Effect of silencing Ras homolog family member C on proliferation, invasion, and migration of salivary adenoid cystic carcinoma

doi:10.7518/hxkq.2024.2024092

Abstract

Abstract:

Objective This study aimed to investigate the effects of silencing Ras homolog family member C (RhoC) on the proliferation, apoptosis, invasion, migration, and epithelial-mesenchymal transition (EMT) of salivary adenoid cystic carcinoma (SACC) and its molecular mechanisms. Methods A total of 27 SACC lesions and normal salivary gland tissues that were surgically resected at Qingdao Municipal Hospital from January 1, 2019 to March 1, 2024 were selected, and the expression levels of RhoC were detected by Western blot and immunohistochemistry. Three small interfering RNA (siRNAs) were designed to target the RhoC gene sequence, transfected into SACC-LM and SACC-83 cell lines, and evaluated for transfection efficiency. The protein expression levels of RhoC, Rho-associated protein kinase-1 (ROCK1), p38 mitogen-activated protein kinase (p38MAPK), phosphorylated-p38MAPK (p-p38MAPK), twist family bHLH transcription factor 1 (TWIST1), E-cadherin, N-cadherin, and Vimentin were compared using Western blot. CCK-8 assay, flow cytometry, transwell invasion assay, and wound healing assay were conducted to assess the differences in cell proliferation, apoptosis, invasion, and migration abilities among the groups. Bioinformatics methods were also used to predict possible upstream micro RNAs (miRNAs) of RhoC and their expression levels in SACC. Moreover, dual-luciferase reporter gene experiments were performed to verify the binding sites of miR-138-5p and RhoC. Results RhoC was highly expressed in SACC (P<0.05). After silencing RhoC, the test group showed a significant decrease in the expression level of ROCK1, p-p38MAPK, TWIST1, N-cadherin, and Vimentin, as well as a significant increase in the expression level of E-cadherin (P<0.05). No significant difference in the expression level of p38MAPK was observed (P>0.05). The cell proliferation, invasion, and migration ability decreased in the test group, whereas the apoptosis rates significantly increased (P<0.05). miR-138-5p was lowly expressed in SACC, and miR-138-5p mimic can significantly downregulated the luciferase activity of 293T cells after transfection with a RhoC wild-type plasmid (P<0.05). Conclusion RhoC is highly expressed in SACC, and RhoC silencing may target the downstream ROCK1/p38MAPK/TWIST1 signaling pathway, thereby inhibiting the proliferation, invasion, migration, and EMT of SACC while promoting its apoptosis. On the contrary, miR-138-5p is lowly expressed in SACC and is a potential upstream gene of RhoC, and there may be binding sites between the two genes.

Key words: salivary adenoid cystic carcinoma, Ras homolog family member C, Rho-associated coiled-coil containing protein kinase 1, epithelial-mesenchymal transition, miR-138-5p

CLC Number:

R782

Yu Wenyuan, Zhao Peng, Shao Yu, Xu Yong, Xu Jin, Xie Lei, Yu Chenghao, He Qiuping, Chen Zhenggang. Effect of silencing Ras homolog family member C on proliferation, invasion, and migration of salivary adenoid cystic carcinoma[J]. West China Journal of Stomatology, 2024, 42(6): 723-734.

Figures/Tables 14

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siRNA	序列
siRNA-RhoC 1#	GUGCCUUUGGCUACCUUGA
siRNA-RhoC 2#	CTACTGTCTTTGAGAACTA
siRNA-RhoC 3#	GACTGCTGTAGGCCTTCTT
siRNA-NC	UUCUUCGAACGUGUCACGU

项目		0 h	24 h	48 h	72 h
SACC-LM	空白对照组	0.59±0.08	0.83±0.06	1.54±0.22	1.73±0.14
	阴性对照组	0.60±0.11	0.78±0.08	1.46±0.16	1.66±0.13
	实验组	0.55±0.10	0.69±0.10*^#	1.14±0.23*^#	1.38±0.19*^#
SACC-83	空白对照组	0.50±0.04	0.66±0.06	1.37±0.10	1.49±0.10
	阴性对照组	0.52±0.05	0.63±0.08	1.40±0.10	1.53±0.13
	实验组	0.49±0.06	0.56±0.11*^#	1.08±0.11*^#	1.24±0.10*^#

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