Preliminary Study On Function And Mechanism Of LncRNA PIK3CD-AS1 In Renal Cancer Cells

Part ?: Study on the effect of PIK3CD-AS1 on the invasion of renal cancer cells and related mechanismObjective: To study the effect of PIK3CD-AS1 on the invasion ability of renal cancer cells,and to explore possible mechanisms,and provide reference for the diagnosis and prognosis of renal clear cell carcinoma.Methods: We first used real-time quantitative PCR to detect whether the constructed PIK3CD-AS1(referred to as PIK)cells that were stable and highly expressing PIK3CD-AS1 still overexpress PIK or not.Then,PIK cells were used as the experimental group and PEGFP cells were used as the control group for cell invasion experiments.According to the results of the bioassay analysis,p53 was selected as the target protein.Then extract the total protein of PIK cells and PEGFP cells,and use Western blot detect the expression of p53 in the two cells.Results: The results of real-time quantitative PCR showed that PIK cells still overexpressed PIK relative to PEGFP cells;PIK cells had significantly less invasive ability than PEGFP cells;Western blot results showed that PIK cells expressed p53 protein significantly higher than PEGFP cells.Conclusion: PIK3CD-AS1 can inhibit the invasion and migration of renal cancer cells.The possible mechanism is that PIK3CD-AS1 promotes the expression of p53 protein in renal cancer cells.Part ? Studies on the role of DFFA in pik3cd-as1 in inhibiting the development and progression of renal cancer cellsObjective: The previous research result of this research group is that PIK cells express DFFA less than PEGFP cells.In-depth study of the role of DFFA in PIK cell proliferation,invasion,and apoptosis,and search for related mechanisms,and provide a theoretical basis for future targeted therapy of kidney cancer.Methods: Plasmid transfection and drug screening were used to construct 769 PPIK + DFFA + cells stably high expressing DFFA.Real-time quantitative PCR was used to verify the expression of DFFA in 769 PPIK + cells after transfection screening.769 PPIK + DFFA + cells stably high expressing DFFA were used as experimental groups.769 PPIK + DFFAcells transfected with the empty plasmid were used as a control group.Perform cell proliferation,cell invasion,and apoptosis experiments on the two groups of cells.Extract total protein of two groups of cells,And using Western blot to detect the expression of p53 and pAKT(Ser 473)protein in the two groups of cells.Results: Real-time quantitative PCR test results showed that 769 PPIK + DFFA +cells expressed DFFA significantly higher than 769 PPIK + DFFA-cells;769PPIK + DFFA + cells had significantly greater proliferation and invasion capabilities than 769 PPIK + DFFA-cells,and the apoptosis rate of 769 PPIK + DFFA + cells was significantly lower than that of 7769 PPIK + DFFA-cells.The expression of p53 protein in 769 PPIK + DFFA + cells was significantly lower than that in 769 PPIK + DFFA-cells,and the expression of pAKT protein in 769 PPIK + DFFA + cells was significantly higher than that in 769 PPIK + DFFA-cells.Conclusion: After transfection and drug screening,769 PPIK + DFFA +cells with stable overexpression of DFFA were successfully constructed;DFFA promoted the proliferation and invasion and migration of 769 PPIK + cells,and reduced its apoptosis rate.The possible mechanism is that DFFA inhibited the expression of p53 protein in 769 PPIK + cells And promote its expression pAKT protein.Part ? The expression of 1M gene around PIK3CD-AS1Objective: To further analyze the possible mechanism of PIK3CD-AS1 regulating renal cancer cell function.Methods: In order to further analyze the possible mechanism of PIK3CD-AS1 regulating renal cancer cell function,we searched genes within 1M of PIK3CD-AS1 and design upstream and downstream primers.Using 769 PPIK + cells as the experimental group and 769PPIK-cells as the control group,total RNA was extracted,and the expression of genes around PIK3CD-AS1 in both cells was detected by real-time quantitative PCR the amount.Results: The expression of SLC25A33 in 769 PPIK + cells was 7.6 times that in 769PPIK-(t = 10.1,P <0.05);the expression of SPSB1 in 769 PPIK + cells was 2.3 times that in 769PPIK-(t = 7.7,P <0.05);The expression of H6 PD in 769 PPIK + cells was 1.8 times that in769PPIK-(t = 5.1,P <0.05).No significant changes were found in the other genes.Conclusion: Among the genes around PIK3CD-AS1,SLC25A33,SPSB1 and H6 PD,were found to increase with the high expression of PIK3CD-AS1.Among them,the increase of SLC25A33 was the most significant.It may be related to the regulation of PIK3CD-AS1 on the function of renal cancer,it Can be