementioned RNA–protein and RNA–RNA inter- calculated as the survival time specifically free of distant
actions to suppress miR-503-5p expression. These find- metastasis. The log-rank test was used to examine the
ings broaden the ceRNA landscape of TINCR and survival difference between distinct patient groups. All
enhance our understanding of the complex regulatory statistical tests were two-sided, and P < 0.05 was con-
network through which lncRNAs influence their target sidered statistically significant. Gene ontology (GO)-term
genes. Moreover, the study has uncovered a previously enrichment and Kyoto Encyclopedia of Genes and Gen-
unrecognized positive-feedback loop in the STAT3– omes (KEGG) pathway analyses of these genes were
TINCR–EGFR signaling axis in tumorigenesis, and this performed using DAVID as previously described50,51
feedback loop could thus represent a new target in
pharmacological strategies for treating human breast Breast-tissue specimens and clinical assessments
cancer. Eligible patients with a histological diagnosis of breast
cancer who had received neither chemotherapy nor
Materials and methods radiotherapy before surgical resection were recruited to
Public data access and analysis this study. Informed consent was obtained from all
Genome-wide expression profiles of TINCR and clinical patients. In total, 125 breast-cancer tissues and 125 nor-
pathology information were downloaded from the data- mal tissues were obtained from Harbin Medical Uni-
bases of The Cancer Genome Atlas (TCGA) (https://tcga- versity Cancer Center (HMUCC). For RNA extraction,
data.nci.nih.gov/), Gene Expression Profiling Interactive fresh tissue was collected from patients with breast cancer
Analysis (GEPIA) (http://gepia.cancer-pku.cn/), Cancer and normal controls and stored at −80 °C immediately
Cell Line Encyclopedia (CCLE) (https://portals. after resection. Two independent senior pathologists
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 13 of 16
confirmed the pathological diagnosis and molecular sub- FBS for 14 days. Colonies were fixed with 4% methanol for
type of each cancer tissue. This study conformed to the 30 min, and 500 μL of 0.5% crystal violet (Catalog #: 332488,
clinical research guidelines and was approved by the Sigma-Aldrich, St. Louis, MO, USA) was added to each well
research ethics committee of Harbin Medical University for 30 min to visualize the colonies for counting.
Cancer Hospital. We obtained written informed consent
from all patients. Invasion assays
Cells in serum-free RPMI-1640 or DMEM medium were
Cell culture, plasmid construction, and transfection placed in the BRAND® Insert with Matrigel (Catalog #:
Breast-cancer cell lines (UACC-812, MDA-MB-231, and BR782806, Sigma-Aldrich, USA). RPMI-1640 or DMEM
4T1) were obtained from the Chinese Academy of Sciences medium containing 10% FBS was added to the lower
Cell Bank and Cellbio (China) and cultured and stored chamber. After incubating at 37 °C for 24 h, the noninvad-
according to their instructions. Mycoplasma testing was ing cells that remained in the top chamber were removed
performed before the experiments. For transfection, cells with a cotton swab, and the cells that had migrated to the
were seeded in six-well dishes the night before to obtain underside of the membrane were fixed with 100% methanol
60–70% confluence for plasmid transfection and 80% con- for 30 min, air-dried, stained with 0.5% crystal violet,
fluence for microRNA (miRNA) or siRNA transfection; on imaged, and counted under a light microscope.
the following day, cells were transfected using Lipofecta-
mine 2000 (Invitrogen), according to the manufacturer’s Wound-healing assay
instructions. At 48 h post transfection, cells were harvested Cells were plated in six-well culture plates and culti-
for quantitative reverse-transcription-PCR (qRT-PCR) vated to achieve over 90% confluence in RPMI-1640 or
analysis. For lentiviral transduction, lentiviruses were used DMEM medium containing 5% FBS. A vertical wound per
to infect 5 × 105 cells seeded in six-well plates by using well was created using a 10-µL pipette tip. After two
4–6 μg/mL polybrene (107689, Sigma-Aldrich), after which washes with PBS to eliminate cell debris, the cells were
the infected cells were selected using 1 μg/mL puromycin reincubated in RPMI-1640 or DMEM medium containing
(Catalog Number 540411, Calbiochem, USA). Stable 0.1% FBS. Images were captured at the indicated time to
knockdown cell lines were identified using qRT-PCR. measure the size of the remaining wound.
