MIR-147B REGULATED PROLIFERATION
AND APOPTOSIS OF GASTRIC CANCER CELLS
BY TARGETING CPEB2 VIA THE PTEN PATHWAY
Tao K.1,2, Dong J-H.2, Wang D.1, Li F.2†#, Zhang Z-T.#1*
*Corresponding Author: Zhong-Tao Zhang, MD, Email: sxzhangzhongtao@sina.com, ORCID ID: 0000-0002-1184-2591
#: Zhong-Tao Zhang and Feng Li contributed equally to the article
†: Co-corresponding author: Feng Li, Email: sxlifengwobuxin@sina.com, ORCID: 0000-0002-7322-422X
page: 10
|
|
RESULTS
miR-147b is up-regulated in GC cells and tissues
The differentially expressed miR-147b was screened
out in 3 pairs of samples by the miRNA chip (Fig. 1A), which
was found three times higher than that of adjacent tissues in
three random pairs of gastric cancer samples (Fig. 1B). QPCR
further detected the expression of miR-147b in 50 pairs
of gastric cancer and adjacent tissues, and miR-147bwasfound
to be highly expressed in the gastric cancer tissues
(0.53±0.02 vs 1.35±0.03, P<0.05) (Fig. 1C). The expression
of miR-147b in each GC cell line was recorded as Normal
cell0.99±0.01, SGC-7901: 1.55±0.03, BGC-8231.74±0.12,
AGS: 1.47±0.02, MGC-803, 1.8±0.04, MKN-45, 1.44±0.03,
P<0.05) (Fig. 1D, table 1). BGC-823 and MGC-803 cell
lines with increased expression level of miR-147b were
selected for further analysis. After transfection, the relative
gene expression of miR-147b nc in the control group and
miR-147b inhibitor group were found to be 1.00±0.09, and
0.23±0.07 respectively, with a significant difference (t =
3.07, P < 0.05), indicating successful transfection (Fig. 1D).
miR-147b promotes tumor growth
in human cell lines
Scratch analysis showed that the miR-147b inhibitor
group inhibited GC cell growth and reduced cell migration
compared with miR-147b NC (Fig. 1E). In addition, by
flow cytometry, we have also analyzed the apoptotic effect
of miR-147b. As compared with miR-147b NC, miR-147b
inhibitor increased the early apoptosis of MGC-803 cells
[(61.8±0.2)% vs (55.4±0.1)% ]. It increased the early apoptosis of BGC-823 cells [(35.5 ± 0.2)% vs. (23.1 ± 0.2)%] (Fig.
2A). On the other hand, clone formation experiments showed
that themiR-147b inhibitor drastically restricts the proliferation
of BGC-823 and MGC-803 cells (Fig. 2B, table 2).
CCK-8 proliferation experiment showed that themiR-147b
inhibitor significantly inhibited the growth of BGC-823 and
MGC-803 cells, and inhibition rate was more prominent as
the time increases (t = 2.78, P < 0.05) (Fig. 2C and D, table 3).
miR-147b affects the expression of CPEB2
in GC cells
We searched the target gene CPEB2 that can bind to
miR-147b through the target scan database, and we mapped
the base pairs that miR-147b crosses with the 3 ‘untranslated
region of the predicted target gene CPEB2 (Fig. 3A).
We selected MGC-803 with relatively high endogenous
CPEB2 content to detect whether the miR-147b inhibitor
of miR-147b NC affects CPEB2 expression. The double
luciferase reporter vectors of CPEB2 wild-type and mutant
were further constructed (Fig.3A). The above vectors were
co-transfected with NC and miR-147b inhibitor into MGC-
803 cells, respectively. It was found that CPEB2 was the
target gene directly affected by miR-147b. In the presence
of CPEB23 ‘- UTR, this activity produced about 20% (Fig.
3B), indicating that the expression of miR-147b inhibitor
accelerated the activity of CPEB23’-UTR. Western blot
showed that the expression of CPEB2 in MGC-803 cells
in miR-147b inhibitor group and miR-147b NC+CPEB2
group showed higher concentration as compared to negative
control group (Fig. 3C). These results suggest that CPEB2
is the target of miR-147b and is negatively regulated by it.
