Downregulation of METTL7B Inhibits Proliferation of Human Clear Cell Renal Cancer Cells In Vivo and In Vitro

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most aggressive urologic tumor, and its incidence and diagonosis have been continuously increasing. Identifying novel molecular biomarker for inhibiting the progression of ccRCC will facilitate developing new treatment strategies. Although methyltransferase-like 7B (METTL7B) was identified as a Golgi-associated methyltransferase, the function and mechanism of METTL7B in ccRCC development and progression has not been explored. METTL7B expression were significantly upregulated in ccRCC tissues (n = 60), which significantly associated with TNM classification, tumor size, lymph node metastasis, and poor prognosis for ccRCC patients. Functional studies showed downregulation of METTL7B inhibited cell proliferation, migration in vitro, and xenograft tumor formation in vivo. In addition, METTL7B knockdown promoted cell cycle arrest at G0/G1phase and induced cellular apoptosis. Taken together, downregulation of METTL7B inhibits ccRCC cell proliferation and tumorigenesis in vivo and in vitro. These findings provide a rationale for using METTL7B as a potential therapeutic target in ccRCC patients.

Keywords: METTL7B; ccRCC; methyltransferase; proliferation; tumorigenesis.

Figure 1

METTL7B is highly expressed in human ccRCC tissues. (A) The expressions of METTL7B in ccRCC tissues and normal renal tissues were analyzed based on TCGA database. Data are presented as the mean ± SD. (B) The expression of METTL7B mRNA were evaluated in 60 pairs of ccRCC tissues and normal renal tissues by qRT-PCR. Data are presented as the mean ± SD of three independent experiments. (C) The expressions of METTL7B in ccRCC and paired normal renal tissues were tested by IHC staining. (D) METTL7B staining scores in ccRCC tumors and the corresponding non-tumor tissues (n = 60). (E) Kaplan–Meier survival curves showed that METTL7B expression level was negatively correlated with prognosis prediction of ccRCC analyzed. *p < 0.05.

Figure 2

Knockdown of METTL7B inhibits the proliferation of ccRCC cells in vitro. 796-p and ACHN cells transfected with sh-METTL7B (shMETTL7B) or sh-Control (shNC) for 48 h. The transfection efficiency confirmed by qRT-PCR (A) and Western blot (B). (C) The living cell population was analyzed using Trypan Blue dye exclusion assay. (D) the cell viability were measured using MTT assay. Data are presented as the mean ± SD of three independent experiments. **p < 0.01, *p < 0.05 vs. shNC group.

Figure 3

Knockdown of METTL7B promotes ccRCC cell cycle arrest and cellular apoptosis. (A) Colony-formation assay of 796-p and ACHN cells transfected with sh-METTL7B (shMETTL7B) or sh-Control (shNC). After incubation for 14 days, colonies were stained and photographed. *P < 0.05, vs. shNC group. (B, C) 796-p and ACHN cells transfected with sh-METTL7B (shMETTL7B) or sh-Control (shNC) were collected and analyzed using flow cytometry for cell apoptosis and cell cycle. Data are presented as the mean ± SD of three independent experiments. **p < 0.01, *p < 0.05 vs. shNC group.

Figure 4

Knockdown of METTL7B inhibited G0/G1 realted protein expression. 796-p and ACHN cells transfected with sh-METTL7B (shMETTL7B) or sh-Control (shNC) for 48 h. The total protein was extracted and subjected to SDS-PAGE, followed by western blot analysis. Beta-actin was used as an internal control. Data are presented as means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs shCtrl group.

Figure 5

Knockdown of METTL7B inhibits ccRCC invasion and migration. 796-p and ACHN cells transfected with sh-METTL7B (shMETTL7B) or sh-Control (shNC) for 48 h. Invasion and migration assays were measured using Transwell champers. (A) Knockdown of METTL7B could inhibit cell migration in 796-p and ACHN cells. (B) Knockdown of METTL7B could inhibit cell invasion in 796-p and ACHN cells. Data represent mean ± S.D. of three independent experiments. Scale bar = 100 μm, *P < 0.05, **P < 0.01 vs shCtrl group.

Figure 6

Knockdown of METTL7B inhibited EMT in ccRCC cells. A significant reduction of vimentin and N-cadherin but increase E-cadherin was detected in shMETTL7B cells compared to shNC cells by Western blotting. Beta-actin was used as an internal control. Data are presented as means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs shCtrl group.

Figure 7

Knockdown of METTL7B inhibits xenograft tumor formation in vivo. (A) Representative xenograft tumors for indicated cells were shown. (B) METTL7B knockdown significantly reduced xenograft tumor growth in male nude mice by tumor volume examination. (C) Depletion of METTL7B significantly suppressed xenograft tumor weights. *P < 0.05 vs shCtrl group.

Figure 8

Knockdown of METTL7B inhibits the expression of Ki67 and PNCA in xenograft tumor tiusses. IHC staining were used to measured the expression of Ki67 and PNCA in xenograft tumor tiusses. The intensities of Ki-67 and PCNA were both decreased in xenograft tumors from shMETTL7B-transfected 796-p cells. *P < 0.05 vs shCtrl group.