MiR-338-5p can promote the proliferation, migration and invasion of bladder cancer cells by targeting TSHZ3

doi:10.11958/20221910

Abstract

Abstract:

Objective To investigate the mechanism of miR-338-5p regulating the proliferation, migration and invasion of bladder cancer cells by targeting TSHZ3. Methods The expression of miR-338-5p in 33 samples of bladder cancer tissue and paracancerous tissue was detected by qRT-PCR. Bladder cancer T24 and UM-UC-3 cells were transfected with mimics-NC, miR-338-5p mimics, inhibitor-NC and miR-338-5p inhibitor, and the transfection efficiency was detected by qRT-PCR. CCK-8 assay was used to detect the effect of overexpression or knockdown of miR-338-5p on the proliferation of bladder cancer cells. Transwell assay was used to detect the effect of miR-338-5p on migration and invasion of bladder cancer cells. The potential target genes of miR-338-5p were predicted by TargetScan, miRDB and Targetminer databases, and TSHZ3 was chosen for the target gene. The targeting relationship between miR-338-5p and TSHZ3 was verified by dual-luciferase reporter gene assay and Western blot assay. miR-338-5p mimics and TSHZ3 overexpression plasmids were co-transfected in bladder cancer cells, and the proliferation, migration and invasion of cells were detected by CCK-8 and Transwell assays. Western blot assay was used to detect the overexpression of miR-338-5p or TSHZ3 against Wnt/ β-catenin signaling pathway. Results miR-338-5p was significantly upregulated in bladder cancer (P<0.05). Overexpression of miR-338-5p increased the proliferation, migration and invasion of bladder cancer cells (P<0.05), while transfection of miR-338-5p inhibitor decreased the proliferation, migration and invasion of bladder cancer cells (P<0.05). Bioinformatics analysis and the dual-luciferase reporter gene assay showed that there was a targeting relationship between miR-338-5p and TSHZ3. The rescue experiment showed that co-transfection of miR-338-5p mimics and TSHZ3 overexpression plasmid partially eliminated the promoting effect of overexpression of miR-338-5p on the proliferation, migration and invasion of bladder cancer cells (P<0.05). Western blot results showed that overexpression of miR-338-5p significantly decreased the expression level of TSHZ3 protein and increased Wnt3a and β-catenin protein expression levels (P<0.05). In addition, overexpression of TSHZ3 reduced Wnt3a and β-catenin protein expression levels (P<0.05). Conclusion miR-338-5p is upregulated in bladder cancer, and promotes the proliferation, migration and invasion of bladder cancer by targeting TSHZ3 expression. The mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

Key words: urinary bladder neoplasms, cell proliferation, cell movement, neoplasm invasiveness, miR-338-5p, TSHZ3, Wnt/β-catenin

CLC Number:

R737.14

Figures/Tables 17

Fig.1 The expression of miR-338-5p in normal urothelial cells and bladder cancer cells

Fig.2 Transfection efficiency of overexpression and inhibition of miR-338-5p

Fig.3 Effect of overexpression of miR-338-5p on cell proliferation detected by CCK-8 assay

Fig.4 Effect of inhibition of miR-338-5p on cell proliferation detected by CCK-8 assay

Tab.1 Comparison of the number of migrated and invaded cells between the four groups

组别	T24
组别	迁移	侵袭
mimics-NC组	136.00±3.61	138.34±3.51
miR-338-5p mimics组	200.67±4.51	202.67±8.50
t	19.400^＊＊	12.110^＊＊
inhibitor-NC组	327.00±6.56	305.67±8.02
miR-338-5p inhibitor组	252.33±6.51	211.33±8.33
t	14.000^＊＊	14.132^＊＊
组别	UM-UC-3
组别	迁移	侵袭
mimics-NC组	237.00±5.57	291.67±7.02
miR-338-5p mimics组	387.67±5.13	468.33±3.06
t	34.645^＊＊	39.950^＊＊
inhibitor-NC组	344.33±5.03	375.00±9.00
miR-338-5p inhibitor组	146.00±7.21	159.67±4.51
t	39.064^＊＊	37.051^＊＊

Fig.5 Effects of overexpression of miR-338-5p on migration and invasion of bladder cancer cells (crystal violet staining, ×200)

Fig.6 Effects of inhibition of miR-338-5p on migration and invasion of bladder cancer cells (crystal violet staining, ×200)

Fig.7 Nucleotide sequences complementary to miR-338-5p in the 3'-UTR of TSHZ3

Tab.2 Comparison of the luciferase activity between the two groups

组别	T24		UM-UC-3
组别	TSHZ3-wt	TSHZ3-mut	TSHZ3-wt	TSHZ3-mut
mimics-NC组	1.00±0.25	0.94±0.05	1.06±0.07	1.02±0.11
miR-338-5p mimics组	0.54±0.07	0.91±0.03	0.49±0.04	0.96±0.04
t	10.473^＊＊	1.067	12.173^＊＊	0.888

Fig.8 Effect of overexpression of miR-338-5p on TSHZ3 mRNA expression

Fig. 9 Effect of overexpression of miR-338-5p on TSHZ3 protein level

Fig.10 The mRNA expression of TSHZ3 in normal urothelial cells and bladder cancer cells

Fig.11 Cell viability detected by CCK-8 assay in each group

Tab.3 Comparison of the number of migrated and invaded cells between the three groups

组别		T24
组别		迁移	侵袭
mimics-NC组		144.33±4.51	151.00±6.00
miR-338-5p mimics组		309.67±5.51^a	356.33±8.50^a
miR-338-5p mimics+TSHZ3组		204.67±7.02^b	214.67±9.50^b
F		630.003^＊＊	500.471^＊＊
组别	UM-UC-3
组别	迁移		侵袭
mimics-NC组	173.67±5.03		101.00±6.00
miR-338-5p mimics组	332.67±4.73^a		284.33±6.51^a
miR-338-5p mimics+TSHZ3组	255.00±7.94^b		213.00±6.56^b
F	514.087^＊＊		633.505^＊＊

Fig.12 Migration and invasion of each group of cells (crystal violet staining, ×200)

Fig.13 Effect of overexpression of miR-338-5p on the protein level of Wnt3a and β-catenin

Fig.14 Effect of overexpression of TSHZ3 on the protein level of Wnt3a and β-catenin

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