LncRNA HCG18 Promotes Clear Cell Renal Cell Carcinoma Progression by Targeting miR-152-3p to Upregulate RAB14

Abstract

Background: Long noncoding RNAs (lncRNAs) have been regarded as crucial regulators in many cancers, including clear cell renal cell carcinoma (ccRCC). This research aimed to explore the biological role and molecular mechanism of lncRNA HCG18 in ccRCC.

Materials and methods: The expression levels of HCG18, miR-152-3p and RAB14 were examined by RT-qPCR. Cell viability, migration and invasion were examined by CCK-8 and transwell assays. Luciferase reporter and RIP assays were adopted to verify the interaction between miR-152-3p and HCG18 or RAB14.

Results: It was found that HCG18 expression was highly expressed in ccRCC tissues and cells, and patients with high expression of HCG18 had a short overall survival time. Moreover, HCG18 depletion attenuated ccRCC cell viability, migration and invasion. In addition, miR-152-3p was confirmed as a downstream target of HCG18 and was inversely regulated by HCG18, and RAB14 was a target of miR-152-3p. Functional assays demonstrated that miR-152-3p silencing or RAB14 addition abolished the inhibitory effects of HCG18 knockdown on ccRCC progression.

Conclusion: The results of the present study indicated that HCG18 accelerated the development and progression of ccRCC by upregulating RAB14 via sponging miR-152-3p, suggesting a potential therapeutic target for patients with ccRCC.

Keywords: HCG18; RAB14; clear cell renal cell carcinoma; miR-152-3p.

Figure 1

HCG18 is overexpressed in ccRCC and knockdown of HCG18 represses the tumorigenesis of ccRCC. (A and B) RT-qPCR assay was applied to detect the expression of HCG18 in ccRCC tissues (n=32) and cell lines. (C) The ROC curve of serum HCG18 for diagnosing ccRCC. (D) Kaplan–Meier method was used to assess the association between HCG18 expression and overall survival rate in ccRCC patients. (E) RT-qPCR assay was applied to detect the transfection efficiency of shNC and shHCG18 in Caki-1 and 786-O cells. (F) CCK-8 assay was used to measure cell proliferation in Caki-1 and 786-O cells after knocking down HCG18. (G and H) Transwell assay was performed to assess cell migration and invasion in Caki-1 and 786-O cells transfected with shHCG18. (I and J) In vivo experiments the tumor growth rate in mice of ccRCC cells transfected with shHCG18. *p < 0.05.

Figure 2

HCG18 is a molecular sponge for miR-152-3p in ccRCC. (A) Binding sequences between HCG18 and miR-152-3p were predicted by starBase website. (B) Luciferase reporter assay was adopted to verify the binding ability between HCG18 and miR-152-3p in 293T cells. (C and D) RIP assay was used to analyze enrichment of HCG18 and miR-152-3p in Caki-1 and 786-O cells of anti-Ago2 group compared with anti-IgG group. (E) RT-qPCR assay was performed to measure the expression of miR-152-3p in ccRCC tissues. (F) Pearson’s correlation analysis showed the correlation between HCG18 and miR-152-3p in ccRCC tissues. (G) RT-qPCR analysis was applied to detect miR-152-3p expression in Caki-1 and 786-O cells transfected with shHCG18. *p < 0.05.

Figure 3

HCG18 sponges miR-152-3p and positively modulates RAB14 expression in ccRCC. (A) Binding sequences between miR-152-3p and RAB14 were predicted by starBase website. (B) RT-qPCR assay showed the expression of RAB14 in ccRCC tissues. (C) RT-qPCR assay was applied to assess the expression of RAB14 in Caki-1 and 786-O cells transfected with shHCG18. (D and E) Pearson’s correlation analysis showed the correlation between RAB14 and miR-152-3p or HCG18 in ccRCC tissues. (F) RT-qPCR assay was applied to assess the expression of miR-152-3p in Caki-1 and 786-O cells transfected with miR-152-3p inhibitor. (G) RT-qPCR assay showed RAB14 expression in Caki-1 and 786-O cells transfected with shNC, shHCG18, and shHCG18+miR-152-3p inhibitor. *p < 0.05.

Figure 4

HCG18 plays an oncogenic role in ccRCC by regulating the miR-152-3p/RAB14 axis. (A–C) CCK-8 and transwell assays were used to test the proliferation, migration and invasion abilities in Caki-1 and 786-O cells transfected with shNC, shHCG18, and shHCG18+miR-152-3p inhibitor. (D) RT-qPCR assay showed the expression of RAB14 in Caki-1 and 786-O cells transfected with pcDNA3.1/RAB14. (E–G) CCK-8 and transwell assays were used to measure the proliferation, migration and invasion abilities in Caki-1 and 786-O cells transfected with shNC, shHCG18, and shHCG18+pcDNA3.1/RAB14. *p < 0.05.