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Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1  

 

CTMJ | traditionalmedicinejournals.com   

 

The role of mta2 expression in bladder cancer cells and its regulation 

mechanism 
Dr.K.Nagarsee 

1
, Dr.Nadeem 2, G.Rajani 3,Aszad Hussain Barbhuyan 

4,
 

Assistant professor 
1,2,3,4

, 
Department of Pharmacy, 

Samskruti College of Pharmacy, 
Kondapur (V), Ghatkesar (M) Medchal Dist, Telangana, India. 

 

 
Abstract 
Researching the role of the mta2 gene in bladder cancer and its possible therapeutic applications is the goal of this 

research.Methods: The T24 bladder cancer cells were transfected with a viral vector to induce overexpression of the 

metastasis-associated gene 2 (mta2), whereas the EJ bladder cancer cells were transfected with si-mta2. Under the 

two mta2 expression settings, cell lines were tested for their invasiveness and migratory capability in vitro using 

Matrigel and Transwell methods, respectively.End result: Data from transwell migration experiments showed that 

T24 bladder cancer cell lines were much more able to migrate when mta2 protein was overexpressed, but EJ cell 

lines were much less able to migrate when mta2 was knocked down (p < 0.01). In the matrigel invasion experiment, it 

was shown that the invasive capability of the T24 bladder cancer cell line was greatly increased by overexpressing 

the mta2 protein, whereas the invasive capacity of the EJ bladder cancer cell line was dramatically decreased (p < 

0.01) by knocking down mta2. In the T24 bladder cancer cell line, which overexpresses mta2, the levels of E-cadherin 

and N-cadherin were lower than in the cd511b-transfected and untransfected groups, respectively. In addition, 

compared to cells that were transfected with si-NC or not transfected, the E-cadherin protein expression in the EJ 

bladder cancer cell line with mta2 knockdown was noticeably greater (p < 0.01).In conclusion, mta2 knockdown 

blocks bladder cancer cell lines' ability to proliferate, migrate, and invade by preventing proteins involved in the 

epithelial-mesenchymal transition from doing their jobs.Reproductive process, migration, bladder cancer, mta2 

 

 

INTRODUCTION 

 
The most frequently diagnosed tumor of the urinary 

tract is bladder carcinoma which is usually located on 

the mucosal surface and ranks amongst the 10 most-

often diagnosed human neoplasms [1]. The age of 

onset of bladder cancer is relatively broad: it occurs 

almost at any given age, even in children. However, 

the occurrence of bladder cancer slowly rises as a 

function of age. There have been increases in 

incidence of bladder cancer in recent times, due to 

factors such as heightened use of sundry chemical 

products, tobacco use, and aging human populations 

[2]. At present, radical surgery and adjuvant treatment 

are used to mitigate the signs of bladder carcinoma, 

but studies have revealed that postoperative bladder 

cancer patients still experience high degrees of local 

recurrence, distant metastasis and poor prognosis [3]. 

The etiology of bladder cancer is complex. Two clear 

risk factors are exposure to aromatic amines and 

smoking, but little is known about some unpredictable 

biological behaviors within the cancer tissue or 

signaling routes associated with its progression [4]. 

Therefore, in order to efficiently carry out timely 

diagnosis and treatment of bladder tumor patients, 

there is need to identify new molecular markers for 

evaluation of its prognosis. 

Some investigations have revealed that mta2, one of 

the Metastasis-Associated Genes (MTAs), is up-

regulated in ovarian cancer, hepatocellular carcinoma, 

bladder cancer and other malignant tumors, and is 

intimately linked to cancer cell migratory and 

invasive potential [5]. However, the number of reports 

on relative expression of mta2 in bladder carcinoma, 

and its role, is limited. Thus, this research was aimed 

at determining mta2 expression in bladder carcinoma 

cells, as well as the influence of mta2 overexpression 

and mta2 silencing on their multiplication, 

invasiveness and migratory potential. This was to 

identify the relevance of mta2, and its possible 

regulatory mechanism in the progression of bladder 

carcinoma. 

