Long Non-coding RNA SNHG12, a New Therapeutic Target, Regulates miR-199a-5p/Klotho to Promote the Growth and Metastasis of Intrahepatic Cholangiocarcinoma Cells

Abstract

Background: Small nucleolar RNA host gene 12 (SNHG12) is a newly identified long non-coding RNA (lncRNA) whose involvements have been explored in several cancers. Our study aimed to explore the functions of SNHG12 on intrahepatic cholangiocarcinoma (ICC) progression and its interaction with miR-199a-5p and Klotho. Methods: RT-PCR was performed to examine the expressions of SNHG12, miR-199a-5p and Klotho in ICC cells. Cell counting kit-8 (CCK-8), colony formation assays and transwell assays were applied to analyze the proliferation, migration and invasion of ICC cells. Luciferase assays, RIP assays and RNA pull-down assays were carried out to demonstrate the direct binding relationships among SNHG12, miR-199a-5p and Klotho. The xenograft nude models were applied to test the effects of SNHG12 on ICC tumor growth. Results: The expression of SNHG12 and Klotho was distinctly increased in ICC cells, while miR-199a-5p expressions were decreased. Functionally, the silence of SNHG12 inhibited the proliferation and metastasis of ICC cells, while miR-199a-5p overexpression exhibited an opposite result. Mechanistically, Knockdown of SNHG12 significantly suppressed the expressions of miR-199a-5p by sponging it, and then increased Klotho expression. The final in vivo experiments suggested that the silence of SNHG12 distinctly inhibited tumor growth. Conclusion: Our findings indicated that SNHG12 inhibited cell proliferation and metastasis process of ICC cells through modulating the miR-199a-5p/Klotho axis and it is expected to become a potential therapeutic target for ICC.

Keywords: Klotho; intrahepatic cholangiocarcinoma; lncRNA SNHG12; metastasis; miR-199a-5p.

Figure 1

Increased SNHG12 expression and its oncogenic roles in ICC cells. (A) Higher levels of SNHG12 were observed in ICC specimens than normal specimens by amazing TCGA datasets. (B) RT-PCR determined the expressions of SNHG12 in ICC cells and BEC cells. (C) SNHG12 expression was examined in TFK-1 and CCLP1 cells transfected with sh-NC, sh-SNHG12-1 or sh-SNHG12-2. (D) Cell proliferation analysis. TFK-1 and CCLP1 cells were detected for 24, 48 and 72 h after transfection. (E) Proliferation of TFK-1 and CCLP1 cells transfected with sh-SNHG12-1 or sh-SNHG12-2 as determined by colony formation assays. (F,G) Transwell assays were used to detect cellular invasion and migration in SNHG12-knockdown TFK-1 and CCLP1 cells. *p < 0.05, **p < 0.01.

Figure 2

Overexpression of SNHG12 promoted the proliferation and metastasis of TFK-1 and CCLP1 cells. (A) RT-PCR assay was used to detect SNHG12 level in SNHG12 overexpression plasmids transfected TFK-1 and CCLP1 cells. (B,C) Overexpression of SNHG12 distinctly promoted the proliferation of TFK-1 and CCLP1 cells. (D,E) Transwell assay indicated the migrated and invaded cell number in TFK-1 and CCLP1 cells cells. **p < 0.01.

Figure 3

miR-199a-5p overexpression promoted the proliferation and metastasis of TFK-1 and CCLP1 cells. (A) Relative SNHG12 levels in nuclear and cytosolic fractions of TFK-1 and CCLP1 cells. (B) Bioinformatics analysis to select relevant miRNAs. (C) The histogram of miR-199a-5p expressions in four ICC cells and BEC cells. (D) RT-PCR examined the expressions of miRNA-199a-5p in TFK-1 and CCLP1 cells transfected with miR-NC or miR-199a-5p mimics. (E) CCK-8 assays revealed that OD values of TFK-1 and CCLP1 cells were distinctly declined when transfected with miR-199a-5p mimics. (F) Cell clone number was distinctly decreased when transfected with miR-199a-5p mimics. (G,H) Transwell assays of migration and invasion of TFK-1 and CCLP1 cells after treatment with miR-199a-5p mimics or miR-NC. **p < 0.01.

Figure 4

Knockdown of miR-199a-5p reversed the distinct suppression of SNHG12 knockdown on the ICC progression. (A) RIP assay to confirm the coexistence of SNHG12 and miR-199a-5p in TFK-1 and CCLP1 cells. (B) Schematic outlining the predicted binding sites between miR-199a-5p and SNHG12. (C) miR-199a-5p mimics markedly reduced luciferase activity in SNHG12-wild not in SNHG12-Mut in TFK-1 and CCLP1 cells. (D–G) Rescue experiments were used to determine the efficacy of miR-199a-5p knockdown on SNHG12 down-regulation via CCK-8, colony formation assay and transwell assays. **p < 0.01.

Figure 5

Klotho is a target gene of miR-199a-5p. (A) Four ICC cells exhibited an increased expression of Klotho compare with BEC cells using RT-PCR. (B) RT-PCR for the expression of Klotho in TFK-1 and CCLP1 cells transfected with sh-NC, sh-SNHG12-1, pcDNA3.1 or pcDNA3.1/SNHG12. (C) RIP assays to verify the coexistence of three molecules. (D) RNA pull-down assay to test enrichment of SNHG12 and Klotho pulled down by miR-199a-5p. (E) Schematic representation of the predicted target site for miR-199a-5p in SNHG12. (F) Luciferase reporter assay in TFK-1 and CCLP1 cells, co-transfected with the reporter plasmid and the indicated miRs. **p < 0.01.

Figure 6

The upregulation of Klotho reversed functional depletion caused by SNHG12 knockdown. (A) CCK-8 assays were applied to determine the proliferation of TFK-1 and CCLP1 cells cotransfected with sh-NC, sh-SNHG12-1 or sh-SNHG12-1 +pcDNA3.1/Klotho. (B) Colony formation assays were used to detect the proliferation of TFK-1 and CCLP1 cells cotransfected with sh-NC, sh-SNHG12-1 or sh-SNHG12-1 +pcDNA3.1/Klotho. (C–E) Transwell assays were applied to determine the migration and invasion of TFK-1 and CCLP1 cells cotransfected with sh-NC, sh-SNHG12-1 or sh-SNHG12-1 +pcDNA3.1/Klotho. (E) Tumors derived from mice in two different groups were presented. (F,G) Tumor volume and weight in the xenograft mice from the SNHG12-knockdown group and the control group. **p < 0.01.