Objectives: 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).
Conclusions: 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.
目的: 探讨沉默Ras同源物家族成员C（RhoC）对唾液腺腺样囊性癌（SACC）增殖、凋亡、侵袭、迁移和上皮-间充质转化（EMT）的影响及其分子机制。方法: 选择2019年1月1日—2024年3月1日于青岛市市立医院手术切除的SACC病灶和正常唾液腺组织各27例，通过蛋白免疫印迹（Western blot）和免疫组织化学法检测RhoC的表达水平。针对RhoC基因序列设计3条小干扰RNA（siRNA），转染至SACC-LM和SACC-83细胞系中并评估转染效率。通过Western blot比较RhoC、Rho相关卷曲螺旋蛋白激酶1（ROCK1）、p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化p38MAPK（p-p38MAPK）、扭曲家族bHLH转录因子1（TWIST1）、E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）的蛋白表达水平。CCK-8实验、流式细胞术、Transwell侵袭实验、伤口愈合实验分别检测各组细胞增殖、凋亡、侵袭和迁移能力的差异。生物信息学方法预测RhoC可能的上游微小RNA（miRNA）及其在SACC中的表达情况，双荧光素酶报告基因实验验证二者的结合位点。结果: RhoC在SACC中的表达显著上升（P<0.05）。沉默RhoC后，实验组ROCK1、p-p38MAPK、TWIST1、N-cadherin、Vimentin的表达显著下降，E-cadherin的表达显著上升（P<0.05）；p38MAPK的表达无明显差异（P>0.05）；细胞增殖、侵袭和迁移能力显著下降，凋亡率显著上升（P<0.05）。miR-138-5p在SACC中低表达，miR-138-5p micmic可以显著下调转染RhoC野生型质粒后293T细胞的荧光素酶活性（P<0.05）。结论: RhoC在SACC中高表达，沉默RhoC可能靶向下游ROCK1/p38MAPK/TWIST1信号通路从而抑制SACC的增殖、侵袭、迁移和EMT，同时促进其凋亡。miR-138-5p在SACC中低表达，是RhoC潜在的上游基因，二者可能存在结合位点。.
Keywords: Ras homolog family member C; Rho-associated coiled-coil containing protein kinase 1; epithelial-mesenchymal transition; miR-138-5p; salivary adenoid cystic carcinoma.