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To investigatethe expression of miR-378a-3p in CC cells, we performed RT-qPCR analysis to determine the relative miR-378a-3p levels in the serum of 48 pairs of patients with CC and healthy controls. As shown in Figure 1A, serum miR-378a-3p expression was decreased in CC patients compared to that in normal subjects. Similarly, the analysis of the expression of miR-378a-3p in tissues from CC patients and their adjacent normal tissues revealed that miR-378a-3p expression was markedly decreased in the CC group (Figure 1B).
Figure 1. MiR-378a-3p is downregulated in cervical cancer (CC) serum and tissues. (A) Real-time quantitative polymerase chain reaction analysis was performed to evaluate the relative expression levels of miR-378a-3p in 48 tissue sample pairs of patients with CC and healthy control subjects. (B) The expression levels of miR-378a-3p in CC tissues and adjacent normal tissues were measured. *P < 0.05 and **P < 0.01 versus the control group indicate that the difference was significant.
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The relationship between CC clinicopathological features and serummiR-378a-3p expression shown in Table 1 revealed that miR-378a-3p expression was closely related to tumor size, tumor-node-metastasis (TNM) stage, and lymph node metastasis. For the CC patient samples, patients with low miR-378a-3p expression had larger tumor sizes; more tumors in stages II, III, and IV; and lymph node metastasis. However, miR-378a-3p expression was not associated with age or pathological grading.
Variable No. of patients (n = 48) miR-378a-3p expression χ2 P High (n = 19) Low (n = 29) Age (years) ≤ 50 13 5 8 0.009 0.923 > 50 35 14 21 Tumor size (cm) ≤ 4 20 12 8 5.976 0.015 > 4 28 7 21 TNM stage I–II 18 11 7 5.581 0.018 III–IV 30 8 22 Pathological grading Well (I) 3 3 0 5.141 0.077 Moderate (II–III) 34 14 20 Poor (IV) 11 3 8 Lymph node metastasis Negative 23 13 10 5.298 0.022 Positive 25 6 19 Table 1. Relevance between serum miR-378a-3p expression and clinical pathologic characteristics of cervical cancer
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The relationship between serum miR-378a-3p expression and the overall survival (OS) of patients with CC was analyzed using the Kaplan-Meier method. Patients with CC were separated into high and low miR-378a-3p expression groups. The results showed that low miR-378a-3p expression was related to poor OS in CC patients (Figure 2A). Moreover, the survival analysis of patients, based on miR-378a-3p expression using Kaplan-Meier Plotter visualization software[23] and The Cancer Genome Atlas database, showed that the mortality rates of patients with low miR-378a-3p expression were significantly higher compared to those with high expression (Figure 2B).
Figure 2. Analysis on the overall survival (OS) of cervical cancer (CC) patients with different miR-378a-3p expression levels. (A) The relationship between serum miR-378a-3p expression and the OS of 48 CC patients was analyzed by the Kaplan-Meier method. (B) OS analysis of miR-378a-3p was performed using Kaplan-Meier Plotter visualization software based on The Cancer Genome Atlas database (N = 306). The figure was downloaded from Kaplan-Meier Plotter.
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After transfection with miR-378a-3p mimics, CCK-8, cell colony formation, and scratch wound healing assays were used to investigate the effect of miR-378a-3p expression on CC cell proliferation. Compared to the NC group, the viability of SiHa and HeLa cells in the miR-378a-3p mimic group was significantly decreased at 48 h (Figure 3A). The results of the cell colony formation assay revealed that the number and size of the colonies of the transfected SiHa and HeLa cells in the mimic group were suppressed (Figure 3B). In addition, the migration ability was dramatically inhibited after transfection with miR-378a-3p mimics (Figure 3C).
Figure 3. Overexpression of miR-378a-3p inhibits cervical cancer (CC) cell proliferation. (A) The optical density values in SiHa and HeLa cells in the miR-378a-3p mimic and NC groupswere determined by the Cell Counting Kit-8 assay. (B) The cell colony formation assay was performed to assess the proliferation of SiHa and HeLa cells. (C) The wound healing assay was performed to evaluate the migration of SiHa and HeLa cells. *P < 0.05 versus the control group indicate that the difference was significant.
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Mice were subcutaneously injected with HeLa cells transfected with lentivirus-packaged miR-378a-3p mimics, and the tumor volume was measured every 7 days for 28 days. The results showed that the tumor volumein the miR-378a-3p mimic group was lower than that in the NC group (Figure 4A). We also measured the tumor weight in the two groups, and the results revealed that the tumor weight was dramatically reduced with overexpression of miR-378a-3p (Figure 4B). In addition, after injection of miR-378a-3p mimics, the isolated tumor tissues had a higher level of miR-378a-3p expression (Figure 4C). Moreover, immunohistochemical analysis of Ki-67 expression in isolated tumor tissues showed that Ki-67 expression was significantly decreased in the miR-378a-3p group (Figure 4D).
Figure 4. MiR-378a-3p inhibits tumor growth in vivo. (A) Tumor volume was measured every 7 days. (B) Tumors were isolated after 28 days of HeLa cell injection, and images for representative mice and tumors were captured (left). The tumor weight was also calculated (right). (C) The expressions levels of miR-378a-3p in isolated tumors were determined. (D) The Ki-67 expression level was determined by immunohistochemical analysis. *P < 0.05 and **P < 0.01 versus the small interfering RNA negative control group indicate that the difference was significant.
MicroRNA-378a-3p Downregulation as a Novel Biomarker with Poor Clinical Outcomes in Cervical Cancer
doi: 10.3967/bes2021.026
- Received Date: 2020-08-11
- Accepted Date: 2021-01-18
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Key words:
- Cervical cancer /
- miR-378a-3p /
- Biomarker /
- Prognosis /
- Tumor growth
Abstract:
Citation: | ZHANG Liang, WU Zhen An. MicroRNA-378a-3p Downregulation as a Novel Biomarker with Poor Clinical Outcomes in Cervical Cancer[J]. Biomedical and Environmental Sciences, 2021, 34(3): 213-221. doi: 10.3967/bes2021.026 |