qRT-PCR Western blotting
The total RNA was isolated from cells and tissue sam- Cells were lysed in lysis buffer containing 150 mmol/L
ples by using Trizol reagent (Invitrogen) according to the NaCl, 1% Triton X-100, 5 mmol/L EDTA, 5000 U/mL
manufacturer’s protocols, and 0.5 μg of the RNA was aprotinin, 20 mg/mL leupeptin, 1 mmol/L phe-
reverse-transcribed into cDNA by using a High-Capacity nylmethylsulfonylfluoride, 2 mmol/L sodium orthovana-
cDNA Reverse Transcription Kit (Applied Biosystems, date, 50 mmol/L NaF, 5% glycerol, 10 mmol/L Tris-HCl
USA). The SYBR Green PCR Master Mix Kit (Applied (pH 7.4), and 2% SDS. Next, protein concentrations were
Biosystems) was used to quantify RNA levels, with measured using a protein assay kit (Catalog #: 5000001,
GAPDH or U6 serving as an internal control. qRT-PCR Bio-Rad, Richmond, CA), and then equal amounts of
was performed on a 7500 FAST Real-Time PCR System protein were separated using SDS-PAGE and electro-
(Applied Biosystems). blotted onto nitrocellulose membranes, which were
blocked (overnight at 4 °C) with 5% nonfat milk in 0.1%
Cell-viability assays Tween-20/TBST. The membranes were immunoblotted
The viability of treated cells was quantified using the with primary antibodies from Cell Signaling Technology
Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, (CST) against EGFR (#2085), p-EGFR (#11862), JAK2
Kumamoto, Japan) assay, according to the manufacturer’s (Catalog #: 3230), p-JAK2 (Catalog #: 3771), STAT3
instructions. Briefly, cells were seeded in 96-well micro- (Catalog #: 9139), and p-STAT3 (Catalog #: 9145), washed
titer plates at a density of 3–5 × 103 cells/well with 100 μL with Tween-20/PBS, and incubated with horseradish
of the medium, CCK-8 solution was added to each well, peroxidase-conjugated secondary antibodies for 1 h. After
and the plates were incubated at 37 °C for 60 min. Next, washing with Tween-20/PBS, protein bands on the
the 450-nm absorbance of each cell suspension was membranes were visualized using an enhanced chemilu-
measured using a microplate reader, with a medium minescence detection system (Western Lightning, Perkin-
containing 10% CCK-8 serving as a control. Elmer, Norwalk, CT, USA).
Colony-formation assays Animal experiments
In total, 1000× cells were plated in a six-well plate and Animal experiments were approved by the Medical
cultured in RPMI-1640 or DMEM medium containing 10% Experimental Animal Care Commission of Harbin
Official journal of the Cell Death Differentiation AssociationWang et al. Cell Death and Disease (2021)12:83 Page 14 of 16
Medical University. Six-to-eight-week female Balb/c mice Bisulfite-sequencing PCR (BSP)
were obtained from Beijing Vital River Laboratory Animal To measure the methylation levels on the miR-503
Technology Company. Approximately 5 × 104 cells locus promoter, BSP was conducted as described in our
transfected with 4T1-Scramble or 4T1-Sh Tincr shRNA previous study53. Briefly, online tools were used to analyze
were suspended in 200 μL of serum-free medium and the CpG islands in the miR-503 locus promoter, and CpG
injected directly into the right mammary fat pad. Gefitinib sites at the 5ʹ end of the promoter, within the sequence
(Catalog #: S1025, Selleck) was administered orally for ranging from nucleotide −310 to 92, were selected for
7 days at a dose of 50 mg/kg52. Tumor growth was mea- BSP analysis. Genomic DNA was isolated and modified
sured using calipers once every 3 days, and tumor volume with bisulfite, and the bisulfite-treated DNA was PCR-
was calculated as tumor volume = 1/2 (length × width2). amplified. Subsequently, the PCR products were separated
After the mice were euthanized at the endpoint, the on gels, and the bands of the correct size were excised,
tumors induced by the injections were dissected out and and the PCR products were purified and subcloned into
weighed. pTG19-T vector; positive clones were obtained through
ampicillin antibiotic selection, and ten positive clones
Immunohistochemistry were subject to DNA sequencing at Generay Biotech Co.,
Paraffin-embedded tissue sections of 100 breast-cancer Ltd. (Shanghai, China).