 

EXPERIMENTAL 
 

Materials 

 

Chongqing Youbao Biotechnology Co. Ltd was the 

supplier of bladder cancer cell lines (24, EJ and J82), 

mta2 interference lentivirus and plasmid required. 

 

Handling of cells 

 

The bladder cancer cells were maintained in RPMI-



Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1  

 

CTMJ | traditionalmedicinejournals.com   

1640 medium containing 10 % FBS. The experiments 

were carried out using logarithmic growth phase cells. 

The T24 bladder cancer cells were infected with a 1:1 

(v:v) mixture of bovine serum-free medium and virus 

solution, in line with lentivirus transfection kit 

instructions. The plasmid DNA was incorporated into 

Escherichia coli, followed by 12-h culturing at 37 oC, 

after which it was subjected to extraction. Then, 

transfection of bladder cancer cells with the mta2 was 

done in line with the steps indicated in the transfection 

instructions. 

Western blot assay 

 

Total protein was extracted from bladder cancer cells 

by adding RIPA buffer solution and shaking on ice for 

lysis for 20 min. The lysed cell samples were 

centrifuged for 10 min, and the supernatant was 

collected. The protein contents of the supernatants 

were determined using the BCA method. Then, each 

protein sample was subjected to SDS-PAGE, followed 

by transfer to polyvinylidene difluoride membrane. 

The membrane was incubated fat-free milk solution 

to block non-specific binding of the blot. Then, it was 

incubated with mta2-specific primary antibodies 

overnight at 4 oC, followed by incubation with a 

secondary antibody at room temperature for 1 h. 

Luminescent substrates or staining agents were used 

for color development to observe the expression level 

of the target protein. 

 

MTS colorimetry 

 

Bladder cancer cells transfected with mta2 

overexpression and low expression were cultured to 

logarithmic growth stage. The cells were evenly 

inoculated in 96-well plates, with approximately 5000 

cells per well. After incubating the well plates in the 

incubator for 24 h, MTS reagent was added to each 

well, and the wells were incubated for 1-4 h. Then, the 

absorbance of each well was read in a multi- channel 

ELISA reader and recorded. Using the absorbance 

values, the proliferation capacity of cells in each 

group was calculated, and the differences between the 

mta2 overexpression and low-expression groups were 

determined. 

 

Matrix invasion and Transwell migration 

 

The upper chamber of Transwell was evenly coated 

with Matrigel, while the lower chamber contained 

equivalent amount of cell culture medium. The 

bladder cancer cells transfected with mta2 

overexpression and low expression were made into 

single-cell suspensions, and the cell suspension was 

added to the pores covered by Matrigel or Transwell. 

The well plates were incubated in the incubator for 24 

- 48 h. Then, the migrated cells on the upper 

surface were removed with washing solution or cotton 

swab. For the Matrigel invasion experiment, cell 

fixation was performed using repair solution, and then 

cells were stained with specific staining agents. For 

the Transwell migration experiment, direct staining 

was done, and the stained well plates were examined 

under a microscope. The number of invading or 

migrating cells was counted, and the differences 

between different MTA2 expression groups were 

obtained. 

 

Statistical analysis 

 

The SPSS 22.0 statistical software package was used 

to perform statistical analysis of the recorded data. 

The counting data are presented as (n (%)). Groups 

were compared using χ²-test. Quantitative data are 

presented as mean ± standard deviation (SD). Paired 

data were compared between groups using 

independent sample t-test, while non-normal 

distribution data were compared using rank sum tests. 

Statistical significance was considered at p < 0.05. 

 

RESULTS 

 
mta2 protein expression in bladder cancer cells 

There was significantly lower amount of mta2 protein 

expression in T24 than in EJ bladder cancer cell lines, 

and the mta2 expression level in EJ bladder cancer 

cell line was significantly higher than that in other 

bladder cancer cell lines (p < 0.05). These results are 

shown in Table 1. Therefore, T24 and EJ bladder 

cancer cells were chosen for use in follow-up studies. 

