| Renal cell carcinoma(RCC)is a malignant tumor of urinary system originating from renal tubular epithelium,which has the characteristics of high mortality and high recurrence rate.RCC has become the second largest tumor of urinary and reproductive system after bladder cancer in China.Recurrence and metastasis are the main causes of renal cancer treatment failure.Therefore,early assessment of the risk of renal cancer recurrence and metastasis and finding an effective therapeutic target are urgent clinical needs.For this reason,it is particularly important to deeply explore the pathogenesis of RCC.The recurrence and metastasis of renal cancer are closely associated with various biological behaviors,including proliferation,invasion,migration,and tumor angiogenesis of renal cancer cells.CAV-1,as a significant structural protein of the cell membrane,plays a multifaceted role in the development and progression of different types of tumors.It not only influences the biological behavior of tumor cells but also impacts angiogenesis.However,the precise mechanism by which it operates in renal cancer remains unclear.This research aims to enhance our understanding of kidney cancer recurrence and metastasis.In recent years,mi RNAs have emerged as crucial players in cancer development by targeting m RNA.Among them,mi R-203 has demonstrated evident tumor-suppressive effects in breast cancer,lung cancer,prostate cancer,and rectal cancer.Nevertheless,its specific mechanism of action in renal cell carcinoma(RCC)remains poorly elucidated.Currently,ultrasonic perfusion has proven to be an effective clinical method for assessing the risk of recurrence and metastasis in patients,as well as evaluating the efficacy of anti-vascular targeted therapies.Nevertheless,there is a notable lack of correlation studies examining the relationship between ultrasonic perfusion and CAV-1 expression levels in renal cancer.Such correlation studies would contribute to a deeper understanding of the role and value of each contrast-enhanced ultrasound parameter in assessing the biological behavior of tumors.In this study,low expression of mi R-203 and high expression of CAV1 were found in postoperative specimens of RCC patients.As a structural protein,CAV1 played an important role in tumor proliferation,migration and tumor neovascularization.The differences in the expression levels of mi R-203 and CAV-1gene targets and PI3K/AKT signaling pathway were found for the first time by bioinformatics.The related in vitro experiments were carried out in 112 postoperative pathological tissues of RCC patients diagnosed by ultrasound puncture,which once again confirmed the role of mi R-203 in regulating CAV-1 in interstitial transition,proliferation,migration,invasion and apoptosis of RCC.Finally,we constructed a nude mouse model of RCC,and observed the effect of mi RNA-203 on tumor blood supply and the expression of CAV-1 by contrast-enhanced ultrasound from the perspective of ultrasound molecular imaging,which further revealed the potential of mi R-203 as a therapeutic target for RCC,and provided a theoretical basis for evaluating the risk of metastasis and recurrence.The existence of targets of mi R-203 and CAV-1 genes was identified through the comprehensive application of bioinformatics,cell experiments and animal experiments,it was confirmed that mi R-203 could regulate the interstitial transformation,proliferation,migration,invasion and apoptosis of RCC by down-regulating the expression of CAV-1 and inhibiting the PI3K/AKT signaling pathway,with the potential to be applied in the clinical prediction,diagnosis and treatment of RCC.Finally,a nude mouse model of renal cancer was constructed,the effects of mi R-203 on RCC tumor blood supply and regulatory effect on CAV-1 were observed by contrast-enhanced ultrasound from the perspective of ultrasonic molecular imaging,thus further revealing the potential of mi R-203 as a therapeutic target for renal cancer and providing a new perspective for clinical assessment of the risk of renal cancer metastasis and recurrence.Methods:1.The target genes were screened by Gene Expression Omnibus(GEO).RCC gene expression primitive chips(GSE53757,GSE14762,GSE77199 and GSE6344)were downloaded,and abnormally expressed mi RNA was analyzed.RCC related genes were searched by Dis Ge NET,and the interaction network between pre-selected cross DEGs and known disease genes was generated.It was speculated that there was correlation between CAV-1 