POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway

Abstract

Aim: The most common type of cancer that leads to death worldwide is lung cancer. Despite significant surgery and chemotherapy improvements, lung cancer patient's survival rate is still poor. The RNA polymerase I subunit D (POLR1D) gene can induce various cancers. A current study reported that POLR1D plays a vital role in cancer prognosis. However, its biological function in the development of lung cancer remains unclear.

Methods: Reverse transcription PCR (RT-PCR) measured the relative POLR1D protein expression level in lung cancer cell lines. Lung cancer cell proliferation, migration, and invasion were analyzed by performing cell counting kit-8 (CCK-8), and transwell. The phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/AKT) signaling pathway-related protein expressions were examined by Western blotting assay.

Results: POLR1D protein expression was elevated in lung cancer. Lung cancer cell loss-of-function tests showed that POLR1D silencing could attenuate cell viability both in SK-MES-1 and in H2170 cells. Furthermore, silencing POLR1D inhibited SK-MES-1 and H2170 cells proliferation, migration, and invasion. Moreover, SK-MES-1 and H2170 cells' migration and invasion capacity were potentially suppressed by the knockdown of POLR1D. The progression of multiple cancers has been implicated in the PI3K/AKT pathway. Here, we observed that POLR1D silencing suppressed lung cancer progression by inhibition of the PI3K-Akt pathway.

Conclusions: The study speculated that POLR1D might provide a new potential therapeutic possibility for treating lung cancer patients via targeting PI3K/AKT.

Keywords: AKT; Cell proliferation; Lung cancer; PI3K; POLR1D.

Fig. 1

Expression of POLR1D in lung cancer cells. a The mRNA expression of POLR1D in human embryonic lung fibroblasts (HFL1) and five-lung cancer cell lines (H2170, SK-MES-1, H226, PC-9, H1975) by RT‑qPCR. b The representative bands of POLR1D protein in six-lung cancer cell lines. c Quantification of POLR1D protein expression determined by Western blot. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 vs. HFL1 group

Fig. 2

Inhibition of POLR1D in lung cancer cells by siRNA suppresses cell proliferation. a Two siRNAs targeting POLR1D mRNA (POLR1D-si1 and POLR1D-si2) were introduced into SK-MES-1 and H2170 cells for transient knockdown of POLR1D. The levels of POLR1D protein were detected by Western blotting in SK-MES-1 and H2170 cells. Expression was normalized against endogenous GAPDH levels. b, c Quantification of POLR1D protein levels SK-MES-1 and H2170 cells. d The levels of POLR1D protein were detected by Western blotting in H2170 cells. e, f The cell growth rate was suppressed by POLR1D knockdown in SK-MES-1 and H2170 cells detected by CCK-8 assay 72 h after transfection. The information shown is the mean of three separate experiments (mean ± SD). ***P < 0.001 vs. Control group

Fig. 3

Downregulation of POLR1D inhibits the migration of lung cancer cells. The effects of POLR1D deficiency on cell migration were investigated by Transwell assay in (a) SK-MES-1 and (b) H2170 cells (scale bar: 50 μm). The relative migrated cells were quantified in (c) SK-MES-1 and (d) H2170 cells. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001 vs. PBS group

Fig. 4

Downregulation of POLR1D inhibits the invasion of lung cancer cells. Knockdown of POLR1D on cell invasion was investigated by Transwell assay in (a) SK-MES-1 and (b) H2170 cells (scale bar: 50 μm). The relative invaded cells were quantified in (c) SK-MES-1 and (d) H2170 cells. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001 vs. PBS group

Fig. 5

Effects of POLR1D silencing on proteins of EMT and PI3K-AKT pathway. a Representative gel blots depicting levels of cleaved caspase-3 (normalized to GAPDH), E-cadherin (normalized to GAPDH), N-cadherin (normalized to GAPDH), phosphorylated PI3K (p-PI3K) and total PI3K (normalized to total PI3K), phosphorylated AKT (p-AKT) and total AKT (normalized to total AKT). These protein blots were quantified in (b) SK-MES-1 cells and c H2170 cells. Data represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001 vs. PBS group