 
 

mta2 expressions in selected cell lines transfected 

with mta2 

 

In the present study, mta2 was successfully 

transferred into T24 bladder cancer cell line. The mta2 

protein expression level in T24 bladder cancer cells in 

mta2 transfection group was significantly higher than 

those in un-transfected group and cd511b transfection 

group. However, mta2 expression was unchanged, 

irrespective of cd511b transfection status. Moreover, 

mta2 of EJ bladder cancer was effectively silenced, 

resulting in significantly lower mta2 expression levels 

in EJ bladder cancer cells than in un- transfected cells. 

However, mta2 expression was comparable in si-NC 

transfected and un- transfected cells. These results are 

presented in Table 2 and Table 3. 

 

Multiplication of overexpression and low- 

expression mta2 cell groups 

 

The absorbance readings of T24 bladder cancer cells 

in mta2 transfection group were significantly higher 

than those in the transfected and cd511b transfection 

groups at different times (p < 0.01). In contrast, 

absorbance values were not changed by cd511b 



Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1  

 

CTMJ | traditionalmedicinejournals.com   

transfection status. However, the absorbance of EJ 

bladder cancer cell line was significantly reduced in 

si-mta2-transfected cells, when compared with the un-

transfected and si-mta2 transfection groups. The si-

mta2 transfection had no marked effect on 

absorbance. These results are presented in Table 4. 

 
 

 

 
 

Migratory potential of overexpression and under-

expression mta2 cell groups 

 

In the in vitro migration experiment, the population of 

T24 bladder cancer cells that crossed the basement in 

mta2 transfection group was significantly higher than 

the corresponding numbers in the un-transfected and 

cd511b transfection groups. However, cd511b 

transfection status did not affect mta2 expression. 

Significantly lower number of EJ bladder cancer cells 

crossed the basement membrane in si-mta2-

transfected cells than in un-transfected and si-NC 

transfection groups. 

 

The si-NC transfected and transfected cells did not 

differ in mta2 expression. These data are presented in 

Table 5 and Table 6. 

 

 

 
Invasiveness of overexpression and under- 

expression of mta2 cell groups 

 

In the invasion experiment, the population of T24 

bladder cancer cells that crossed the membrane in 

mta2 transfection group was significantly higher, 

relative to the un-transfected and cd511b transfected 

groups. However, cd511b transfection had no effect on 

mta2 expression. The population of EJ bladder cancer 

cells that crossed membrane was significantly reduced 

in si-mta2 transfection cells, relative to un- transfected 

and si-NC transfection cells (p < 0.01). The si-NC 

transfection had no effect on mta2 expression. These 

results are presented in Table 7 and Table 8. 

 

 
Levels of EMT-associated proteins in 

overexpression and under-expression mta2 cell 

groups 

 

E-Cadherin protein level in T24 bladder cancer cells 

was significantly down-regulated in mta2 transfection 

cells, relative to the un-transfected and cd511b 

transfection groups, but N-cadherin protein was 

significantly up-regulated in mta2 transfection cells, 

relative to un-transfected and cd511b transfection 

cells. The cd511b transfection had no marked effect 

on the expressions of the two proteins. These data are 

presented in Table 9. 

 

 



Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1  

 

CTMJ | traditionalmedicinejournals.com   

 
 

There was significantly higher E-cadherin protein 

expression level in si-mta2 transfection EJ bladder 

cancer cells than in un-transfected and si-NC 

transfection cells, but N-cadherin protein was 

expressed significantly lowly in si- mta2 transfection 

cells, relative to the corresponding levels in un-

transfected and si- NC transfection cells. However, si-

NC transfection did not affect the expression levels of 

the two proteins. These data are presented in Table 10. 