and disease genes.After gene enrichment analysis,PI3K/AKT signaling pathway related to abnormal mi RNA expression was found for the first time,and then the expression level difference between mi R-203 and CAV-1gene targets and PI3K/AKT signaling pathway was verified again by mi RNA targeted scanning.2.The expression patterns of mi R-203 and CAV-1 and their correlation with clinical stage were determined by RCC postoperative samples.112 cases of RCC diagnosed by ultrasound puncture and treated by related surgery were collected,and their cancer tissues and adjacent normal tissues were taken.Immunohistochemistry(IHC)and fluorescence real-time quantitative PCR(q RT-PCR)were used to detect the difference of CAV-1 expression in RCC tissue and normal tissue,at the same time,the correlation between the expression of mi R-203 and CAV-1 and the stage and differentiation of lymph node metastasis in RCC patients was analyzed based on pathological parameters.3.The mechanism of mi R-203,CAV-1 and PI3K/AKT signaling pathways in RCC was further verified by cell experiments.The cell line with the highest expression of CAV-1 in human renal clear cell adenocarcinoma was screened by RT-PCR and WB.Detected the expression of mi R-203,CAV-1,PI3 K,AKT,β-catenin,N-cadherin,bcl-2,E-cadherin and Bax to determine the mechanism of mi R-203,CAV-1 and PI3K/AKT signaling pathway in RCC.After the cell line was cultured and transfected,MTT assay,scratch assay,Transwell migration assay and flow cytometry were performed to analyze the proliferation,migration,invasion,cycle distribution and apoptosis of RCC cells induced by mi R-203,and to determine whether mi R-203 has potential as a biomarker for evaluating RCC.4.Contrast-enhanced ultrasound was used to evaluate the value of mi R-203/CAV-1 in predicting metastasis and recurrence of RCC.The nude mice model of 786-O RCC was established.After tumor formation,they were randomly divided into mi R-203 agomir group and mi R-203 agomir NC group.The tumor volume was measured and recorded,and the growth curve was drawn.The effect of mi R-203 argomir on blood supply of tumor was observed by contrast-enhanced ultrasound before death.Tumor tissue was taken for HE staining,and the expression of CAV-1 was detected by immunohistochemistry.The correlation between quantitative parameters of contrast-enhanced ultrasound and CAV-1 expression was analyzed,and whether it can be applied to metastasis and recurrence of RCC was evaluated.Results:1.CAV-1 was the target gene of mi R-203.The bioinformatics prediction website(micro RNA.org)and double luciferase reporter gene analysis were used to confirm whether CAV-1 is the direct target gene of mi R-203.Bioinformatics prediction showed that there was a specific binding region between CAV-1 gene sequence and mi R-203 sequence,which confirmed that CAV-1was the target gene of mi R-203.The results of double luciferase reporter gene analysis showed that the luciferase activity of mi R-203 mimic/wt-CAV-1co-transfection group was lower than that of negative control(NC)group(P<0.05),while the luciferase activity of CAV-1 mutant 3?UTR was not significantly different(P>0.05),which confirmed that CAV-1 was the target of mi R-203.The results of the above two studies confirmed that mi R-203 can specifically bind to CAV-1,and CAV-1is the direct target gene of mi R-203.2.CAV-1 was over-expressed in RCC tissues.The positive expression rate of CAV-1 in RCC tissues and adjacent normal tissues was detected by IHC to investigate the effect of CAV-1 on RCC.CAV-1 cells with positive staining all have linear yellow-brown cell membrane.Adjacent normal tissues showed uniform distribution and regular arrangement of blood vessels,while RCC tissues showed uneven distribution and random arrangement of blood vessels.Some RCC microvessels are even anastomosed into a network,losing the intrinsic vascular lumen structure.The positive rate of CAV-1 in RCC tissues was significantly higher than that in adjacent normal tissues(P<0.05).3.The expression of mi R-203 was down-regulated and that of CAV-1 was up-regulated in RCC.RT-q PCR and WB analysis were used to determine the expression of mi R-203 and CAV-1 in RCC tissues and adjacent normal tissues.The results showed that the expression of mi R-203 in RCC was significantly lower than that in adjacent normal tissues,while the levels of CAV-1 gene and protein were significantly higher in RCC tissues than those in adjacent normal tissues(P<0.05).4.The low expression