Upregulation of miR-345-5p Suppresses Cell Growth of Lung Adenocarcinoma by Regulating Ras Homolog Family Member A (RhoA) and Rho/Rho Associated Protein Kinase (Rho/ROCK) Pathway
Lung cancer remains one of the most prevalent and deadly cancers worldwide, with lung adenocarcinoma being a significant histological subtype of non-small cell lung carcinoma (NSCLC). Despite advancements in diagnosis and treatment, the 5-year survival rate for lung adenocarcinoma is only 15%. This highlights the urgent need for further research into the molecular mechanisms underlying this disease to improve therapeutic strategies. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have emerged as critical players in cancer progression. Among these, miR-345-5p has been implicated in various cancers, but its role in lung adenocarcinoma remains poorly understood. This study investigates the biological function and molecular mechanisms of miR-345-5p in lung adenocarcinoma, focusing on its interaction with the RhoA and Rho/ROCK signaling pathway.
The study began by examining the expression levels of miR-345-5p in lung adenocarcinoma tissues and cell lines. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), researchers found that miR-345-5p expression was significantly lower in lung adenocarcinoma tissues compared to adjacent normal tissues (0.241 ± 0.095 vs. 1.000 ± 0.233, t = 19.247, P < 0.001). Similarly, miR-345-5p expression was reduced in several lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) compared to normal human bronchial epithelial cells (BEAS-2B). These findings suggest that miR-345-5p may function as a tumor suppressor in lung adenocarcinoma.
To explore the functional role of miR-345-5p, researchers conducted a series of experiments to assess its effects on cell proliferation, apoptosis, migration, and invasion. Overexpression of miR-345-5p in PC-9 and H1650 cells significantly inhibited cell proliferation, as evidenced by colony formation assays and 5-ethynyl-20-deoxyuridine (EdU) incorporation assays. Specifically, the number of colonies formed by PC-9 cells decreased from 103.446 ± 12.132 to 47.684 ± 6.892 (t = 5.666, P = 0.005), and the percentage of EdU-positive cells dropped from 54.173 ± 5.479 to 23.174 ± 3.166 (t = 9.769, P < 0.001). Similar results were observed in H1650 cells, indicating that miR-345-5p suppresses lung adenocarcinoma cell proliferation.
In addition to inhibiting proliferation, miR-345-5p overexpression promoted apoptosis in lung adenocarcinoma cells. Flow cytometry analysis revealed that the percentage of apoptotic PC-9 cells increased from 2.550 ± 0.421 to 14.931 ± 2.653 (t = 12.287, P < 0.001), while H1650 cells showed an increase from 3.112 ± 0.655 to 16.507 ± 3.052 (t = 10.409, P < 0.001). These results suggest that miR-345-5p induces apoptosis in lung adenocarcinoma cells, further supporting its tumor-suppressive role.
The study also investigated the effects of miR-345-5p on cell migration and invasion. Wound healing assays demonstrated that miR-345-5p overexpression significantly reduced the migratory capacity of PC-9 and H1650 cells. The wound closure rate for PC-9 cells decreased from 54.511 ± 6.859 to 21.272 ± 3.559 (t = 6.823, P = 0.002), and for H1650 cells, it decreased from 67.508 ± 6.532 to 32.831 ± 4.557 (t = 6.567, P = 0.003). Similarly, transwell invasion assays showed that miR-345-5p overexpression reduced the number of invaded cells, with PC-9 cells decreasing from 46.325 ± 7.026 to 22.315 ± 4.736 (t = 33.755, P < 0.001) and H1650 cells decreasing from 52.115 ± 7.849 to 19.774 ± 5.936 (t = 6.538, P = 0.003). These findings indicate that miR-345-5p inhibits both migration and invasion of lung adenocarcinoma cells.
To elucidate the molecular mechanisms underlying miR-345-5p’s tumor-suppressive effects, researchers identified RhoA as a potential target of miR-345-5p. RhoA, a member of the Rho GTPase family, is known to promote cancer cell proliferation, migration, and invasion. Using bioinformatics tools and RNA pulldown assays, researchers confirmed that miR-345-5p directly binds to RhoA. Luciferase reporter assays further validated this interaction, showing that miR-345-5p mimics significantly reduced the luciferase activity of RhoA wild-type (WT) vectors in PC-9 and H1650 cells (PC-9: 0.492 ± 0.051 vs. 1.000 ± 0.136, t = 33.469, P < 0.001; H1650: 0.411 ± 0.047 vs. 1.000 ± 0.164, t = 16.000, P < 0.001). No significant change was observed in RhoA mutant (Mut) vectors, confirming the specificity of the interaction.
The study also demonstrated that miR-345-5p overexpression downregulated RhoA expression at both the mRNA and protein levels in PC-9 and H1650 cells. Specifically, RhoA mRNA levels decreased from 1.000 ± 0.127 to 0.321 ± 0.047 in PC-9 cells (t = 8.536, P < 0.001) and from 1.000 ± 0.156 to 0.398 ± 0.054 in H1650 cells (t = 4.429, P = 0.044). These results indicate that miR-345-5p negatively regulates RhoA expression in lung adenocarcinoma cells.
To further investigate the functional relationship between miR-345-5p and RhoA, researchers performed rescue experiments. Overexpression of RhoA reversed the inhibitory effects of miR-345-5p on cell proliferation, migration, and invasion. For example, in PC-9 cells, the number of colonies increased from 47.684 ± 6.892 to 103.446 ± 12.132 upon RhoA overexpression (P = 0.004), and the percentage of EdU-positive cells rose from 23.174 ± 3.166 to 54.173 ± 5.479 (P = 0.004). Similar results were observed in H1650 cells, confirming that RhoA is a critical downstream effector of miR-345-5p in lung adenocarcinoma.
The study also explored the role of the Rho/ROCK signaling pathway in mediating the effects of miR-345-5p. RhoA is a key regulator of the Rho/ROCK pathway, which is involved in various cellular processes, including cell movement, proliferation, and adhesion. Western blot analysis revealed that miR-345-5p overexpression downregulated the protein levels of RhoA, ROCK1, and ROCK2 in PC-9 and H1650 cells. Conversely, miR-345-5p inhibition increased the expression of these proteins. Treatment with CCG-1423, a RhoA inhibitor, further confirmed the involvement of the Rho/ROCK pathway in miR-345-5p-mediated suppression of lung adenocarcinoma cell growth.
In conclusion, this study demonstrates that miR-345-5p functions as a tumor suppressor in lung adenocarcinoma by downregulating RhoA and inhibiting the Rho/ROCK signaling pathway. Upregulation of miR-345-5p suppresses cell proliferation, migration, and invasion while promoting apoptosis in lung adenocarcinoma cells. These findings provide new insights into the molecular mechanisms underlying lung adenocarcinoma and suggest that miR-345-5p could be a potential therapeutic target for this disease.
doi.org/10.1097/CM9.0000000000001804
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