Hu et al. BMC Medical Genetics(2020) 21:2
https://doi.org/10.1186/s12881-019-0940-6

RESEARCH ARTICLE

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lncRNA TINCR sponges miR-214-5p to
upregulate ROCK1 in hepatocellular
carcinoma
Min Hu1* , Yaowu Han1, Ying Zhang2, Yuanfeng Zhou2 and Lin Ye3

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Abstract

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Background: Our preliminary bioinformatics analysis showed that lncRNA TINCR may absorb miR-214-5p by serving
is sponge, while miR-214-5p targets ROCK1. This study aimed to investigate the interactions among these 3 factors
in hepatocellular carcinoma (HCC).
Methods: Expression of TINCR, ROCK1 and miR-214-5p in HCC and non-tumor tissues was detected by performing
qPCR. The correlations among TINCR, ROCK1 and miR-214-5p in HCC tissues were analyzed by performing linear
regression. Overexpression experiments were performed to analyze gene interactions. Cell proliferation was
analyzed by CCK-8 assay.

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Results: We found that TINCR and ROCK1 were upregulated, while miR-214-5p was downregulated in HCC. TINCR
and ROCK1 were positively correlated, while TINCR and miR-214-5p were not significantly correlated. In HCC cells,
TINCR overexpression is followed by ROCK1 overexpression, while miR-214-5p overexpression induced thedownregulation of ROCK1. In addition, TINCR and miR-214-5p did not affect the expression of each other. TINCR
and ROCK1 overexpression led to increased rate of cancer cell proliferation, while miR-214-5p played an opposite
role and reduced the effects of TINCR overexpression. Therefore, TINCR sponges miR-214-5p to upregulate ROCK1
in HCC, thereby promoting cancer cell invasion and migration.

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Keywords: Hepatocellular carcinoma, lncRNA TINCR, miR-214-5p, ROCK1

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Background
Incidence of hepatocellular carcinoma (HCC) ranks top
places all over the world [1]. With the popularization ofrisk factor screening program and the application of prevention regimens, incidence of HCC has dropped significantly in many regions, such as parts of China and Japan[2]. However, an increasing trend in both incidence andmortality rates of HCC has been observed in many European countries and North America owing to the highprevalence of hepatitis B virus (HBV) and hepatitis C virus(HCV) infections [3]. In spite of the considerable numberof risk factors identified for HCC, pathogenesis of this disease remains poorly understood [4, 5], which is a big challenge in the development of novel therapeutic targets.

* Correspondence: pdmnzwhj4623501@126.com
1
Department of Pathology, Anhui University of Chinese Medicine, Hefei, 1
Qianjiang Road, Hefei 230012, Anhui province, China
Full list of author information is available at the end of the article

ROCK1, or Rho-associated protein kinase 1, acts at thedownstream of small GTPase RhoA to mediate the formation of contractile force [6]. Oncology studies have shownthat ROCK1 is usually overexpressed during cancer development and ROCK1 overexpression promotes cancer development by regulating cancer cell behaviors, such as
proliferation, invasion and migration [7, 8]. In effect, inhibition of ROCK1 is novel therapeutic choice for cancer [7,8]. It is known that, the expression of ROCK1 can be regulated by certain miRNAs [9, 10], and the function of miRNAs can be attenuated by lncRNA sponges [11]. In a
recent study, Zhang et al. reported that miR-214-5p inhibits
osteosarcoma cell proliferation by directly targeting ROCK1[12]. TINCR has been reported to be an oncogenic lncRNA
in many types of cancers including HCC [13]. Our bioinformatics analysis showed that TINCR may sponge miR-2145p. This study was performed to investigate the interactions
among TINCR, miR-214-5p and ROCK1 in HCC.

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Hu et al. BMC Medical Genetics(2020) 21:2

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Methods

detect the expression of miR-214-5p with U6 as endogenous control. All experiments were performed intriplicate manner and expression levels were normalizedusing 2-ΔΔCT method.

Patients and specimens

Form December 2016 to December 2018 a total of 137
HCC patients were admitted by Anhui University of Chinese Medicine. From those patients our study selected 60
cases (gender: 37 males and 23 females; age: 33 to 67
years; mean: 49.9 ± 6.3 years) to serve as research subjects.
Inclusion criteria: 1) No therapies for any clinical disorders were performed within 100 days; 2) new HCC cases.
Exclusion criteria: 1) patients with other clinical disorders;2) patients with recurrent HCC; 3) patients with previous
history of malignancies. The HCC patients were educatedwith the experimental principle and all of them involvedin this study provided signed informed consent. All patients were diagnosed by histopathological biopsy. During
the operation of biopsy, non-tumor and HCC tissues werecollected from each patient. All tissue specimens wereconfirmed by at least 3 experienced pathologists. This
study has been approved by Ethics Committee of Anhui
University of Chinese Medicine.

