Long Intergenic Non-Coding RNA LINC01088 Facilitates Malignant Phenotypes And Immune Escape Of Colorectal Cancer

Background and objectives:Colorectal cancer(CRC)is one of the most common cancers with the third leading incidence and second highest mortality worldwide.In-depth understanding of the molecular mechanisms of colorectal cancer is beneficial to guide the diagnosis and treatment of cancer.Long non-coding RNAs are RNA transcripts longer than 200 nucleotides,including antisense long non-coding RNAs,intronic long non-coding RNAs,and intergenic long non-coding RNAs(LincRNAs).Long non-coding RNAs play important roles in a variety of physiological or pathological processes such as organismal development and disease.Intergenic long non-coding RNA LINC01088 is located on chromosome 4q21.21,whose biological function in colorectal cancer progression and immune escape remains unclear.Investigating the biological function of LINC01088 in colorectal cancer gains new insights into the molecular mechanisms of colorectal cancer progression and provides potential targets for colorectal cancer diagnosis and treatment.Methods:Part I.To explore the expression of LINC01088 in colorectal cancer tissues,paracancerous tissues and cell lines and its relationship with clinical parameters of patients with colorectal cancer1.1 Thirty-six surgically resected tumor tissues and paired para-cancerous tissues from colorectal cancer patients were collected.Human colorectal cancer cell lines HCT116,Lo Vo, Caco2,SW480 and human normal intestinal epithelial cells CCD841Co N were cultured.Total RNA was extracted from tissues or cells using Trizol reagent.After reverse transcription,qPCR assay was performed to test the expression levels of LINC01088 in tumor tissues and CRC cells.1.2 To determine the distribution of LINC01088 in CRC cells,colorectal cancer cell lines were collected.After nucleoplasmic separation,LINC01088 levels in the nucleus and cytoplasm were detected by qPCR analysis.1.3 To construct a fluorescent probe targeting LINC01088,RNA-FISH assay was performed to evaluate the subcellular localization of LINC01088 in CRC tissues and cells.Part II 2.To explore the effect of LINC01088 on biological behavior of colorectal cancer cells2.1 Cell functional assays were performed in vitro.Cell viability of CRC cells was assessed by CCK-8 assay,the proliferative ability of CRC cells was tested by colony formation assay, and cell cycle distribution of CRC cells was measured by flow cytometry.The migratory and invasive abilities of CRC cells were tested by Transwell migration and invasion assays.2.2 In vivo,we constructed a subcutaneous xenograft tumor model and lung metastasis mouse model.2.3 To explore the role of LINC01088 in immune escape of colorectal cancer,human peripheral blood CD8~+T cells were extracted,expanded and cultured.CD8~+T cells were co-cultured with CRC cells.Crystalline violet staining,supernatant lactate dehydrogenase(LDH)assay and PI staining were performed to observe the killing effect of CD8~+T cells on CRC cells.2.4 Fresh samples from surgically resected colorectal cancer tissues were collected,and intestinal crypts were isolated by physical mincing as well as biological digestion and cultured into intestinal organoids by commercialized reagents.During the long-term culturing process,intestinal organoids were digested and passaged.Intestinal organoids were identified by immunofluorescence.2.5 To further investigate the function of LINC01088 in intestinal organoids,intestinal organoids were infected with sh-LINC01088 lentivirus as well as control lentivirus,and the growth of intestinal organoids with LINC01088 knockdown was observed microscopically. The effect of LINC01088 knockdown on apoptosis of intestinal organoids was measured by PI staining.Part III 3.To explore downstream targets of LINC01088 in CRC cells3.1 Screening of down-regulated microRNAs that bind to LINC01088 in colorectal cancer by public databases.3.2 RNA immunoprecipitation(RIP)and qPCR experiments were performed to verify whether LINC01088 binds to the above microRNAs.Subsequently,qPCR analysis was used to test potential up-regulated microRNAs in CRC cells with LINC01088 knockdown.3.3 The role of the 9 target