used for further research.Part ? The Role and Mechanism of SLC25A33 in PIK3CD-AS1 Inhibiting Renal Cancer Cell Function and Its Upstream and Downstream Relationship with DFFAObjective: This study found that in 1M genes around PIK3CD-AS1,SLC25A33 was expressed in PIK cells 7.6 times as much as PEGFP.It may be related to the regulation of renal cancer cell function by PIK3CD-AS1.Further study of SLC25A33 may find a more complete PIK3CD-AS1 Regulate the mechanism of renal cancer cell function.And try to study the relationship between SLC25A33 and DFFA.Methods: 769 PPIK + cells were transfected with siRNA-SLC25A33,then 769 PPIK +cells were down-regulated to express SLC25A33.Real-time quantitative PCR was used to verify the expression of SLC25A33 in769 PPIK + cells after transfection;769PPIK+ siRNASLC cells that down-regulated SLC25A33 were used as the experimental group.769 PPIK + siRNA-ctrl cells stained with siRNA-ctrl were used as a control group.Cell proliferation,cell invasion,and apoptosis experiments were performed according to the experimental methods;total protein of two cells was extracted,and p53 and pAKT protein expression of the two cells were detected by Western blot;PCR was used to detect the expression of DFFA in769PPIK+ siRNA-SLC cells and769 PPIK + siRNA-ctrl cells.Results: The results of real-time quantitative PCR showed that 769PPIK+ siRNA-SLC cells expressed SLC25A33 significantly lower than 769 PPIK + siRNA-ctrl cells;769PPIK+ siRNASLC cells had stronger proliferation and invasion capabilities than 769 PPIK + siRNA-ctrl cells and The apoptosis rate of 769PPIK+ siRNA-SLC cells is less than 769 PPIK + siRNA-ctrl cells;Western blot results show that 769PPIK+ siRNA-SLC cells express p53 protein significantly lower than 769 PPIK + siRNA-ctrl cells,and 769PPIK+ siRNA-SLC cells express pAKT protein Higher than 769 PPIK + siRNA-ctrl cells;769PPIK+ siRNA-SLC cells express DFFA significantly higher than 769 PPIK + siRNA-ctrl cells.Conclusion: Decreasing the expression of SLC25A33 promotes the proliferation and invasion and migration ability of 769 PPIK + cells,and reduces its apoptosis rate.The possible mechanism is to reduce the expression of SLC25A33 to inhibit the expression of p53 protein and promote the expression of pAKT protein in 769 PPIK + cells.DFFA may be the downstream gene of SLC25A33.Part ? Study the relationship between p53 and pAKT and PIK3CD-AS1's inhibition of the development of renal cancerObjective: This study found that PIK3CD-AS1 may be related to p53 and pAKT protein inhibition of renal cancer cell function,and DFFA promoted 769PPIK+ cell function is also related to p53 and pAKT protein.After reducing and excluding the effects of p53 or pAKT as much as possible,through the cell proliferation,invasion,and apoptosis experiments,we further explore the relationship between p53 and pAKT and PIK3CD-AS1 in inhibiting the development of renal cell carcinoma.Methods: siRNA-p53 was transfected into 769PPIK-,769PPIK+,769PPIK+DFFA-,769PPIK+ DFFA+ cells,and siRNA-pAKT was also transfected into the above four cells;the four cells transfected with siRNA-p53 were used for proliferation,invasion,apoptosis experiment,the four cells transfected with siRNA-pAKT were also subjected to proliferation,invasion and apoptosis experiments.Results: Before and after transfection of siRNA-p53,the proliferation and invasion ability of 769PPIK+ cells changed from significantly lower than 769PPIK-cells to significantly higher than 769PPIK-cells,and the apoptosis rate changed from significantly higher than 769PPIK-cells to significantly lower than 769PPIK-cells.769PPIK+DFFA+ cell proliferation and invasion ability changed from significantly stronger than 769PPIK+DFFA-cell to no difference with 769PPIK+DFFA-cell,and apoptosis rate changed from significantly lower than 769PPIK+DFFA-cell to no Obvious difference.Before and after transfection of siRNA-pAKT,the proliferation and invasion ability of 769PPIK+ cells changed from significantly lower than 769PPIK-cells to significantly higher than 769PPIK-cells,and the apoptosis rate changed from significantly higher than 769PPIK-cells to significantly lower than 769PPIK-cells.769PPIK+DFFA + cell proliferative ability changed from significantly stronger than 769PPIK+DFFA-cell to lower than 769PPIK+DFFA-cell,invasive ability changed from significantly stronger than 769PPIK+DFFA-cell to no significant difference from 769PPIK+DFFA-cells,the apoptosis rate changed from significantly lower than 769PPIK+DFFA-cells to no significant difference from 769PPIK+DFFA-cells.Conclusion: p53 and pAKT may be important downstream links of PIK3CD-AS1 / DFFA axis regulating renal cancer cell function.