Inhibition Of Pokemon By ShRNA Mediated RNA Interference In RCC Cell OS-RC-2

ObjectiveTo investigate the correlation between the expression and clinical pathology features of Pokemon protein in renal cell carcinoma(RCC); By constructing shRNA recombinant plasmid, it is aimed to explore the effect of recombinant plasmid on the proliferation and apoptosis of Pokemon oncogene of OS-RC-2cells.Methods1. The expression of Pokemon protein was detected by using immunohistochemical techniques (S-P) in64cases of primary RCC.20cases of adjacent noncancerous kidney tissue and20cases of benign tumor of kidney, and the correlation between clinical and histopathologic records were retrospectively analyzed.2. Construction and identification of recombinant plasmid expressing shRNA targeting Pokemon oncogene.3. The recombinant plasmid were transfected into OS-RC-2cells with LipofactamineTM2000: The expression of Pokemon mRNA was detected with Real-time quantitative PCR.4. Methyl thiazolyl tetrazolium (MTT) was applied to analyse cell proliferation of OS-RC-2cells after transfection5. Fluorescence Activated Cell Sorter was applied to study cell apoptosis of OS-RC-2cells after transfection.Results1. Pokemon protein was located in the nuclear and plasma of RCC cells. The rate of strong positive Pokemon protein expression was71.9%in RCC, which was significantly higher than in adjacent noncancerous kidney tissue (30%) and benign tumor of kidney (35%)(P<0.05).2. Pokemon protein expression in RCC had relationship with tumor grading, but it did not show any significant correlation with patients’ sex, age, clinical stage, pathology types and lymph node metastasis.3. shRNA recombinant plasmid were constructed correctly as proved by restriction analysisand by DNA sequencing.4. We found that the transfect efficiency is about42%at24hours post-transfection. At the same time.cell growths were significantly repressed and the characteristic morphology of transfected cells changes greatly against control group. The results of real-timequantitative PCR showed that the Pokemon mRNA expression levels have obviously downregulated compared with the control group, the inhibitory rate is about65.8%.5. MTT assay indicated that OS-RC-2transfection could obviously inhibit cell proliferation.6. Transfection with Pokemon shRNA recombinant plasmid could induce cell apoptosis of OS-RC-2cells, the early apoptosis rate is about40.25±1.48%.ConclusionsStrong positive Pokemon expression may play an important role in RCC occurrence and renal cell carcinoma worsen,The recombinant plasmids Transfecting shRNA targeting Pokemon gene were successfully constructed, which could specifically downregulate the expression of Pokemon at mRNA levels.The recombinant plasmids containing Pokemon shRNA inhibit proliferation and induce apoptosis of OS-RC-2cells,respectively.The current results may be helpful in delineating molecular mechanism of Pokemon tumorigenicity and providing a new approach for the treatment of RCC in the future.