LncRNA CASC19 promotes the growth and glycolysis of colorectal cancer cells and tumor metastasis in mice

Abstract

It has been reported that lncRNA CASC19 is abnormally highly expressed in colorectal cancer (CRC) progression, suggesting that it may regulate the occurrence and metastasis of CRC, but its specific mechanism is still unclear. To further explore the effect of CASC19 on CRC, we overexpressed or knocked down CASC19 in HR4838 cells. The results of Transwell invasion assay and cell clonogenic assay showed that CASC19 promoted cell invasion and proliferation. Flow cytometry results showed that CASC19 inhibited cell apoptosis. In addition, by detecting glucose uptake, lactate content and ATP production, it was found that CASC19 promoted glycolysis, while CASC19 silencing had the opposite effect. Interestingly, small nuclear ribonucleoprotein polypeptide A (SNRPA) is an RNA binding protein of CASC19. Overexpression of SNRPA promoted tumor cell invasion, proliferation, glycolysis, and inhibits apoptosis, while SNRPA silencing has the opposite effect. Moreover, SNRPA overexpression reversed the inhibitory effect of CASC19-sh on invasion, proliferation and glycolysis of HR4838 cells and the promoting effect on apoptosis, which was mediated by activating the Wnt/β-catenin pathway. In the subcutaneous transplantation tumor model of BALB/c nude mice, we observed that the tumor growth of CASC19 knockdown mice was slower, and the tumor weight and volume were smaller, which was related to the low expression of CASC19 and SNRPA. In conclusion, our results showed that CASC19 promoted the growth and glycolysis of CRC cells and tumor metastasis in mice by upregulating SNRPA, which may provide a new molecular marker for the diagnosis and treatment of CRC.

Keywords: Colorectal cancer; Glycolysis; LncRNA CASC19; SNRPA.

Fig. 1

CASC19 expression in CRC tissues and cell lines. (A) Online biological database (http://gepia.cancer-pku.cn/index.html). (B) RT-qPCR was used to detect the expression of CASC19 in 30 pairs of adjacent and cancerous tissues from CRC patients. (C) RT-qPCR was used to detect the expression of CASC19 in CRC cell lines SW1463, SW837 and HR8348, and human normal colonic epithelial cell line FHC. (D) Survival analysis of CRC patients with different expression levels of CASC19. N = 5. *P < 0.05

Fig. 2

Effect of CASC19 on HR8348 cells. The HR8348 cells were transfected with CASC19-OE or CASC19-sh. (A) The transfection efficiency of CASC19-OE or CASC19-sh. (B) Cell migration (Transwell assay). (C) Cell proliferation (Plate clonogenic assay). (D) Cell apoptosis. (E) Glucose uptake. (F) Lactic acid production. (G) ATP level. (H&I&J) The protein expression of glycolytic molecules HK2 and LDHA. N = 5. *P < 0.05

Fig. 3

SNRPA is an RNA binding protein of CASC19. (A) Online biological database (http://starbase.sysu.edu.cn/). (B&C) The binding relationship of CASC19 and SNRPA was validated by RNA IP assay and RNA pull down. (D) The expression of SNRPA in adjacent and CRC tissues was detected by immunohistochemistry. (E) The protein expression of SNRPA in CRC cell lines and human normal colonic epithelial cell line FHC. (F) Correlation analysis of the expression of CASC19 and SNRPA in 30 CRC tissues. N = 5. *P < 0.05

Fig. 4

Effect of SNRPA on HR8348 cells. The HR8348 cells were transfected with SNRPA-OE or SNRPA-sh. (A) The transfection efficiency of CASC19-OE or CASC19-sh. (B) Cell migration (Transwell assay). (C) Cell proliferation (Plate clonogenic assay). (D) Cell apoptosis. (E) Glucose uptake. (F) Lactic acid production. (G) ATP level. (H&I&J) The protein expression of glycolytic molecules HK2 and LDHA. N = 5. *P < 0.05

Fig. 5

CASC19 affects HR8348 cell growth and glycolysis through SNRPA. The HR8348 cells were transfected with CASC19-sh alone or together with SNRPA-OE. (A&B) The protein expression of SNRPA. (A&C) The protein expression of β-catenin and c-Myc. (D) Cell migration (Transwell assay). (E) Cell proliferation (Plate clonogenic assay). (F) Cell apoptosis. (G) Glucose uptake. H. Lactic acid production. (I) ATP level. (J&K&L) The protein expression of glycolytic molecules HK2 and LDHA. N = 5. *P < 0.05

Fig. 6

The effect of CASC19 on tumor formation in vivo. (A) Representative images of tumor tissues at day 35. (B) Growth curve of transplanted tumor on days 7, 14, 21, 28 and 35. (C) The weight of transplanted tumor at day 35. (D) The expressions of CASC19. (E) Immunohistochemical images of SNRPA and Ki67. (F) The protein expression of glycolytic molecules HK2 and LDHA. (G) The protein expression of β-catenin and c-Myc. (H) Pyruvate content. N = 5. *P < 0.05