Long noncoding RNA SNHG5 promotes 5-fluorouracil resistance in colorectal cancer by regulating miR-26b/p-glycoprotein axis

Figure 1 Upregulated SNHG5 expression is associated with 5-fluorouracil resistance in colorectal cancer. A and B: The expression levels of SNHG5 were assessed in collected samples [normal tissues = 16, colorectal cancer (CRC) tissues = 36] and The Cancer Genome Atlas database; C: The expression of SNHG5 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC cell lines (LOVO, HT-29, HCT-8, and SW116) compared to normal colorectal epithelial cell line; D: The expression levels of SNHG5 were examined in the 5-fluorouracil (5-Fu)-sensitive and 5-Fu-resistant CRC groups; E and F: SNHG5 expression levels in HT-29 and HT-29/5-Fu or LOVO and LOVO/5-Fu cells were assessed by qRT-PCR. ^bP < 0.01. 5-Fu: 5-fluorouracil; CRC: Colorectal cancer.

Figure 2 SNHG5 overexpression enhances 5-fluorouracil resistance in colorectal cancer cells. A: The expression of SNHG5 was assessed in LOVO and HT-29 cells which were transfected with SNHG5 and scrambled control lentivirus; B and C: The viability of LOVO and HT-29 cells treated with different concentrations of 5-fluorouracil (5-Fu) was evaluated, and the IC50 of 5-Fu in colorectal cancer cells was calculated; D: The apoptosis rate of stable cells treated with 5-Fu was analyzed via annexin-V fluorescein 5-isothiocyanate/propidium iodide staining assays (left) and quantified (right); E: Western blot analysis was performed to assess the expression of apoptosis markers (Bax and Bcl-2), with β-actin serving as an internal control (left), and densitometric analysis of the Western blot signals is shown (right). ^bP < 0.01. Con: Control; 5-Fu: 5-fluorouracil.

Figure 3 SNHG5 knockdown suppresses 5-fluorouracil resistance in 5-fluorouracil-resistant colorectal cancer cells. A: The expression of SNHG5 was assessed in 5-fluorouracil (5-Fu)-resistant colorectal cancer (CRC) cells (LOVO/5-Fu, HT-29/5-Fu) which were transfected with SNHG5-short hairpin RNA and negative control lentivirus; B and C: The viability of LOVO/5-Fu and HT-29/5-Fu cells treated with 5-Fu at different concentrations was evaluated, and the IC₅₀ of 5-Fu-resistant CRC cells was calculated; D: The apoptosis rate of stable cells treated with 5-Fu was analyzed using annexin-V fluorescein 5-isothiocyanate/propidium iodide staining assays (left) and quantified (right); E: Western blot analysis was performed to assess the expression of markers of apoptosis (Bax and Bcl-2), with β-actin as an internal control (left), and densitometric analysis of Western blot signals is shown (right). ^bP < 0.01. ^cP < 0.001. shSNHG5: SNHG5-short hairpin RNA; shNC: Negative control-short hairpin RNA; 5-Fu: 5-fluorouracil.

Figure 4 SNHG5 promotes 5-fluorouracil resistance in colorectal cancer in vivo. A: Tumor growth after treatment with 5-fluorouracil was assessed using the following formula: V = 1/2 × a² × b, where a is tumor length and b is tumor width; B: Representative images of trimmed tumors; C: Tumor weight was calculated; D: Ki-67 expression was evaluated in tumor tissues by immunohistochemistry (scale bar = 400 μm). ^aP < 0.05. ^bP < 0.01. Con: Control; shSNHG5: SNHG5-short hairpin RNA; shNC: Negative control-short hairpin RNA; 5-Fu: 5-fluorouracil.

Figure 5 SNHG5 directly binds to miR-26b in colorectal cancer. A: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze SNHG5 expression in the nuclear and cytoplasmic fractions of LOVO and HT-29 cells; B: A Venn diagram was used to identify potential miRNAs that may target the binding region of SNHG5; C: The expression levels of miR-26b were evaluated via qRT-PCR in LOVO and HT-29 cells transfected with SNHG5 and control or long noncoding RNAs SNHG5 small interfering RNA (siRNA), control siRNA; D: A dual-luciferase assay was used to assess the seed-matching sites or mutation sites between SNHG5 and miR-26b in HEK-293T cells; E: Anti-argonaute 2 RNA immunoprecipitation analyses were performed using LOVO and HT-29 cells transfected with miR-26b, and the enrichment of SNHG5 was analyzed via qRT-PCR. ^bP < 0.01. Si-NC: Control small interfering RNA; Si-SNHG5: SNHG5 small interfering RNA; WT: Wild type; Mut: Mutant; miR-NC: MiR-control; NS: No significance; CMV: Cytomegalovirus; Ago-2: Argonaute 2.

Figure 6 SNHG5 promotes colorectal cell resistance to 5-fluorouracil by sponging miR-26b. The stable cells were transfected with miR-26b or miR-control (miR-NC), after 24 hours, and then treated with 5-fluorouracil. A: Cell viability was analyzed using cell counting kit 8 assays; B: The apoptosis rate of stable cells was analyzed using annexin-V fluorescein 5-isothiocyanate/propidium iodide staining assays (left) and quantified (right); C: Western blot analysis was performed to assess the expression of markers of apoptosis (Bax and Bcl-2), with β-actin serving as an internal control (left), and a densitometric analysis of Western blot signals is shown (right). ^bP < 0.01. ¹P compared with the control + miR-NC group or the negative control-short hairpin RNA (shNC) + miR-NC group. ²P compared with the control + miR-26b group or the shNC + miR-26b group. ³P compared with the SNHG5 + miR-NC group or the SNHG5-short hairpin RNA + miR-NC group. miR-NC: MiR-control; Con: Control; shSNHG5: SNHG5-short hairpin RNA; shNC: Negative control-short hairpin RNA; 5-Fu: 5-fluorouracil.

Figure 7 SNHG5 contributes to 5-fluorouracil resistance in part by regulating the miR-26b/p-glycoprotein axis. A and B: The expression levels of miR-26b and p-glycoprotein (Pgp) were evaluated in the colorectal cancer tissues; C and D: Pearson’s correlation analysis was performed to analyze the correlation between SNHG5 and miR-26b, SNHG5 and Pgp; E: Western blotting was performed to assess the expression levels of Pgp in LOVO and HT-29 cells transfected with control + miR-control (miR-NC), SNHG5 + miR-NC, control + miR-26b, or SNHG5 + miR-26b. Densitometric analysis of the Western blot signals is shown; F: Western blotting was performed to assess the expression levels of Pgp, Bax and Bcl-2 in LOVO cells transfected with negative control-short hairpin RNA (shNC) + vector, SNHG5-short hairpin RNA (shSNHG5) + Vector, shNC + Pgp, or shSNHG5 + Pgp. Densitometric analysis of the Western blot signals is shown. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. miR-NC: MiR-control; Con: Control; shSNHG5: SNHG5-short hairpin RNA; shNC: Negative control-short hairpin RNA; 5-Fu: 5-fluorouracil; Pgp: P-glycoprotein.