LncRNA SNHG7 sponges miR-216b to promote proliferation and liver metastasis of colorectal cancer through upregulating GALNT1

Abstract

Accumulating evidence suggests long noncoding RNAs (lncRNAs) play an important role in cancer progression. However, the function of lncRNA SNHG7 in colorectal cancer (CRC) remains unclear. In this study, SNHG7 expression was significantly upregulated in CRC tissues, especially in aggressive cases. In accordance, high level of SNHG7 was observed in CRC cell lines compared to normal colon cells. Furthermore, SNHG7 overexpression promoted the proliferation, migration, and invasion of CRC cell lines, while SNHG7 depletion inhibited invasion and cell viability in vitro. Mechanistically, knockdown of SNHG7 inhibited GALNT1 and EMT markers (E-cadherin and Vimentin). Importantly, SNHG7 directly interacted with miR-216b and downregulation of miR-216b reversed efficiently the suppression of GALNT1 induced by SNHG7 siRNA. Moreover, overexpression of SNHG7 significantly enhanced the tumorigenesis and liver metastasis of SW480 cells in vivo. SNHG7 positively regulated GALNT1 level through sponging miR-216b, and played an oncogenic role in CRC progression. Together, our study elucidated the role of SNHG7 as an miRNA sponge in CRC, and shed new light on lncRNA-directed diagnostics and therapeutics in CRC.

Fig. 1. The differential expression of SNHG7 in CRC tissues and cell lines and subcellular location.

a The differential expression of SNHG7 in CRC samples (n = 48) and adjacent normal colon tissues (n = 48) was shown. b The differential expression of SNHG7 in CRC patients with liver metastasis (n = 23) and without metastasis (n = 25) was analyzed. c Kaplan−Meier analyses of the correlations between SNHG7 level and overall survival of 48 patients with CRC (p = 0.033; log-rank test). The median of the dataset is selected as the cutoff point between “High SNHG7” and “Low SNHG7”. d The differential levels of SNHG7 in CRC cell lines and FHC cell were examined. e Cellular localization of SNHG7 in CRC cells was shown. GAPDH and U6 served as a cytoplasmic and nuclear localization marker, respectively. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05

Fig. 2. Knockdown of SNHG7 inhibited cell proliferation, migration and invasion, and promoted apoptosis of CRC cells in vitro.

a The level of SNHG7 transfection with siSNHG7 or siControl was analyzed by qRT-PCR. b Growth curves of SW620 and LOVO cells after transfection with siSNHG7 or siControl were determined via CCK-8 assays. c Colony formation assays showed that knockdown of SNHG7 inhibited CRC cell proliferation. d Suppression of SNHG7 expression attenuated the proliferation of CRC cells by EdU assay. Scale bars = 50 μm. e The levels of cleaved PARP, cleaved caspase-7, and cleaved caspase-3 following SNHG7 silenced in CRC cells were determined via western blot. f Flow cytometry assay showed that silencing of SNHG7 increased the rate of apoptosis in CRC cells. g Flow cytometry assay showed that siSNHG7 resulted in S arrest in CRC cells. The cell cycle distribution was exhibited. h siSNHG7 resulted in a slower closing of scratch wound by wound-healing assay. Scale bars = 50 μm. i Transwell invasion assay was measured and the results were expressed as the number of invaded cells per field. Scale bars = 20 μm. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05

Fig. 3. Overexpression of SNHG7 promoted CRC cells proliferation, migration and invasion, and inhibited apoptosis in vitro.

a The expression of SNHG7 in CRC cell lines transfection with pcDNA/SNHG7 or pcDNA/Control was analyzed by qRT-PCR. b The proliferative ability of SW480 and HCT-116 cells transfected with pcDNA/SNHG7 or pcDNA/Control was performed by CCK-8 assay. Colony formation (c) and EdU assay (d) were performed in SW480 and HCT-116 cells transfected with pcDNA/SNHG7 or pcDNA/Control. Scale bars = 50 μm. e The expression of cleaved PARP, cleaved caspase-7, and cleaved caspase-3 was analyzed by western blot. f The apoptotic rates of cells were detected by flow cytometry. g The cell cycle progression of SW480 and HCT-116 cells was evaluated after transfection with pcDNA/SNHG7 or pcDNA/Control using Flow cytometry. h Wound scratch assay in pcDNA/SNHG7 or pcDNA/Control transfected SW480 and HCT-116 cells was shown. Scale bars = 50 μm. i Transwell invasion assay in pcDNA/SNHG7 or pcDNA/Control transfected SW480 and HCT-116 cells was shown. Scale bars = 20 μm. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05

