School of Stomatology, Shandong Second Medical University, Weifang 261053, China.
Dept. of Stomatology, Qing-dao Hospital,University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao 266071, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2024 Dec 1;42(6):723-734. doi: 10.7518/hxkq.2024.2024092.
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.
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.
RhoC was highly expressed in SACC (<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 (<0.05). No significant difference in the expression level of p38MAPK was observed (>0.05). The cell proliferation, invasion, and migration ability decreased in the test group, whereas the apoptosis rates significantly increased (<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 (<0.05).
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.
本研究旨在探讨沉默 Ras 同源家族成员 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。
选取 2019 年 1 月 1 日至 2024 年 3 月 1 日青岛市立医院手术切除的 27 例 SACC 病变和正常唾液腺组织，采用 Western blot 和免疫组织化学法检测 RhoC 的表达水平。设计了 3 条针对 RhoC 基因序列的小干扰 RNA（siRNA），转染 SACC-LM 和 SACC-83 细胞系，并评估转染效率。采用 Western blot 比较 RhoC、Rho 相关蛋白激酶 1（ROCK1）、p38 丝裂原激活蛋白激酶（p38MAPK）、磷酸化 p38MAPK（p-p38MAPK）、卷曲螺旋家族 bHLH 转录因子 1（TWIST1）、E-钙黏蛋白、N-钙黏蛋白和波形蛋白的蛋白表达水平。通过 CCK-8 检测、流式细胞术、Transwell 侵袭实验和划痕愈合实验评估各组细胞增殖、凋亡、侵袭和迁移能力的差异。还采用生物信息学方法预测 RhoC 的可能上游 microRNAs（miRNAs）及其在 SACC 中的表达水平。此外，进行双荧光素酶报告基因实验验证 miR-138-5p 与 RhoC 的结合位点。
RhoC 在 SACC 中高表达（<0.05）。沉默 RhoC 后，实验组 ROCK1、p-p38MAPK、TWIST1、N-钙黏蛋白和波形蛋白的表达水平显著降低，E-钙黏蛋白的表达水平显著升高（<0.05）。p38MAPK 的表达水平无显著差异（>0.05）。实验组细胞增殖、侵袭和迁移能力下降，凋亡率显著升高（<0.05）。miR-138-5p 在 SACC 中低表达，转染 RhoC 野生型质粒后，miR-138-5p 模拟物可显著下调 293T 细胞的荧光素酶活性（<0.05）。
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。相反，miR-138-5p 在 SACC 中低表达，是 RhoC 的潜在上游基因，两者之间可能存在结合位点。
