The Study On Roles Of MicroRNA-200a-3p Target Regulated SPAG9 In The Biological Behavior Of Renal Cell Carcinoma

Objective:In this study, we aimed to detect the role of mi R-200a-3p and its regulation mechanism on cell proliferation, migration, invasion, apoptosis and cell cycle in renal cell carcinoma, to explore a new target in treating the renal cell carcinoma. Methods:1. The relative mi R-200a-3p expression levels in distinct kidney tumors, their adjacent normal tissues, HK-2, 786-O and ACHN were analyzed by real-time PCR. 2. We overexpressed mi R-200a-3p by transfecting the cells with mi R-200a-3p mimics versus the negative control(mi R-NC) in 786-O and ACHN cells. Then we observed the cell proliferation, colony formation, cell cycle, cell apoptosis, migration and invasion. To identify antitumorigenic roles of mi R-200a-3p in RCC in vivo, ACHN cells were subcutaneous injected into nude mice until tumor formation. mi R-200a-3p versus mi R-NC were infected into tumor respectively and then tumor growth were evaluated. 3. We used different mi RNA target-predicting bioinformatics such as Target Scan, Pictar, mi Randa to identify potential effector(s) of mi R-200a-3p. Then luciferase reporter assay, quantitative real time PCR and western boltting were used for targets validation. 4. quantitative real time PCR and immunohistochemistry were used to determine the expression of SPAG9 in distinct renal cell carcinoma, normal tissues, HK-2, 786-O and ACHN. 5. Finally, functional studies were performed when the target gene were knockdown using si RNA technique. RCC cells were transfected with a synthesized si RNA for SPAG9(si-SPAG9) versus the negative control(si-NC). Then we observed the cell proliferation, colony formation, cell cycle, cell apoptosis, migration and invasion. A rescue experiment was performed by co-transfecting with mi R-200a-3p mimics(versus the negative control) and pc DNA3.1-SPAG9(versus the negative control) into RCC cells. 6. Tumor phenotypes related genes protein(p21, cyclin D1, bcl-2, Bax, MMP9) levels were assessed by Western Blot in RCC cells overexpressing mi R-200a-3p(versus versus the negative control), and transfecting with si-SPAG9(versus the negative control) and co-transfecting with mi R-200a-3p mimics(versus the negative control) and pc DNA3.1-SPAG9(versus the negative control). Results:1. mi R-200a-3p was the most remarkably downregulated mi RNA in renal tumor tissues, 786-O and ACHN. q RT-PCR showed that mi R-200a-3p was downregulated in 53.2%(P<0.001), 73.1%(P<0.05) and 82.6%(P<0.01) in renal tumor tissues, 786-O and ACHN. 2. mi R-200a-3p expression was elevated up to 1067- and 889-fold in 786-O and ACHN cells, respectively. The cellular proliferation analyses showed that overexpression of mi R-200a-3p suppressed RCC cell proliferation(P<0.05), colony formation(P<0.05), caused cell cyclearrest at G0/G1 phase and decreased the S phase population(P<0.05), promoted cell apoptosis(P<0.01). Moreover, overexpression of mi R-200a-3p markedly impaired RCC cell migration and invasiveness compared to mi R-NC(P<0.05). In vivo, there was a more significant decrease in tumor size upon transfecting of mi R-200a-3p(P<0.01). 3. SPAG9 was validated as a direct target for mi R-200a-3p and mi R-200a-3p suppressed the target expression on both m RNA and protein levels in vitro and in vivo. 4. SPAG9 expression was elevated up to 4-, 4.21-and 6.25-fold in 786-O and ACHN cells, respectively. Immunohistochemistry show the coincident results were obtained. 5. Knockdown of SPAG9 in RCC cells attenuated cell growth(P<0.05), colony formation(P<0.05), induced G0/G1 cell-cycle arrest(P<0.05), promoted cell apoptosis(P<0.01) and suppressed cell migration(P<0.05) and invasion(P<0.05), similarly to the phenotypic alterations upon mi R-200a-3p overexpression. Importantly, the roles of mi R-200a-3p was effectively reversed by pc DNA3.1-SPAG9 in 786-O. 6. overexpression of mi R-200a-3p and Suppression of SPAG9 resulted in an attenuation of cyclin D1, bcl-2 and MMP-9. overexpression of mi R-200a-3p and Suppression of SPAG9 resulted in a raise of p21 and Bax. In addition, the roles of mi R-200a-3p was effectively reversed by pc DNA3.1-SPAG9. Conclusion:Mi R-200a-3p can be used as a potential marker in renal cell carcinoma cancer diagnosis. Mi R-200a-3p is an important tumor suppressive gene, it suppresses tumor proliferation, migration, invasion and induces apoptosis in renal cell carcinoma. Mi R-200a-3p exerts an antineoplastic effect on renal cell carcinoma by targeting SPAG9.