of mi R-203 and the overexpression of CAV-1 were correlated with the stage of RCC.The expression of mi R-203 and CAV-1 in RCC and adjacent normal tissues were detected by RT-q PCR.The expression of mi R-203 in RCC was significantly lower than that in adjacent normal tissues(P<0.05).The expression of mi R-203 and CAV-1in RCC patients was correlated with lymph node metastasis stage and differentiation degree(P<0.05),but not with age,gender and tumor size(P>0.05).The expression of mi R-203 in poorly differentiated stage Ⅲ/Ⅳ RCC patients was significantly lower than that in highly differentiated stage Ⅰ/Ⅱ RCC patients,while the expression of CAV-1 showed an opposite trend.5.Human renal clear cell adenocarcinoma cell line 786-O was selected for in vitro experiment.Compared with ACHN,OS-RC-2 and ketr-3 cell lines,the expression of CAV-1m RNA and protein in human renal clear cell adenocarcinoma cell line 786-O increased significantly(P<0.05).Human renal clear cell adenocarcinoma cell line786-O was selected for follow-up experiments.6.mi R-203 inhibits CAV-1 and PI3K/AKT signaling pathways in RCC.The expressions of mi R-203,CAV-1,PI3 K,AKT,β-catenin,N-cadherin,bcl-2,E-cadherin and Bax were detected by RT-q PCR and western blot.RT-q PCR and western blot analysis showed that there was no significant difference in gene expression between blank group and NC group(P>0.05).Compared with blank group and NC group,mi R-203 expression increased significantly in mi R-203 mimics group and mi R-203 mimics+si RNA-CAV-1 group(P<0.05),decreased significantly in mi R-203 inhibitor group(P<0.05),but did not change significantly in si RNA-CAV-1group(P>0.05).In mi R-203 inhibitor group,the m RNA and protein expressions of CAV-1,PI3 K,phosphorylated AKT,β-catenin,N-cadherin and bcl-2 were significantly increased(P<0.05),while the expressions of E-cadherin and Bax were significantly decreased(P<0.05).The results of mi R-203 mimics,si RNA-CAV-1 and mi R-203 mimics+si RNA-CAV-1 groups were contrary(P<0.05).There was no significant difference between mi R-203 mimics group and si RNA-CAV-1 group(P>0.05).Compared with mi R-203 mimics+si RNA-CAV-1 group,the m RNA and protein levels of E-cadherin and Bax were significantly increased(all P<0.05),while the expressions of CAV-1,PI3 K,phosphorylated AKT,β-catenin,N-cadherin and bcl-2 were significantly decreased(all P<0.05).Finally,the expression of EMT-related factors E-cadherin and N-cadherin was detected by immunofluorescence,and the results still support the above contents.7.mi R-203 inhibited the proliferation of RCC cells through CAV-1.MTT showed that there was no significant difference in viability among the groups after 24 hours of transfection(P>0.05),but there was significant difference in viability among the groups after 48 hours and 72 hours of transfection(P<0.05).There was no significant difference in cell viability between the blank group and the normal control group at each time point(P>0.05),but the cell viability of mi R-203 inhibitor group at 48 hours and 72 hours was significantly higher than that of the blank group and the normal control group(P<0.05).To the other three groups(mi R-203 mimics group,si RNA-CAV-1 group and mi R-203 mimics+si RNA-CAV-1group),cell viability decreased significantly at 48 hours and 72 hours(P<0.05).The results showed that mi R-203 inhibited the proliferation of RCC cells by targeting CAV-1.8.mi R-203 inhibits RCC cell migration through CAV-1.Cell migration was measured by scratch test.There was no significant difference in migration ability between blank group and NC group at 0 and 24 hours(P>0.05).Compared with blank group and NC group,cell migration in mi R-203 inhibitor group increased significantly(P<0.05),while cell migration in mi R-203 mimics group,si RNA-CAV-1 group and mi R-203 mimics+si RNA-CAV-1 group decreased significantly(P<0.05).Compared with mi R-203 mimics group,the migration of mi R-203 mimics+si RNA-CAV-1 group decreased significantly(P<0.05).The results showed that mi R-203 inhibited the migration of RCC cells by inhibiting CAV-1.9.mi R-203 inhibits RCC cell invasion through CAV-1.Transwell test showed that there was no significant difference in the number of cells transferred from upper chamber to lower chamber between blank group and NC group(P>0.05).Compared with blank group and NC group,the number of metastatic cells in mi R-203 inhibitor group increased significantly(P<0.05),while the number of metastatic cells in mi R-203 mimics group,si