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RNA-RNA interaction prediction

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In vitro experiments in this study were performed using
HCC cell lines H1581 (Cat# ATCC® CRL-5878™) andSNU-475 (ATCC® CRL-2236™) from ATCC (USA). Cells
were cultivated at 37 °C with 5% CO2 in RPMI-1640
medium (10% FBS) before use. PcDNA3.1 vectors expressing TINCR and ROCK1 were constructed by RIBOBIO (Guangzhou, China). MiR-214-5p mimic andnegative control miRNA were also from RIBOBIO.
H1581 cells were harvested and were counted, followed
by transfection of 10 nM pcDNA3.1 vector expressing
TINCR or ROCK2, or 10 nM empty pcDNA3.1 vector(negative control, NC), or 30 nM miR-214-5p mimic, or
30 nM negative control miRNA (NC) into 106 cells
through lipofectamine 2000 (Invitrogen, USA)-mediatedmethod. Cells with no transfections were also included
to serve was control (C) group. The interval between
subsequent experiments and transfection was 24 h.

H1581 cells were collected and mixed with RIPA solution (RIBOBIO) with a ratio of 105 cells per 1 ml solution. Total proteins were extracted following theprotocol provided by RIBOBIO. Protein samples were
quantified using BCA kit (RIBOBIO), followed by denaturing in boiled water for 5 min. Following 12% SDSPAGE gel electrophoresis, gel transfer (PVDF membrane) and blocking (2 h in 5% non-fat milk at room
temperature) were performed. Following that, primary
antibodies of rabbit GAPDH (1: 1500, ab37168, Abcam)
and RCOK1 (1: 1200, ab66320, Abcam) were used toblot the membranes at 4 °C for 15 h. After that, HRP
goat anti-rabbit (IgG) secondary antibody (1:1400;
ab6721; Abcam) was used to further blot the membranes
for 2 h at 24 °C. All signal developments were performed
using ECL solution (Sangon, Shanghai, China). Image J
v1.46 software was used to normalize all gray values.

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HCC cells and transient transfections

Western blot analysis

RNA extractions and qPCR

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Non-tumor and HCC tissues were ground. H1581 cells
were harvested and counted. To extract total RNAs,
0.015 g tissue or 106 cells were mixed with 1 ml Trizol
reagent (Invitrogen, USA). To retain miRNA, 80% ethanol was used to participate and wash RNAs. MMLV Reverse Transcriptase (Lucigen, USA) and QuantiTectSYBR Green PCR Kit (Qiagen, Shanghai, China) wereused to perform total RNA reverse transfections andprepare qPCR mixtures to detect the expression of
TINCR and ROCK2 with GAPDH as endogenous control. miScript II RT Kit (QIAGEN) and miScript SYBRGreen PCR Kit (QIAGEN) were used to perform miRNA
reverse transcriptions and prepare qPCR mixtures to

The interaction between TINCR and miR-214-5p was performed using online program IntaRNA (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp). Sequence of TINCR
was used as the long sequence and sequence of miR-2145p was used as the short sequence. All other parameters
were default.
Cell proliferation analysis

H1581 and SNU-475 cells were harvested and counted. 1
ml RPMI-1640 medium (10% FBS) was mixed with 5 ×
104 cells to prepare single cell suspensions. The cells were
cultivated in a 96-well plate (0.1 ml per well) at 37 °C in a
5% CO2 incubator. Three replicate wells were set for eachexperiment. To monitor cell proliferation, 10ul CCK-8 solution (Sigma-Aldrich) was added at 2 h before the end ofcell culture. Cell collection was performed every 24 h for a
total of 4 days. After the termination of cell culture OD
values at 450 nm were measured. OD value of Control
group was set to “100”, all other time pointes and othergroups were normalized to this value.
Statistical analysis

Data from 3 biological replicates were used to calculatemean values, which were used in all data analyses. Correlations were analyzed by linear regression. Differencesin gene expression levels and cell proliferation rates
among aforementioned different cell transfection groups
were analyzed by ANOVA (one-way) and post hoc
Tukey test. Differences in levels of gene expression(2020) 21:2

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Hu et al. BMC Medical Genetics

hybridization energy is − 22.7 kcal/mol.
TINCR can be a sponge of miR-214-5p.