Fig. 4. SNHG7 acted as a ceRNA by sponging miR-216b and regulated GALNT1 expression indirectly.

a The differential expression of GALNT1 in CRC tissues and adjacent normal colon tissues was examined. Expressional levels of GALNT1 in CRC cell lines and FHC cell. b Pearson’s correlation curve identified positive correlation between SNHG7 and GALNT1 in CRC tissues. c The expressional levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with siSNHG7 were evaluated by qRT-PCR and western blot. d The levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with pcDNA/SNHG7 were evaluated by qRT-PCR and western blot. e The predicted binding sites of miR-216b to the SNHG7 sequence were shown. f The differential expression of miR-216b in CRC tissues and adjacent normal colon tissues was analyzed. g The levels of miR-216b was investigated in CRC cell lines and human normal colon cell line by qRT-PCR. h Pearson’s correlation curve revealed the negative relevance between SNHG7 and miR-216b expression. i RNA-IP was performed in SW620 and SW480 cells transfected with NC mimic and miR-216b mimic. SNHG7 expression was detected using qRT-PCR. j Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing SNHG7-Wt or SNHG7-Mut transcript were analyzed. k The predicted binding sites of miR-216b to the GALNT1 sequence were shown. l Pearson’s correlation curve revealed the negative relevance between GALNT1 and miR-216b levels. m Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing GALLNT1-Wt or GALNT1-Mut transcript were performed. n The levels of GALNT1 transfected with miR-216b inhibitor or siSNHG7 in SW620 cell were analyzed by qRT-PCR and western blot. o The levels of GALNT1 transfected with miR-216b mimic or pcDNA/SNHG7 in SW480 cells were analyzed by qRT-PCR and western blot. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05

Fig. 5. MiR-216b reversed the promoting effect of SNHG7 on the growth and metastasis of CRC cells.

a and b Functional assays identified the phenomenon of SNHG7 and GALNT1 regulated each other to compete for the binding of miR-216b by EdU assay, colony formation, transwell invasion assay in SW620 and SW480 cell lines. Scale bars = 50 μm (EDU) and 20 μm (transwell invasion). c The levels of E-cadherin, Vimentin in SW620 cells cotransfected siSNHG7 with miR-216b inhibitor and siGALNT1 was analyzed by western blot. d The levels of E-cadherin, Vimentin in SW480 cells cotransfected pcDNA/SNHG7 with miR-216b mimic and GALNT1 were examined by western blot. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05

Fig. 6. Ectopic expression of SNHG7 promoted CRC growth and liver metastasis in vivo.

a Tumors collected from mice were exhibited. b Tumor weight of mouse was measured. c Tumor volume curve of mouse upon LV-SNHG7 or LV-NC treatment was analyzed. d Immunohistochemical staining of Ki-67 was used to assess proliferation, and apoptosis was detected using Tunnel kit. e Representative sections of liver tumors (n = 6 in each group) were shown. The blue arrows indicated the tumor nodules. Overexpression of SNHG7 significantly increased the metastasis of SW480 cells to the liver. f The liver and spleen sections were shown via HE staining (scale bar = 200 μm). g The number of metastasis in the liver was determined. h As assessed by qRT-PCR, the levels of GALNT1 and Vimentin mRNA were increased and the level of E-cadherin was decreased, respectively, in liver metastasis originating from mice in the SW480-LV-SNHG7 group compared with that in the SW480-LV-NC group. The error bars in all graphs represented SD, and each experiment was repeated three times. *p < 0.05