RESUMEN
Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P<0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group (P<0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group (P<0.05) but higher than the miR-143 mimic group significantly (P<0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all P<0.05). In the tissue samples, the miR-143 expression in the cervical cancer group was significantly lower than that of the cervical intraepithelial neoplasia group (0.32±0.06 vs. 0.93±0.17, P<0.05); whereas the K-ras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, P<0.05). Conclusions: In vitro, miR-143 can inhibit the proliferative activity of HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.
Asunto(s)
Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Genes ras , MicroARNs/fisiología , Neoplasias del Cuello Uterino/patología , Regulación hacia Abajo , Femenino , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Proteínas ras/metabolismoRESUMEN
Ligament geometry is crucial to surgical treatment success in anterior cruciate ligament (ACL) injury. This study aimed to optimize the MRI technique to elucidate the geometry of the patellar ligament (PL) and ACL in vivo. A 1.5-T superconducting MRI system with a special surface coil and fast spin echo was used to acquire high-resolution T1-weighted images (H-T1WI) of the ACL. The sagittal plane angle was 10° to 15° towards the inner side of the vertical line of the tangent line axis of the femoral intercondylar fossa. The H-T1WI images of the PL were centered at the lower margin of the patella and the center of the tibial tuberosity. The lengths of the PL and ACL were measured using a Radworks 5.1 workstation. ACL and PL lengths were compared between left and right knees and between genders, and left PL length measurements obtained separately by three doctors underwent correlation analysis. The quality of the images satisfied the clinical measurement requirements. The duration of sagittal image acquisition was 2 min and 25 s. The average PL length was 42.20 ± 4.21 and 40.15 ± 4.00 mm, and the average ACL length was 36.98 ± 4.12 and 35.80 ± 4.67 mm, in male and female subjects, respectively. The intraclass correlation coefficients of the PL lengths obtained by the three specialists were greater than 0.997. This MRI technique provides highly stable and repeatable in vivo data of PL and ACL geometry relevant to ACL reconstruction surgery with PL grafts.