 

 
DISCUSSION 
 

Malignant tumor is one of the most common diseases 

threatening people's lives at present. The incidence 

and case fatality rate of malignant tumors are rising all 

over the world, and they have also become one of the 

main causes of death among the Chinese population 

[6]. The metastatic and invasive nature of malignant 

tumors is the main cause of poor prognosis in the vast 

majority of patients. Therefore, studying the 

mechanisms underlying the metastatic and invasive 

potential of cancer cells has become an urgent issue 

that needs to be addressed. Bladder carcinoma is one 

of the most frequently seen malignant diseases in 

urinary canal, with high metastasis and invasion, but 

its specific regulatory mechanism is still unclear 

[8].The mta2 protein is a 68-amino acid polypeptide 

with nucleosome remodeling activity, and it belongs 

to one of the mta family members associated with 

tumor metastasis. Studies have revealed that mta2 

protein is well-expressed in certain cancers such as 

cancers of the bladder, colon and esophagus, with 

close association with Tumor Node Metastasis stage, 

metastasis to lymph nodes, and differentiation [9]. 

Thus, mta2 gene is likely to be involved in the 

occurrence and progression of bladder carcinoma. In 

order to investigate the link between mta2 and the 

occurrence of bladder carcinoma, lentivirus-

overexpressing vectors were employed to establish 

mta2 overexpression plasmids so as to generate mta2-

overexpressing T24 cells. In addition, si-mta2 and 

knockdown mta2 were used to transfect EJ cancer 

cells. 

 

Through Matrigel, MTS and Transwell assays, it was 

found that T24 cancer cells overexpressing mta2 

protein had increased proliferation and invasion 

potential. The migratory potential was significantly 

raised in mta2-silenced T24 cells, relative to cd511b-

transfected and un- transfected cells, but the invasive, 

proliferative and migratory potential of mta2-

knockdown EJ cells were suppressed. Thus, mta2 may 

participate in the bladder carcinoma development via 

its effect on bladder cancerous cell invasiveness and 

proliferation. Therefore, it is important to unravel the 

regulatory process through which mta2 influences the 

above biological characteristics of bladder carcinoma 

cells. 

 

The reduction in adhesion between cells and epithelial-

mesenchymal transition (EMT) is an important reason 

for the metastatic potential of cancer cells [10]. 

Therefore, the regulation of mta2 may be related to the 

invasiveness and migratory capacity of bladder 

carcinoma cells. It is known that E-cadherin is a 

Ca2+-reliant adhesion protein which regulates the 

structural intactness of cells of the epithelium, 

maintains tight connections between cells, and 

prevents cell proliferation and invasion [11]. In 

contrast, N- cadherin mainly exists in interstitial cells 

and is loosely connected. Therefore, E-cadherin 

upregulation and N-cadherin downregulation were 

used as crucial indices for evaluating EMT 

transformation in this study. The experimental results 

showed that knocking down mta2 upregulated the 

expression of E-cadherin and downregulated the 

expression of N-cadherin. Overexpression of mta2 

had the opposite effect, with downregulation of E-

cadherin expression and upregulation of N-cadherin 

expression. This suggests that mta2 may promote the 

invasiveness and spread of bladder carcinoma cells 

by regulating EMT transformation. Tumor metastasis-

associated gene 2 (mta2) plays an important role in 

bladder cancer [12,13]. This gene plays a key role in 

regulating the multiplication, invasiveness and 

migration of bladder carcinoma cells. By regulating 

the expression levels of E-cadherin, N-cadherin and 

other related proteins, mta2 affects the metastatic 

potential of bladder carcinoma [14,15]. 

 

CONCLUSION 
 

Overexpression of mta2 enhances the multiplication, 

invasiveness and migration of bladder carcinoma cells, 

but silencing mta2 significantly reduces their 

multiplication, migratory potential and invasiveness. 

Mta2 exhibits these properties through a mechanism 

that is related to the suppression of EMT. Therefore, 

targeting mta2 may be a treatment approach in 

developing a suitable therapy against bladder 

carcinoma. Further studies on the regulatory 

mechanism of mta2 may reveal its exact mechanism 

in the pathogenesis of this disease. 

 

 



Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1  

 

CTMJ | traditionalmedicinejournals.com   

REFERENCES 
 

1. Chang SS, Boorjian SA, Chou R, Clark PE, 

Daneshmand S, Konety BR, Pruthi R, Quale 

DZ, Ritch CR, Seigne JD, et al. Diagnosis 

and treatment of non-muscle invasive 

bladder cancer: AUA/SUO guideline. J Urol 

2016; 196(4): 1021-1029. 