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The interactions among TINCR, ROCK1 and miR-214-5p in
H1581 cells

TINCR and ROCK1 expression vectors as well as miR214-5p mimic were transfected into H1581 cells. At 24 hpost-transfection, expression levels of TINCR, ROCK1 andmiR-214-5p were significantly upregulated Compared to
NC and C groups (Fig. 4a, p < 0.05). In addition, TINCR

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between non-tumor and HCC tissues were exploredusing paired t test. With the median expression level of
TINCR in HCC as cutoff value, the 60 patients were divided into high and low TINCR level group (n = 30).
Chi-squared test was performed to analyze the relationship between patients’ clinical data and levels of TINCR
expression in HCC. p < 0.05 was statistically significant.

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Fig. 1 Expression of TINCR, ROCK1 and miR-214-5p was altered in HCC. Expression of TINCR (a), ROCK1 (b) and miR-214-5p (c) in HCC and nontumor tissues was detected by performing qPCR. All qPCR reactions were performed 3 times. Expression levels were compared between nontumor and HC tissues by paired t test (*, p < 0.05)

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Results
Expression of TINCR, ROCK1 and miR-214-5p was alteredin HCC

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Expression of TINCR, ROCK1 and miR-214-5p in HCC
and non-tumor tissues was detected by performing qPCR.
Expression levels were compared between non-tumor andHC tissues by paired t test. Compared to non-tumor tissues,
expression levels of TINCR (Fig. 1a) and ROCK1 (Fig. 1b)
were significantly higher, whereas expression level of miR214-5p was significantly lower (Fig. 1c) in HCC (p < 0.05).
Chi-squared test showed that levels of TINCR in HCC tissues were significantly correlated with tumor size and TNM
stage (p < 0.05), but not age, gender, histological differentiation, HBV or HCV infections and liver cirrhosis (p < 0.05,
Table 1).
The correlations among TINCR, ROCK1 and miR-214-5p in
HCC

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The correlations among TINCR, ROCK1 and miR-214-5p
in HCC tissues were analyzed by performing linear regression. In HCC tissues, TINCR and ROCK1 were positively
correlated (Fig. 2a), while TINCR and miR-214-5p were notsignificantly correlated (Fig. 2b). In addition, miR-214-5p
and ROCK1 were also not significantly correlated (Fig. 2c).
TINCR may sponge miR-214-5p

The interaction between TINCR and miR-214-5p werepredicted using the aforementioned methods. It can beobserved that TINCR may form base pairing with miR214-5p from position 258 to position 276 (Fig. 3). The

Table 1 Correlation between levels of TINCR expression levels
in HCC and patients’ clinical data
VariablesnHigh (n =30)

Low (n =30)

Chisquarep

Gender
Male

37

17

20

Female

23

13

10

0.63

0.43

0.27

0.60

1.55

0.46

1.67

0.20

0.11

0.74

11.38

0.00075.41

0.02

Age (years)
> 50

28

13

15

< =50

32

17

15

Histological differentiation
Well

14

5

9

Moderate

21

11

10

Poor

25

14

11

HBV or HCV infections
Yes

48

22

26

No

12

8

4

Absence115

6Presence

49

25

24

> 5 cm33

10

23< = 5 cm

27

20

7

Liver cirrhosis

Tumor size

TNM stage
I + II31

11

20

III + IV

29

19

10

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Fig. 2 The correlations among TINCR, ROCK1 and miR-214-5p in HCC. The correlations between TINCR/ROCK1 (a), TINCR/miR-214-5p (b) and miR214-5p/ROCK1 (c) were analyzed by linear regression

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Cell proliferation data were analyzed. Compared to C
and NC groups, TINCR and ROCK1 overexpression led
to increased rate of cancer cell proliferation, while miR214-5p played an opposite role and reduced the effects
of TINCR overexpression (Fig. 5, p < 0.05).

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TINCR promoted H1581 and SNU-475 cell proliferation
through ROCK1 and miR-214-5p

and migration [14]. In a recent study, miR-214-5p was reported to directly target ROCK1 to inhibit cancer cell behaviors [12]. In the present study we also observed the reduced
expression levels of ROCK1 in HCC cells after miR-214-5p
expression. Therefore, miR-214-5p may also target ROCK1
to regulate its expression in HCC. Those data also suggestthat ROCK1 can be targeted by multiple miRNAs.
In this study we observed the upregulated ROCK1and downregulated miR-214-5p in HCC tissues.
Interestingly, we did not observe a significant correlation between miR-214-5p targets ROCK1 in HCC
tissues. This observation suggests the existence of asponge of miR-214-5p. MiRNA sponges only absorb
miRNAs to attenuate their functions but may notaffect their expression levels [15]. Therefore, if asponge exists, the downregulation of miR-214-5p expression may not be significantly correlated with theupregulated ROCK1. The sponge may serve buffer tofinely regulate the levels of functional miRNAs.
TINCR is a well-characterized oncogenic lncRNA
in many types of cancer [13, 16, 17]. In some cases,
TINCR may sponge miRNAs to promote cancerRoverexpression is followed by ROCK1 overexpression, while
miR-214-5p overexpression induced the downregulation ofROCK1 and attenuated the effects of ROCK1 overexpression (Fig. 4b, p < 0.05). In addition, TINCR and miR-214-5p
did not affect the expression of each other (Fig. 4c).