microRNAs in CRC cells was further analyzed by CCK-8,Transwell and co-culture assays.3.4 Dual luciferase reporter gene assay was performed to identify the interaction between LINC01088 and miR-548b-5p,LINC01088 and miR-548c-5p.Part IV.To further investigate molecular mechanisms by which LINC01088 regulates downstream signaling pathways via adsorption of microRNAs in colorectal cancer progression and immune escape4.1 Screening of downstream target genes of miR-548b-5p and miR-548c-5p by public databases DIANA-micro T web server v5.0,miRDB-Micro RNA Target Prediction Database and DIANA-Tar Base v8.4.2 CRC cells were transfected with miR-548b-5p or miR-548c-5p mimics and Western blotting assays were performed to test G3BP1 protein expression.Dual luciferase reporter gene assay was used to verify the interaction between miR-548b-5p and G3BP1 mRNA, miR-548c-5p and G3BP1 mRNA.4.3 qPCR and Western blotting assays were conducted to measure the mRNA and protein levels of G3BP1 in LINC01088-knockdown CRC cells.Further,in vitro functional rescue assays were implemented to overexpress G3BP1 in LINC01088-knockdown CRC cells.The effects of G3BP1 overexpression on CRC cell proliferation,migration,invasion and immune escape were observed by CCK-8,Transwell and co-culture assays.4.4 To investigate the mechanism of LINC01088 in immune escape of CRC,the mRNA levels of programmed cell death ligand 1(PD-L1)and the protein expression of PD-L1 on cell membrane of CRC cells were assessed by qPCR and flow cytometry,respectively.4.5 A subcutaneous-tumor-bearing nude mouse model was established to observe the effects of G3BP1 overexpression on CRC growth and the mRNA levels of PD-L1 in xenograft tumor tissues.Results:Result 1:LINC01088 is significantly highly expressed in colorectal cancer tissues and is distributed in the nucleus and cytoplasm of cells1.1 LINC01088 was significantly highly expressed in colorectal cancer tissues compared with para-cancerous tissues.And the expression levels of LINC01088 in tumor tissues of colorectal cancer patients at tumor node metastasis(TNM)stage III/IV were significantly higher than that of colorectal cancer patients at TNM stage I/II,indicating that the expression levels of LINC01088 increased with the increase of TNM stage.In the in situ tumor tissues from patients with metastatic colorectal cancer,the expression of LINC01088 was significantly higher than that in CRC patients without metastasis.Additionally,the expression of LINC01088 was also significantly higher in the in situ tumor tissues of patients with recurrent colorectal cancer after surgical resection than in patients without recurrence.1.2 Survival analysis showed that patients with high LINC01088 expression in colorectal cancer tissues had a poor prognosis compared to those with low LINC01088 expression in colorectal cancer tissues.1.3 LINC01088 was markedly overexpressed in colorectal cell lines compared to normal intestinal epithelial cells.Nucleo-cytoplasmic fractionation assay showed that LINC01088 was distributed in both nucleus and cytoplasm.1.4 RNA-FISH experiments further confirmed the distribution of LINC01088 in the nucleus and cytoplasm.Result 2:Silencing of LINC01088 suppresses malignant phenotype of CRC cells2.1 LINC01088-knockdown CRC cell lines were successfully generated as verified by qPCR analysis.Interfering with LINC01088 expression by short hairpin RNA(sh RNA) significantly reduced the viability and colony-forming ability of CRC cells.Cell cycle assay revealed that LINC01088 knockdown resulted in G0/G1 phase arrest in CRC cells.2.2 In a nude mouse xenograft model,LINC01088 knockdown significantly suppressed tumor growth.Results from a nude mouse lung metastasis model showed that LINC01088- knockdown CRC cell lines formed fewer lung nodules in mouse lung tissue and that mice transplanted with LINC01088-knockdown CRC cell lines survived longer compared to controls.2.3 Results of co-culture experiments between CD8~+T cells and CRC cells indicated that the killing ability of CD8~+T cells on CRC cells was significantly enhanced after LINC01088 silencing,as shown by a significant decrease in CRC cell survival,along with a significant increase in CRC cell apoptosis.2.4 Intestinal crypt