Testis-specific hnRNP is expressed in colorectal cancer cells and accelerates cell growth mediating ZDHHC11 mRNA stabilization

Abstract

Various heterogeneous nuclear ribonucleoproteins (hnRNPs) have been reported to be associated with cancer cell growth. However, it remains unclear whether hnRNP G-T, which is specifically expressed in the testis, is expressed in tumor cells, and whether hnRNP G-T expressed in colorectal cancer (CRC) cells is associated with tumor progression. We herein report that hnRNP G-T promoted cancer cell growth and stabilized mRNA of ZDHHC11 in CRC. The cell growth was inhibited by transfection of siRNA of hnRNP G-T in cancer cells, but not in non-cancerous epithelial cells. The tumor promotive effect of hnRNP G-T was confirmed in an HCT116 transplanted mouse model. RT-PCR and western blotting indicated the augmentation of hnRNP G-T in CRC in comparison to non-cancerous cells. The downregulation of hnRNP G-T inhibited cancer cell growth and promoted apoptosis in CRC. A transcriptome analysis combined with immunoprecipitation revealed that hnRNP G-T stabilized 174 mRNAs, including ZDHHC11 mRNA. The cell growth was also suppressed by the transfection of siRNA of ZDHHC11 and the mRNA and the protein expression were decreased by the transfection of siRNA of hnRNP G-T. These results suggested that hnRNP G-T promotes the cell growth of CRC by regulating the mRNA of ZDHHC11. Therefore, hnRNP G-T will be highlighted as an effective therapeutic target with less adverse effects in CRC therapy.

Keywords: ATM; ATR; ZDHHC11; colorectal cancer; hnRNP G-T.

FIGURE 1

hnRNP G‐T promotes cell growth in cancer cells, but not non‐cancerous cells. (A) An SRB assay showed significant cell growth in cancer cells (HCT116, SW480, and SUIT‐2), but not non‐cancerous cells (HCEC‐1CT) (n = 5). (B, C) RT‐PCR (B) and western blotting (C) showed the knockdown efficacy of HCT116, SW480, Suit‐2, and HCEC‐1CT cells transfected with siRNA of hnRNP G‐T #1 and #2 at 48 h (n = 3). (D) The size of the HCT116 xenograft tumor was measured daily (n = 5)

FIGURE 2

hnRNP G‐T is overexpressed in colorectal cancer (CRC). (A, B) RT‐PCR (A) and western blots (B) showed that hnRNP G‐T was augmented in HCT116, SW480, and SUIT‐2 cells in comparison to HCEC1CT cells (n = 3). (C) Western blotting showed that hnRNP G‐T was highly induced in the AOM‐DSS carcinogenesis model (n = 7–8). (D) Immunohistochemistry showed cancer cells positive for hnRNP G‐T in the AOM/DSS mouse model. (E) RT‐PCR showed the augmentation of hnRNP G‐T in CRC tissues in comparison to the normal epithelium (n = 18). (F) Immunohistochemistry showed cancer cells positive for hnRNP G‐T in human CRC tissue

FIGURE 3

hnRNP G‐T promotes cell growth and inhibits apoptosis in colorectal cancer cells. (A) Immunocytochemistry showed that ki‐67 positive cells were reduced by the downregulation of hnRNP G‐T. (B) TUNEL staining showed the augmentation of TUNEL‐positive cells by the downregulation of hnRNP G‐T. (C) Western blots showed the induction of cleaved PARP and caspase‐3 in hnRNP G‐T‐knockdown cells

FIGURE 4

The expression of hnRNP G‐T is correlated with the expression of ZDHHC11 and ZDHHC11 was highly expressed in colorectal cancer. (A) An SRB assay showed the cell growth of HCT116 cells by the downregulation of the top five mRNAs. The downregulation of ZDHHC11 was associated with the strong inhibition of cell growth in HCT116 cells (n = 5). (B) The downregulation of ZDHHC11 showed no suppression of cell growth in HCEC‐1CT cells (n = 5). (C, D) RT‐PCR (C) and western blotting (D) showed the knockdown efficacy of HCT116 and HCEC‐1CT cells transfected with siRNA of ZDHHC11 at 24 h (n = 3). (E) RNA‐immunoprecipitation combined with RT‐PCR confirmed the direct binding of hnRNP G‐T and ZDHHC11 mRNA (n = 3) (C). F, G RT‐PCR (F) and western blots (G) showed that the ZDHHC11 mRNA and protein expression were decreased in hnRNP G‐T‐downregulated HCT116 cells (n = 3). (H) The actinomycin D experiment showed that hnRNP G‐T stabilized the ZDHHC11 mRNA in HCT116 cells (n = 3). (I) The growth inhibition rate calculated by the SRB assay showed that growth suppression induced by hnRNP G‐T downregulation recovered with the overexpression of ZDHHC11. The growth inhibition rate (%) = [1 − (OD510 nm of each of the samples on day 3––OD510 nm of each of the samples on day 1)/(OD510 nm of Control on day 3––OD510 nm of Control on day 1)] × 100

FIGURE 5

ZDHHC11 is highly induced in colorectal cancer (CRC). (A, B) RT‐PCR (A) and western blots (B) showed that the ZDHHC11 mRNA and protein expression were decreased in hnRNP G‐T‐downregulated HCTEC‐1CT cells (n = 3). (C) RT‐PCR showed the augmentation of ZDHHC11 in CRC tissues in comparison to the normal epithelium (n = 18). (D) The mRNA expression of ZDHHC11 tends to correlate with the expression of hnRNP G‐T (n = 18)

FIGURE 6

ZDHHC11‐promoted cell growth and inhibited apoptosis in colorectal cancer. (A) Immunocytochemistry showed that ki‐67‐positive cells were reduced by the downregulation of hnRNP G‐T or ZDHHC11. (B) TUNEL staining showed that TUNEL‐positive cells were augmented by the downregulation of hnRNP G‐T or ZDHHC11. (C) Western blots showed the induction of cleaved PARP and cleaved caspase‐3 in hnRNP G‐T or ZDHHC11‐knockdown cells (n = 3)

FIGURE 7

hnRNP G‐T‐ZDHHC11‐promoted cell growth by activating the ATM/ATR pathway and ERK1/2 in colorectal cancer. (A, B) Western blots showed that deactivation of phosphorylated ATM and ATR (A) in hnRNP G‐T or ZDHHC11‐knockdown cells (n = 3) and the deactivation of phosphorylated ERK (B) in hnRNP G‐T or ZDHHC11‐knockdown cells (n = 3)