2. Hamilton JG, Shuk E, Genoff MC, 

Rodríguez VM, Hay JL, Offit K, Robson ME. 

Interest and attitudes of patients with 

advanced cancer with regards to secondary 

germline findings from tumor genomic 

profiling. J Oncol Pract 2017; 13(7): e590-

e601. 

3. Ding W, Hu W, Yang H, Ying T, Tian Y. 

Prognostic correlation between MTA2 

expression level and colorectal cancer. Int J 

Clin Exp Pathol 2015; 8(6): 7173-7180. 

4. Han X, Yang J, Huo Q. Corrective effect of 

Electronics combined with internal 

intervention of Pirarubicin on superior 

bladder cancer. Chin J Epidemiol 2015; 21 

(5): 494-497 

5. Zhou C, Ji J, Cai Q, Shi M, Chen X, Yu Y, 

Zhu Z, Zhang 

6. J. MTA2 enhances colony formation and 

tumor growth of gastric cancer cells through 

IL-11. BMC Cancer 2015; 15: 343. 

7. Liu Z, Liu X, Liang J, Liu Y, Hou X, Zhang 

M, Li Y, Jiang 

8. X. Immunotherapy for hepatocellular 

carcinoma: Current status and future 

prospects. Front Immunol 2021; 12: 765101. 

9. Liu J, Liu W, Mi L, Zeng X, Cai C, Ma J, 

Wang L; Union for China lymphoma 

investigators of the Chinese Society of 

clinical oncology; Union for China leukemia 

investigators of the Chinese Society of 

Clinical Oncology. Incidence and mortality 

of multiple myeloma in China, 2006-2016: 

an analysis of the global burden of disease 

study 2016. J Hematol Oncol 2019; 12(1): 

136. 

10. Huang KY, Petretto E. Cross-species 

integration of single-cell RNA-seq resolved 

alveolar-epithelial transitional states in 

idiopathic pulmonary fibrosis. Am J Physiol 

Lung Cell Mol Physiol 2021; 321(3): L491-

L506. 

11. An JX, Ma MH, Zhang CD, Shao S, Zhou 

NM, Dai DQ. miR-1236-3p inhibits invasion 

and metastasis in gastric cancer by targeting 

MTA2. Cancer Cell Int 2018; 18: 66. 

12. Vu T, Datta PK. Regulation of EMT in 

colorectal cancer: A culprit in metastasis. 

Cancers (Basel) 2017; 9(12): 171. 

13. Kanai Y, Ushijima S, Hui AM, Ochiai A, 

Tsuda H, Sakamoto M, Hirohashi S. The E-

cadherin gene is silenced by CpG 

methylation in human hepatocellular 

carcinomas. Int J Cancer 1997; 71(3): 355-

359. 

14. Guilford PJ, Hopkins JB, Grady WM, 

Markowitz SD, Willis J, Lynch H, Rajput A, 

Wiesner GL, Lindor NM, Burgart LJ, et al. 

E-cadherin germline mutations define an 

inherited cancer syndrome dominated by 

diffuse gastric cancer. Hum Mutat 1999; 

14(3): 249-255. 

15. Priya R, Yap AS. Active tension: the role of 

cadherin adhesion and signaling in 

generating junctional contractility. Curr Top 

Dev Biol 2015; 112: 65-102. 

16. Huntley GW, Benson DL. Neural (N)-

cadherin at developing thalamocortical 

synapses provides an adhesion mechanism 

for the formation of somatopically organized 

connections. J Comp Neurol. 1999; 407(4): 

453-471. 

17. Lozano MD, Panizo A, Toledo GR, Sola JJ, 

Pardo- Mindán J. Immunocytochemistry in 

the differential diagnosis of serous effusions: 

a comparative evaluation of eight 

monoclonal antibodies in Papanicolaou 

stained smears. Cancer 2001; 93(1): 68-72. 