B2 was highly expressed in GC cells
and correlates with prognosis
Quantitative PCR (qPCR) analysis of 50 pairs of
clinical GC tissues and adjoining normal tissue samples
showed that CPEB2 mRNA expression in tumor tissues
was down regulated in 40 patients (80%) (Fig. 4A). The
non-parametric test results of the relationship between
CPEB2 expression and clinicopathological factors showed
that the lower CPEB2 level was related to the GC pTNM
stage (P < 0.05, stages I-II and III-IV, FIG. 4B). In addition,
GC patients with lower CPEB2 expression showed
lower survivability and improved prognosis (Fig. 4C). Immunohistochemical
analysis showed that CPEB2 staining
increased significantly in adjacent normal tissues (Fig. 4D).
H & E staining showed more metastatic nodules in GC
tissues compared with adjacent normal tissues (Fig. 4D).
CPEB2 was found highly expressed in GC cells.
CPEB2 promoted the PTEN pathway
Western blot results showed that the expression
of PTEN protein decreased significantly after reducing
CPEB2 in 803 and 823 cell lines. At the same time, the
expression of tumor suppressor genes p27 and E-cadherin
was inhibited, while the expression of Ki67 and p21 reflecting
tumor activity index increased significantly (Fig. 4E).
|
|
|
|
 |
Number 25
VOL. 25(2), 2022 |
Number 25
VOL. 25 (1), 2022 |
Number 24
VOL. 24(2), 2021 |
Number 24
VOL. 24(1), 2021 |
Number 23
VOL. 23(2), 2020 |
Number 22
VOL. 22(2), 2019 |
Number 22
VOL. 22(1), 2019 |
Number 22
VOL. 22, 2019 Supplement |
Number 21
VOL. 21(2), 2018 |
Number 21
VOL. 21 (1), 2018 |
Number 21
VOL. 21, 2018 Supplement |
Number 20
VOL. 20 (2), 2017 |
Number 20
VOL. 20 (1), 2017 |
Number 19
VOL. 19 (2), 2016 |
Number 19
VOL. 19 (1), 2016 |
Number 18
VOL. 18 (2), 2015 |
Number 18
VOL. 18 (1), 2015 |
Number 17
VOL. 17 (2), 2014 |
Number 17
VOL. 17 (1), 2014 |
Number 16
VOL. 16 (2), 2013 |
Number 16
VOL. 16 (1), 2013 |
Number 15
VOL. 15 (2), 2012 |
Number 15
VOL. 15, 2012 Supplement |
Number 15
Vol. 15 (1), 2012 |
Number 14
14 - Vol. 14 (2), 2011 |
Number 14
The 9th Balkan Congress of Medical Genetics |
Number 14
14 - Vol. 14 (1), 2011 |
Number 13
Vol. 13 (2), 2010 |
Number 13
Vol.13 (1), 2010 |
Number 12
Vol.12 (2), 2009 |
Number 12
Vol.12 (1), 2009 |
Number 11
Vol.11 (2),2008 |
Number 11
Vol.11 (1),2008 |
Number 10
Vol.10 (2), 2007 |
Number 10
10 (1),2007 |
Number 9
1&2, 2006 |
Number 9
3&4, 2006 |
Number 8
1&2, 2005 |
Number 8
3&4, 2004 |
Number 7
1&2, 2004 |
Number 6
3&4, 2003 |
Number 6
1&2, 2003 |
Number 5
3&4, 2002 |
Number 5
1&2, 2002 |
Number 4
Vol.3 (4), 2000 |
Number 4
Vol.2 (4), 1999 |
Number 4
Vol.1 (4), 1998 |
Number 4
3&4, 2001 |
Number 4
1&2, 2001 |
Number 3
Vol.3 (3), 2000 |
Number 3
Vol.2 (3), 1999 |
Number 3
Vol.1 (3), 1998 |
Number 2
Vol.3(2), 2000 |
Number 2
Vol.1 (2), 1998 |
Number 2
Vol.2 (2), 1999 |
Number 1
Vol.3 (1), 2000 |
Number 1
Vol.2 (1), 1999 |
Number 1
Vol.1 (1), 1998 |
|
|