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Discussion
This study mainly investigated the interactions among
TINCR, ROCK1 and miR-214-5p in HCC. We found that
TINCR can upregulate ROCK1 possibly by sponging miR214-5p, which can directly target ROCK1.
Previous studies have identified numerous miRNAs thatcan directly target ROCK1 [9, 10, 12]. In HCC, miR-148b
targets ROCK1 to inhibit cancer cell proliferation, invasion

Fig. 3 TINCR may sponge miR-214-5p. The interaction between TINCR and miR-214-5p was performed using online program IntaRNA. Sequenceof TINCR was used as the long sequence and sequence of miR-214-5p was used as the short sequence. All other parameters were default

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Fig. 4 The interactions among TINCR, ROCK1 and miR-214-5p in HCC cells. TINCR and ROCK1 expression vectors as well as miR-214-5p mimic
were transfected into H1581 cells. At 24 h post-transfection, overexpression of TINCR, ROCK1 and miR-214-5p was confirmed by qPCR (a). The
effects of TINCR and miR-214-5p on ROCK1 overexpression were analyzed by qPCR and western blot (b). The interaction between TINCR andmiR-214-5p was analyzed by qPCR (c). All experiments were repeated 3 times and mean values were presents. NC1, empty vector transfection;
NC2, negative control miRNA transfection (*, p < 0.05)

Fig. 5 TINCR promoted HCC cell proliferation through ROCK1 and miR-214-5p. The effects of TINCR, ROCK1 and miR-214-5p on HCC cellproliferation were analyzed by cell proliferation assay. All experiments were repeated 3 times and mean values were presents. NC1, empty vectortransfection; NC2, negative control miRNA transfection (*, p < 0.05)

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progression. For instance, TINCR sponges miR-375
to regulate PDK1, thereby promoting gastric cancer[16]. In colorectal cancer, TINCR sponges miR-7-5p
to aggregate disease conditions [17]. In this study weshowed that TINCR may sponge miR-214-5p to upregulated ROCK1 in HCC, and the upregulation ofROCK1 by TINCR is involved in the regulation of
HCC cell proliferation. However, other lncRNAs may
also sponge miR-214-5p to participate in thisprocess. Our future studies will explore thispossibility.

Received: 14 October 2019 Accepted: 23 December 2019

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Conclusions
In conclusion, TINCR played an oncogenic role in HCC
by sponging miR-214-5p to upregulate ROCK1.

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Abbreviations
FBS: Fetal bovine serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C
virus; ROCK1: Rho Associated Coiled-Coil Containing Protein Kinase 1;
TINCR: TINCR ubiquitin domain containing
Acknowledgements
Not applicable.

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Authors’ contributions
MH supervised the whole study, designed the concept, analyzed the data,and edited the final manuscript. YWH, YZ, YFZ and LY collected andanalyzed the data, prepared the manuscript. All authors read and approved
the final manuscript.

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Funding
We thank for the financial support from the Anhui Province Returned
Overseas Students Innovation Project; China Postdoctoral Science Fund
Project(2016 M592037) and 2016 Anhui Postdoctoral Fund Project (2016B096)
for the funding to support our purchase of experimental equipment andmaterials.
The funding bodies played no role in the design of the study and collection,
analysis, and interpretation of data and in writing of the manuscript.

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Availability of data and materials
The analyzed data sets generated during the study are available from thecorresponding author on reasonable request.

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Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the Maternity
and Child Care Center of Liuzhou. The research has been carried out inaccordance with the World Medical Association Declaration of Helsinki. All
patients and healthy volunteers provided written informed consent prior totheir inclusion within the study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Pathology, Anhui University of Chinese Medicine, Hefei, 1
Qianjiang Road, Hefei 230012, Anhui province, China. 2Graduate School,
Anhui University of Chinese Medicine, Hefei 230038, Anhui, China.
3
Department of Psychology, Anhui University of Chinese Medicine, Hefei
230012, Anhui, China.

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