was successfully isolated from human colorectal cancer tissue and cultured into mature intestinal organoids.2.5 The intestinal organoids were successfully infected with sh-LINC01088 lentivirus and was observed to express GFP fluorescent protein under fluorescence microscopy.LINC01088 knockdown inhibited the expansion of intestinal organoids and promoted organoids apoptosis.Result 3:LINC01088 directly targets miR-548b-5p and miR-548c-5p3.1 Bioinformatic analysis showed that 35 down-regulated microRNAs were predicted to bind to LINC01088 in colorectal cancer.Results of RIP and qPCR experiments suggested that only 20 microRNAs could be enriched among the above 35 microRNAs.In sh1-LINC01088 CRC cells,qPCR results revealed that 11 microRNAs were significantly overexpressed;meanwhile,13 microRNAs were markedly upregulated in sh2-LINC01088 CRC cells.The two were taken to intersect to obtain a set containing 9 microRNAs:hsa-miR-149-5p,hsa-miR-4778-5p,hsa-miR-548b-5p,hsa-miR-548c-5p,hsa-miR-548d-5p,hsa-miR-548i,hsa- miR-548w hsa-miR-548y,hsa-miR-590-3P.3.2 Hsa-miR-548b-5p(miR-548b-5p)and hsa-miR-548c-5p(miR-548c-5p)were further identified as the main targets of LINC01088 by cellular functional assays.3.3 Dual luciferase reporter gene assay confirmed the direct interaction between LINC01088 and miR-548b-5p,LINC01088 and miR-548c-5p.Result 4:LINC01088 is participated in colorectal cancer progression and immune escape through regulation of microRNAs/G3BP1/PD-L1 signaling pathway4.1 After intersecting the genes screened in the above three databases,the results demonstrated that miR-548b-5p contained 9 downstream target genes;miR-548c-5p contained 41 downstream target genes.miR-548b-5p and miR-548c-5p shared 8 target genes,namely G3BP1,BRWD1,RORA,LY75,CCDC47,DDIT4,UBE4A,TRA2B.4.2 G3BP1 was identified as a downstream target by literature search.CRC cells transfected with miR-548b-5p or miR-548c-5p mimics showed significantly lower G3BP1 protein expression compared to un-transfected cells.Dual luciferase reporter gene assay confirmed the direct interaction between miR-548b-5p and G3BP1 mRNA,miR-548c-5p and G3BP1 mRNA.4.3 In LINC01088-knockdown CRC cells,the mRNA and protein levels of G3BP1 were markedly down-regulated compared to the control group.Rescue assays uncovered that G3BP1 overexpression abrogated the inhibitory effects of LINC01088 knockdown on proliferation,migration,and invasion of CRC cells and reversed the killing effect of CD8+T cells on LINC01088-knockdown CRC cells.4.4 In CRC cells,LINC01088 knockdown contributed to a significant reduction in PD-L1 mRNA and PD-L1 protein expression on CRC cell membrane.4.5 In subcutaneous-tumor-bearing nude mouse model,LINC01088 knockdown significantly restrained CRC cell growth,while G3BP1 overexpression reversed the inhibitory effect of LINC01088 knockdown on CRC growth.4.6 In xenograft tumor tissues,LINC01088 knockdown dramatically downregulated PD-L1 mRNA expression,however,G3BP1 overexpression reversed the PD-L1 mRNA levels caused by LINC01088 knockdown,indicating that PD-L1 expression was regulated by G3BP1.Conclusions:LINC01088 is significantly highly expressed in colorectal cancer tissues compared to para-cancerous tissues and is closely related to TNM stage,metastasis and recurrence in colorectal cancer patients.Colorectal cancer patients with high expression of LINC01088 in tumor tissues have a worse prognosis.LINC01088 is distributed in the nucleus and cytoplasm.Interfering with LINC01088 expression by sh RNA in CRC cells significantly inhibits the proliferative,migratory and invasive capabilities of CRC cells in vitro and in vivo,while silencing of LINC01088 enhances the killing effects of CD8~+T cells on CRC cells in vitro.In addition,LINC01088 knockdown results in slowed growth and increased apoptosis of human colorectal cancer tissues-derived organoids.Furthermore,it was found that highly expressed LINC01088 exerted biological effects through direct binding to miR-548b-5p to miR-548c-5p.G3BP1 is the direct downstream target of miR-548b-5p and miR-548c-5p.G3BP1 indirectly promotes PD-L1 expression.Overall,LINC01088 promotes colorectal cancer progression and immune escape by regulating microRNAs/G3BP1/PD-L1 signaling pathway.