Circular RNA circ-LARP1B contributes to cutaneous squamous cell carcinoma progression by targeting microRNA-515-5p/TPX2 microtubule nucleation factor axis

Abstract

Circular RNAs (circRNAs) have shown pivotal regulatory roles in tumorigenesis and progression. Our purpose was to analyze the role of circRNA La ribonucleoprotein 1B (circ-LARP1B; hsa_circ_0070934) in cutaneous squamous cell carcinoma (CSCC) progression and its associated mechanism. Cell viability, colony formation ability, migration, and invasion were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, colony formation assay, wound healing assay, and transwell invasion assay. Flow cytometry was performed to analyze cell apoptosis and cell cycle progression. Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between microRNA-515-5p (miR-515-5p) and circ-LARP1B or TPX2 microtubule nucleation factor (TPX2). Circ-LARP1B expression was up-regulated in CSCC tissues and cell lines. Circ-LARP1B knockdown suppressed cell viability, colony formation ability, migration, invasion, cell cycle progression, and glycolysis and triggered cell apoptosis in CSCC cells. miR-515-5p was a direct target of circ-LARP1B in CSCC cells, and circ-LARP1B silencing-mediated anti-tumor effects were largely counteracted by miR-515-5p knockdown. miR-515-5p directly interacted with the 3' untranslated region (3'UTR) of TPX2. TPX2 overexpression largely overturned miR-515-5p-mediated anti-tumor effects in CSCC cells. Circ-LARP1B could up-regulate TPX2 expression by sponging miR-515-5p in CSCC cells. Circ-LARP1B knockdown suppressed tumor growth in vivo. In conclusion, circ-LARP1B contributed to CSCC progression by targeting miR-515-5p/TPX2 axis. The circ-LARP1B/miR-515-5p/TPX2 axis might provide novel therapeutic targets for CSCC patients.

Keywords: Cutaneous squamous cell carcinoma; TPX2; circ-LARP1B; miR-515-5p.

Figure 1.

Circ-LARP1B is highly expressed in CSCC tissues and cell lines. (a) The expression of circ-LARP1B was determined in CSCC tissues (n = 38) and adjacent normal tissues (n = 38) by RT-qPCR. (b) The expression of circ-LARP1B in two CSCC cell lines (A431 and HSC-1) and normal human epidermal keratinocytes (NHEK) was measured by RT-qPCR. (c and d) The stability of circ-LARP1B was tested using exonuclease RNase R, and GAPDH was used as the control. The levels of circ-LARP1B and GAPDH were examined in RNase R digestion (RNase R+) group or RNase R- group by RT-qPCR. (e) The subcellular localization of circ-LARP1B was analyzed using the PARIS^TM Kit Protein and RNA Isolation system. GAPDH or U6 was served as the cytoplasmic or nuclear marker, respectively. **P < 0.01.

Figure 2.

Circ-LARP1B silencing suppresses the malignant phenotypes of CSCC cells. (a) RT-qPCR was conducted to analyze the knockdown efficiencies of two siRNAs of circ-LARP1B (si-circ-LARP1B#1 and si-circ-LARP1B#2). (b-m) CSCC cells were transfected with si-NC or si-circ-LARP1B#1. (b) Cell viability was analyzed by MTT assay. (c) The colony formation ability of CSCC cells was analyzed by colony formation assay. (d) Cell apoptosis was measured by flow cytometry. (e) Cell migration ability was assessed by wound healing assay. (f) Cell invasion capacity was detected by transwell invasion assay. (g and h) Cell cycle progression was analyzed by flow cytometry. (i-k) Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. (l) Western blot assay was conducted to determine the protein expression of HK-2 in CSCC cells. (m) The levels of apoptosis-, metastasis-, and proliferation-associated proteins (cleaved caspase 3, MMP2, and cyclin D1) were detected by Western blot assay. *P < 0.05, **P < 0.01.

Figure 3.

miR-515-5p is a direct target of circ-LARP1B in CSCC cells. (a) Circular RNA interactome database predicted the interaction between circ-LARP1B and miR-515-5p. (b) The interaction between circ-LARP1B and miR-515-5p was confirmed by dual-luciferase reporter assay. (c) The target relationship between circ-LARP1B and miR-515-5p was verified by RIP assay. (d) The expression of miR-515-5p in CSCC tissues and cell lines was detected by RT-qPCR. (e) The linear correlation between the expression of circ-LARP1B and miR-515-5p in CSCC tissues was analyzed by Pearson’s correlation coefficient. (f) The overexpression efficiency of circ-LARP1B ectopic expression plasmid in CSCC cells was analyzed by RT-qPCR. (g) The expression of miR-515-5p was detected in CSCC cells transfected with si-NC, si-circ-LARP1B, pLCDH-cir, or circ-LARP1B plasmid by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

