MiR-143-3p Regulates Pulmonary Metastasis Of Molecular Mechanism In Human Osteosarcoma(OS)

Objective: To provide a theoretical basis for exploring mi RNA reexpression in target-suppressing OS metastasis by investigating the effects of mi R-143-3p and its target gene protein on the regulation of human osteosarcoma(OS) biological behavior and the molecular mechanism of lung metastasis.Methods:(1)The expression of mi R-143-3p 、 mi R-411-5p and mi R-195-5p in MG63 and HOS cell was detected by RT-PCR.Moreover, by transfecting MG63 and HOS cell with the above three mature mi RNA(mimics, M), the role of mi RNA in osteosarcoma proliferation, metastasis and invasion was examined through MTT,wound-healing and transwell assays.(2)Average diameters of the different mi RNA mimics/CS nanoparticle suspension concentration were detected by a 3000 HSA Zetasizer. Then the nude mice were tail intravenously injected with mi RNA mimics/CS nanoparticle suspension of various concentrations or 2mg/ml nanoparticle suspension.All were sacrificed at different times. In vivo imaging system showed the lung-targeting of mi RNA mimics/CS nanoparticle suspension by the observation of the accumulated fluorescence intensity in nude mice lungs.(3) The nude mice lung metastases was examined by HE staining following the successful osteosarcoma inoculation in left tibias of nude mice. The rate of tumor formation had been caculated.The model rats divided into two groups(control group and the experimental group) were injected intravenously with negative control mimics/CS nanoparticles suspension and mi R-143-3p mimics/CS nanoparticles suspension with the four weeks administration,respective. Their impact on OS proliferation was preliminarily estimated with the measurement of tumor volume. HE staining displayed lung metastasis in nude mice.(4)The mi R-143-3p target genes(ITGA6, ASAP3, CRELD1 and MSI2) concerning lung metastases were analyzed and screened by Target Scan 6.1, Diana micro T 4.0 andMICRORNA.ORG bioinformatics software. With the mi R-143-3p M transfection into HOS cells, mi R-143-3p target genes expressions were detected with Western blot analysis after protein extraction. And the fresh lung tissues of nude mice in the control group and the experimental group were homogenized, and extracted. Then the result of Western blot in mi R-143-3p target genes expressions was analyzed.Results:(1) RT-PCR results showed a significant decrease in the expression levels of mi R-143-3p MG63 and HOS cells. In the tests of cancer cell proliferation, migration and invasion, MTT experiments displayed mi R-143-3p had no effect on proliferation,the wound-healing assay exhibited the mi R-143-3p suppression on OS metastasis and Transwell assay presented mi R-143-3p could inhibit OS invasion.(2)3000HSA Zetasizer and In vivo imaging system showed the most significant fluorescencent accumulation in lung of MNC/CS nanoparticles suspension at a concentration of 2mg/ml with the particle diameter of 336.9nm and lung targeting.(3)5 weeks after HOS cell implantation in situ, the left tibias of all nude mice appeared mass and swelling, and no lung metastases. At 6 weeks, large mass vacination in left tibias were grown at 100% tumor formation rate. And it showed lung metastases in all nude mice. The nude mice with the MG63 cell inoculation appeared no nude tumor mass and metastasis till the end of experiment. At the end of the administration, compared with the MNC group, M group tumors in situ were not significantly different in average volume, which indicated that the mi R-143-3p inhibitory effect was not obvious. HE staining showed that MNC group had lung metastases and M group almost didn’t,only 1 nude mice had.(4)The mi R-143-3p target genes(ITGA6, ASAP3, CRELD1 and MSI2) concerning lung metastases were analyzed and screened by bioinformatics software. With the mi R-143-3p M transfection into HOS cells, the results of Western blot showed that ASAP3 and MSI2 protein expression significantly decreased while ITGA6 and CRELD1 had no change. And the fresh lung tissues of nude mice in the control group and the experimental group were homogenized, and extracted. Then the result of Western blot showed that ASAP3 and MSI2 protein expression significantly decreased while ITGA6 and CRELD1 had no change.Conclusion:(1) By RT-PCR, MTT, wound-healing and Transwell experiments the antitumor regulator mi R-143-3p was successfully screened. It was preliminarily proved that the mi R-143-3p inhibited OS invasion and metastases with no impact on OS proliferation.(2)3000HSA Zetasizer and in vivo imaging system screened the most significant fluorescencent accumulation in lung of MNC/CS nanoparticles suspension at a concentration of 2mg/ml with the particle diameter of 336.9nm and lung targeting,capable of as the carrier for tail vein injection in vivo.(3)The inhibition of mi R-143-3p mimcs/CS nanoparticles suspension on lung metastasis from OS in vivo indicated that mi R-143-3p suppressed lung metastasis of OS.(4)mi R-143-3p lung metastasis-related target genes were screened by bioinformatics. The analysis of western blot showed with the mi R-143-3p upregulation target proteins ASAP3 and MSI2 reduced in vitro and in vivo while ITGA6 and CRELD1 had no variation.