tissues were deparaffinized in xylene, rehydrated in a
graded series of ethanol solutions, and incubated for Chromatin-immunoprecipitation (ChIP) assay
20 min in 3% H2O2 to block endogenous peroxidase ChIP assays were performed using a ChIP Assay Kit
activity. Next, the sections were heated in target-retrieval (Catalog #: P2078, Beyotime, Shanghai, China), according
solution (Dako) for 15 min in a microwave oven (Oriental to the manufacturer’s protocol with slight modifications.
Rotor) for antigen retrieval. After blocking nonspecific Cells were cross-linked for 10 min with 1% formaldehyde,
binding by incubating sections with 10% goat serum for and after terminating the reaction by adding 0.125 mol/L
2 h at room temperature, the sections were incubated glycine (final concentration), sonicated cell lysates were
overnight at 4 °C with anti-EGFR, anti-JAK2, or anti- prepared, and DNA was immunoprecipitated using an
STAT3 primary antibody. Subsequently, sections were anti-STAT3 antibody (Catalog #: 9139, CST) or anti-
incubated with an appropriate secondary antibody for DNMT1 antibody (Catalog #: GTX116011, GeneTex);
20 min at 37 °C, and binding was visualized using 3,3’- IgG (BD Biosciences, San Diego, CA, USA) served as the
diaminobenzidine tetrahydrochloride. After each treat- negative control. PCR was performed on the immuno-
ment, sections were washed thrice with TBST for 5 min. precipitated DNA to amplify the binding sites, and the
amplified fragments were analyzed on agarose gels.
RNA-FISH assay Chromatin (10%) from before the immunoprecipitation
ViewRNA®Probe (Catalogue Number VA1-3016120, was used as the input control.
Santa Clara) was purchased to perform FISH assay
according to the manufacturer’s protocol. TINCR hybri- Dual-luciferase-reporter assay
dization was carried out in a moist chamber. After The full-length 3ʹ-untranslated regions (UTRs) of
digestion with a working protease solution, slides were human TINCR and EGFR were PCR-amplified and the
incubated with RNase III (AM2290, Life Technologies, fragments were cloned separately into the multiple-
USA) or RNase A (AM2272, Life Technologies) for 2 h. cloning sites in psi-CHECK-2 luciferase miRNA-
Standard immunofluorescence and imaging was per- expression reporter vector. HEK293T cells (plated at
formed by confocal microscopy. ~40–50% confluence) were used for the dual-luciferase-
reporter assays, and Lipofectamine 2000 (Catalog #:
RNA-immunoprecipitation (RIP) assay 11668500, Invitrogen) was used to transfect the cells with
RIP was performed using a Magna RNA-binding pro- 20 μmol/L hsa-miR-503-5p mimic or negative-control
tein immunoprecipitation kit (Catalog #: 17-700 Milli- (NC) mimic and 0.5 mg of the plasmid. Luciferase activ-
pore, Bedford, MA, USA), according to the ities were measured at 48 h post transfection by using a
manufacturer’s instructions. Briefly, cell lysates were dual-luciferase-reporter assay kit (Catalog #: E1910, Pro-
incubated with RIP buffer containing magnetic beads mega, USA) and a luminometer (GloMaxTM 20/20,
conjugated with negative-control normal mouse IgG or Promega, USA).
human anti-DNMT1 antibody. The samples were then
incubated with Proteinase K to isolate the immunopre- Statistical analyses
cipitated RNA. Last, the purified RNAs were extracted The expression of each lncRNA was dichotomized by
and analyzed using real-time PCR to confirm the presence using the median expression as the cutoff to define high
of the binding targets. values (at or above the median) versus low values (below
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