miR-515-5p interference reverses circ-LARP1B absence-induced anti-tumor effects in CSCC cells. (a-k) A431 and HSC-1 cells were transfected with si-NC, si-circ-LARP1B, si-circ-LARP1B + anti-miR-NC or si-circ-LARP1B + anti-miR-515-5p. (a) RT-qPCR was conducted to detect the expression of miR-515-5p in transfected CSCC cells. (b) MTT assay was conducted to assess cell viability. (c) Colony formation assay was used to analyze the colony formation ability of transfected CSCC cells. (d) The apoptosis rate was measured by flow cytometry. (e) Wound healing assay was utilized to analyze the migration ability of CSCC cells. (f) Transwell invasion assay was performed to measure invasion ability of CSCC cells. (g) Cell cycle progression was evaluated by flow cytometry. (h-j) Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. (k) The protein expression of HK-2 was measured in CSCC cells by Western blot assay. *P < 0.05, **P < 0.01.

Figure 5.

miR-515-5p directly interacts with TPX2 3ʹUTR in CSCC cells. (a) The potential binding sites between miR-515-5p and TPX2 3ʹUTR were predicted by Starbase software. (b) Dual-luciferase reporter assay was performed to confirm the target interaction between miR-515-5p and TPX2 in CSCC cells. (c) RIP assay was conducted to verify the interaction between miR-515-5p and TPX2 in CSCC cells. (d) The expression of TPX2 mRNA in CSCC tissues (n = 38) and adjacent non-tumor tissues (n = 38) was determined by RT-qPCR. (e) The level of TPX2 mRNA in NHEK cells and two CSCC cell lines (A431 and HSC-1) was examined by RT-qPCR. (f) Western blot assay was conducted to analyze the protein expression of TPX2 in CSCC tissues and adjacent normal tissues. (g) The protein level of TPX2 in NHEK, A431 and HSC-1 cells was measured by Western blot assay. (h) The linear correlation between the expression of miR-515-5p and TPX2 in CSCC tissues was assessed by Pearson’s correlation coefficient. (i) The overexpression efficiency of miR-515-5p mimics in CSCC cells was analyzed by RT-qPCR. (j and k) Western blot assay was performed to measure the protein expression of TPX2 in A431 and HSC-1 cells transfected with miR-NC, miR-515-5p, anti-miR-NC or anti-miR-515-5p. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

miR-515-5p overexpression-mediated anti-tumor effects in CSCC cells are overturned by the accumulation of TPX2. (a-l) CSCC cells were transfected with miR-NC, miR-515-5p, miR-515-5p + pcDNA or miR-515-5p + TPX2. (a and b) The mRNA and protein expression of TPX2 was examined in CSCC cells by RT-qPCR and Western blot assay. (c) Cell viability was analyzed by MTT assay. (d) The colony formation ability was analyzed by colony formation assay. (e) Cell apoptosis rate was analyzed by flow cytometry. (f and g) Cell migration and invasion abilities were analyzed by wound healing assay and transwell invasion assay. (h) The percentages of CSCC cells in G0/G1 phase, S phase, and G2/M phase were analyzed by flow cytometry. (i-k) Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. (l) Western blot assay was conducted to analyze the protein level of HK-2 in CSCC cells. *P < 0.05, **P < 0.01.

Figure 7.

Circ-LARP1B can up-regulate TPX2 expression by sponging miR-515-5p in CSCC cells. (a-d) CSCC cells were transfected with si-NC, si-circ-LARP1B, si-circ-LARP1B + anti-miR-NC, or si-circ-LARP1B + anti-miR-515-5p. (a and b) The protein level of TPX2 was analyzed by Western blot assay. (c and d) The activity of PI3K/AKT signaling was analyzed by Western blot assay. *P < 0.05, **P < 0.01.

Figure 8.

Circ-LARP1B silencing inhibits tumor growth in vivo. (a) After injection for 7 d, the tumor volume was measured every 5 d as (width² × length)/2. (b) The tumor images in two groups were shown. (c) After injection for 32 d, all nude mice were euthanized, and tumor weight was recorded. (d) IHC assay was conducted to analyze the protein levels of proliferation-, glycolysis-, apoptosis-, and metastasis-associated markers in tumor tissues. (e) The expression of circ-LARP1B, miR-515-5p and TPX2 mRNA was measured by RT-qPCR. (f) Western blot assay was conducted to detect the protein expression of TPX2 in tumor tissues. *P < 0.05, **P < 0.01, ***P < 0.001.