LncRNA LOXL1‐AS1 facilitates the tumorigenesis and stemness of gastric carcinoma via regulation of miR‐708‐5p/USF1 pathway

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  Figure 1. High expression of LOXL1‐AS1 reflected poor prognosis of gastric cancer. A, The expression of LOXL1‐AS1 in gastric cancer tissues (n = 84) and paired non‐cancerous tissues (n = 84) was determined by RT‐qPCR. B, RT‐qPCR analysis of LOXL1‐AS1 level in different TNM stages. C, LOXL1‐AS1 expression in gastric cancer cells (MKN‐45, AGS, SGC7901 and MGC‐803) and normal gastric epithelial cells GES‐1 as detected by RT‐qPCR assay. D, Kaplan‐Meier analysis was used to assess overall survival of gastric cancer patients with high or low LOXL1‐AS1 expression. * P < .05, ** P < .01

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  Figure 2. LOXL1‐AS1 contributed to cell proliferation, migration and EMT in gastric cancer. A, The efficiency of transfection in MKN‐45 and AGS cells was determined by RT‐qPCR. B, The CCK‐8 assay was applied to detect cell viability. C, The EdU assay was also conducted to measure cell proliferation in MKN‐45 and AGS cells. D, Cell migratory capacity was tested by transwell assay. E, Western blot analysis was employed to estimate EMT process through examining the expression E‐cadherin and vimentin. * P < .05, *** P < .001

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  Figure 3. LOXL1‐AS1 promoted the maintenance of CSC characteristics in gastric carcinoma. A, The sphere formation assay was adopted to evaluate the effects of LOXL1‐AS1 depletion on sphere‐forming ability. B, The diameter and quantity of spheres when LOXL1‐AS1 was overexpressed in AGS cells. C, Western blot results of the expression of stem markers (Nanog, SOX2, OCT4) in transfected MKN‐45 and AGS cells. D, CCK8 assay was utilized to assess cell viability after treatment with different doses of cisplatin. * P < .05, ** P < .01

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  Figure 4. USF1 was the target gene of LOXL1‐AS1/miR‐708‐5p. A‐B, LOXL1‐AS1 expression in gastric carcinoma samples and cells was measured by RT‐qPCR assay. C, After transfection, USF1 expression in MKN‐45 and AGS cells was detected by RT‐qPCR. D, The subcellular position of LOXL1‐AS1 was certified by subcellular fractionation analysis. E, The starBase and DIANA databases were applied to carry out the bioinformatics analysis. F, The relationship between LOXL1‐AS1 and candidate miRNAs was evaluated by RNA pull‐down assay using biotinylated LOXL1‐AS1 probe. G‐H, The RT‐qPCR analysis of miR‐708‐5p and LOXL1‐AS1 in transfected MKN‐45 and AGS cells. I, RT‐qPCR and Western blot were implemented to estimate the impacts of miR‐708‐5p on USF1 expression. J, Spearman's correlation between USF1 and LOXL1‐AS1 or miR‐708‐5p in gastric cancer specimens. * P < .05, ** P < .01

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  Figure 5. LOXL1‐AS1 sponged miR‐708‐5p to upregulate the expression of USF1 in gastric carcinoma. A, The speculated binding sites of miR‐708‐5p for LOXL1‐AS1 and USF1 by browsing online bioinformatics websites (starBase and DIANA). B‐C, The luciferase reporter assays were performed to measure the luciferase activities of LOXL1‐AS1 and USF1 in response to miR‐708‐5p mimic or miR‐708‐5p inhibitor. D‐E, The interplay between LOXL1‐AS1, miR‐708‐5p and USF1 was further confirmed by RIP and luciferase reporter assays. F‐G, The regulatory influences of LOXL1‐AS1 and miR‐708‐5p on USF1 mRNA and protein levels were verified by RT‐qPCR assay and Western blot. * P < .05, *** P < .001

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  Figure 6. USF1 activated SOX2 expression at the transcriptional level. A, With the aid of UCSC database, it was found that USF1 might bind with the promoter region of SOX2. B, The binding capability of USF1 to SOX2 promoter was analysed by ChIP assay. C, RT‐qPCR was adopted to evaluate the transfection efficiency. D, The luciferase activity of SOX2 promoter was determined by luciferase reporter assays when USF1 was silenced or overexpressed. E, The regulation of USF1 on SOX2 expression was estimated by RT‐qPCR. * P < .05, ** P < .01, *** P < .001

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  Figure 7. LOXL1‐AS1/miR‐708‐5p/USF1 pathway induced gastric cancer tumorigenesis and development. A, The CCK‐8 assay was employed to examine cell viability after transfection. B, The function of LOXL1‐AS1/miR‐708‐5p/USF1 in cell proliferation was further validated by EdU assay. C, The transwell assay was carried out to evaluate cell migration. D, Western blot was used to detect the expression of EMT‐related proteins E‐cadherin and vimentin. E‐F, Cell stemness was assessed by sphere formation assay and Western blot analysis. G, After transfection, cell viability of MNK‐45 cells was tested by CCK‐8 assay in the presence of different doses of cisplatin. * P < .05, ** P < .01, *** P < .001

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  Figure 8. LOXL1‐AS1 accelerated cell growth of gastric cancer in vivo. A, Images of xenografts from nude mice injected with MNK‐45 cells stably transfected with sh‐NC or sh‐LOXL1‐AS1#1. B, The growth curve of neoplasms was plotted by monitoring tumour volume. C, The tumour tissues from mice were weighted at 4 wk after inoculation. D, The expression of LOXL1‐AS1, miR‐708‐5p and USF1 in xenografts was examined by RT‐qPCR. E, IHC assay was performed to assess the levels of Ki‐67, E‐cadherin, vimentin and SOX2 in neoplasms. F, Lung metastasis was measured in sh‐NC group or sh‐LOXL1‐AS1#1 group. * P < .05, *** P < .001