RNA-CAV-1 group and mi R-203mimics+si RNA-CAV-1 group decreased significantly(P<0.05),and the number of metastatic cells in mi R-203 mimics+si RNA-CAV-1 group was lower than that in mi R-203 mimics group(P<0.05).The results showed that mi R-203 inhibited the invasion of RCC cells through CAV-1.10.mi R-203 blocks RCC cell cycle and promotes apoptosis through CAV-1.Flow cytometry was used to detect the cell cycle distribution and apoptosis of RCC cells under the influence of mi R-203.The results showed that there was no significant difference in cell cycle and apoptosis rate between blank group and NC group(P>0.05).Compared with control group and control group,the proportion of G1 phase cells in mi R-203 mimics,si RNA-CAV-1 and mi R-203mimics+si RNA-CAV-1 groups increased significantly(P<0.05),while the proportion of S phase cells decreased significantly(P<0.05),which indicated that the apoptosis rate increased significantly(P<0.05).Compared with the blank group and the normal control group,the mi R-203 inhibitor group had more cells in S phase and fewer cells in G1 phase,indicating a significant decrease in apoptosis(P<0.05).Compared with mi R-203 mimics+si RNA-CAV-1 group,most of the cells remained in G1 phase and a few in S phase.The results showed that apoptosis increased significantly(P<0.05)in mi R-203 mimics+si RNA-CAV-1 group.In conclusion,up-regulation of mi R-203 can inhibit cell cycle and promote apoptosis by targeting CAV-1.11.mi R-203 showed significant inhibitory effect on RCC xenografts in nude mice.Human renal clear cell adenocarcinoma cell line 786-O was inoculated into nude mice,and the nude mice model of 786-O RCC transplanted tumor was successfully established.Two groups of nude mice were killed 28 days later.The growth curve,tumor weight and gross image showed that the tumor growth in mi R-203 agomir group was significantly inhibited(P<0.05),suggesting that mi R-203 could inhibit the growth of RCC transplanted tumor in vivo.12.Contrast-enhanced ultrasound can visually evaluate the inhibitory effect of mi R-203 on RCC transplanted tumor in nude mice.Contrast-enhanced ultrasonography showed that the tumors in mi R-203 ago group were similar to those in ago NC group(P>0.05),with low echo,oval shape with superficial lobes,clear edges and uneven internal echo,but the tumor volume in mi R-203 ago group was smaller than that in ago NC group(P<0.05).Quantitative analysis of contrast-enhanced ultrasound showed that the rise time(RT),peak time(TTP),mean transit time(m TT)and peak intensity(PI)in mi R-203 ago group were slower than those in NC ago group,and the differences were statistically significant except for RT(P<0.05).13.The quantitative parameters of contrast-enhanced ultrasound were correlated with the expression of CAV-1.Pearson correlation analysis was used to determine whether there was a correlation between quantitative parameters of contrast-enhanced ultrasound and CAV-1 expression.The results showed that there was a negative correlation between TTP,m TT and CAV-1 protein expression(P<0.05),and a positive correlation between PI and CAV-1 protein expression(P<0.05),especially between TTP and PI.There was no significant correlation between the expression of RT and CAV-1(P>0.05).14.Immunohistochemical evaluation of mi R-203 on CAV-1 expression in RCC xenografts in nude mice.The nude mice were sacrificed after contrast-enhanced ultrasound.HE staining showed that in mi R-203 ago group,compared with HE section of tumor in NC ago group,cell density decreased,necrotic area increased and blood vessel density decreased(P<0.05).Immunohistochemistry showed that CAV-1 protein staining was positive in tumor cell membrane.The results showed that the expression of CAV-1 in mi R-203 ago group was significantly lower than that in NC ago group(P<0.05).Conclusions:1.mi R-203 can inhibit the proliferation,migration and invasion of RCC cells by down-regulating the expression of CAV-1 and inactivating the PI3K/AKT signaling pathway,and promote the apoptosis of RCC cells.2.mi R-203 cannot affect the activity of the 3?UTR mutant CAV-1.3.Contrast-enhanced ultrasound can reflect the expression of CAV-1 from the imaging point of view through the change degree of tumor and peripheral invasive microcirculation.It is an effective examination method to evaluate the risk of recurrence and metastasis of RCC and targeted therapy,and has potential for clinical application. |
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