| Background and objects : Hypopharyngeal carcinoma(HSCC)is one of the most malignant head and neck tumors,and because of its insidious location and non-specific initial symptoms,it is often overlooked by patients,and most patients are in the middle and late stages when diagnosed.At present,the main treatments for hypopharyngeal cancer include surgery,radiotherapy,immunotherapy,targeted therapy and so on.However,the treatment effect is not satisfactory.Lnc RNA is a class of long-stranded non-coding RNA,which has been studied in many tumors and plays different roles in different tumors.There is increasing evidence that Lnc RNAs play an important role in tumor development.These include regulation of tumor proliferation,metastasis,invasion,apoptosis,and chemoresistance.However,in hypopharyngeal cancer,Lnc RNAs have been less studied,and the way through which Lnc RNAs affect the progression of hypopharyngeal tumors needs our further study.Programmed cell death 1ligand 1(PD-L1),one of the ligands of PD-1,is mainly expressed on the surface of T lymphocytes and promotes immune escape.In hypopharyngeal cancer,high expression of PD-L1 promotes HSCC cells to evade recognition by the host immune system and accelerates metastasis.In fact,Lnc RNA can regulate PD-L1 expression,thus promoting immune escape of tumors,but the specific mechanism of action in hypopharyngeal cancer is not clear.Methods:(1)Three pairs of cancer and paraneoplastic tissues from patients with hypopharyngeal squamous carcinoma diagnosed by post-surgical pathology were taken to construct gene chips of differential Lnc RNAs,and the differential Lnc RNAs were screened by using the criteria of Pvalue < 0.05,z|log2(Fc)|>2,and the Lnc RNAs were sorted by the higher or lower fold of difference to screen Lnc RNAs;combined with the literature,we identified HOXA11-AS1 and PD-L1 were identified as the next research targets because,HOXA11-AS1 is an antisense Lnc RNA of HOXA11,and HOXA11-AS plays a regulatory role in a variety of human tumors,while HOXA11-AS1 has not been reported in hypopharyngeal cancer,PD-L1 widely mediates immune escape from tumors,and some studies have reported that PD-L1 mediates tumor escape and metastasis in hypopharyngeal cancer tumor escape and metastasis.We first performed qRT-PCR experiments to detect the relative expression of HOXA11-AS1 and PD-L1 in 40 pairs of hypopharyngeal carcinoma and paraneoplastic tissues,normalized the gene expression by GAPDH,processed the data with POWER function,and plotted the dot plot;the median(less than the median is low expression,greater than or equal to the median is high expression)was used as the threshold value for Kaplan-Meier curve survival analysis,correlation analysis of HOXA11-AS1 with PD-L1 using Pearson correlation coefficient,and clinicopathological analysis of HOXA11-AS1.Prediction of HOXA11-AS1 and PD-L1 target proteins using Jaspar and Starbase databases;(2)qRT-PCR was used to detect the expression of HOXA11-AS1,PD-L1,and FOSL1 in nasopharyngeal immortalized epithelial cells(NP69),hypopharyngeal carcinoma cells(Fadu),and human pharyngeal head carcinoma pleural fluid metastasis cell line(Detroit562),and western blot and immunohistochemistry(IHC)were used to detect PD-L1 in cells and The lentiviral cell lines silencing HOXA11-AS1 were constructed and transfected in Fadu cell line and Detroit 562 cell line to examine the silencing efficiency;the expression levels of PD-L1 in control and silenced groups were detected by Western blot and immunofluorescence with or without anti-PD-L1 antibody,and CD8+ T cells were detected using flow cytometry and CD4+ T cells by flow cytometry;interferon-γ concentration by ELISA;CCK-8 for cell growth viability,colony formation assay,cell scoring,transwell assay for its effect on cell proliferation and migration;nucleoplasmic separation assay in Fa Du cells for the expression of HOXA11-AS1 in the nucleus and cytoplasm;(3)qRT-PCR assay to detect FOSL1 expression in hypopharyngeal carcinoma and paraneoplastic tissues,expressed as dot plots,with survival analysis and Kaplan-Meier curves;after silencing FOSL1,the expression of FOSL1 and PD-L1 was detected;chromatin immunoprecipitation(Chi P)and dual luciferase reporter assay were used to verify the FOSL1 and PD-L1 promoter;actinomycin D(ACD)treatment silenced HOXA11-AS1 group and control cells to detect the expression of FOSL1 m RNA;RNA pulldown assay to verify the regulatory relationship between HOXA11-AS1 and FOSL1;RIP assay to detect the binding of PTBP1 to FOSL1 and HOXA11-AS1 construct a silent PTBP1 lentivirus stable strain,qRT-PCR assay to detect the expression of PTBP1,HOXA11-AS1 and FOSL1,and detect the expression of FOSL1 m RNA and HOXA11-AS1 in the control and silent groups treated with actinomycin(ACD);(4)Construct lentiviral stable strains overexpressing HOXA11-AS1 and PTBP1,qRT-PCR and Western assay to detect the expression levels of HOXA11-AS1,FOSL1,PD-L1;introduce the combination of HOXA11-AS1,sh PTBP1,HOXA11-AS1+sh PTBP1 into Fa Du and Detroit cells,and Western blot assays were performed to detect the expression of PTBP1,FOSL1,and PD-L1 in the control NC group,the overexpression HOXA11-AS1 group,the silent PTBP1 group,and the overexpression HOXA11-AS1+sh PTBP1 group;the HOXA11-AS1,sh PD-L1,and HOXA11-AS1+ sh PD-L1 combination was introduced into Fa Du and Detroit cells,and the relative expression of PTBP1,FOSL1 and PD-L1 in NC,HOXA11-AS1,sh-PD-L1 or HOXA11-AS1+sh-PD-L1 cells was detected by Western blot assay;CD8+,CD4+ T cells were detected by flow cytometry percentage;interferon-γ concentration by ELISA;cell activity,colony formation,migration and invasion ability by CCK-8,colony formation,wound healing and Transwell assays;Fa Du and Detroit cells stably expressing sh NC,sh HOXA11-AS11 or sh HOXA11-AS1-2 were injected subcutaneously into NOD-SCID mice to establish a xenograft model.(n=48),which were divided into PBS and PBMCs injection groups,and the pathology,proliferation and PD-L1 expression of transplanted tumors treated with or without PBMCs or PBS were examined by HE,Ki67 and IHC staining;the percentage of Ki67 cells in transplanted tumors was examined;sh NC,sh HOXA11-AS1-1 and sh HOXA11-AS1-2 were calculated graft volume to measure the cytotoxic effect of treatment with or without PBMCs or PBS;in vivo detection of tumor volume and weight after HOXA11-AS1 knockdown;detection of HOXA11-AS levels by RT-q PCR;detection of PD-L1,FOSL1,and PTBP1 expression in tumors after HOXA11-AS1 knockdown by Western Blot assay;and HE and PTBP1 expression in the tumors after HOXA11-AS1 knockdown;HE staining to detect the ability of lung metastasis,tumor pathology and the number of lung metastatic nodes in mice after HOXA11-AS1 knockdown.Results:(1)807 differential Lnc RNAs in the microarray,of which 542 were up-regulated and 265 were down-regulated.(With threshold |log FC|>2and p-value<0.05;further narrowed down to lnc RNAs with nucleotide sequence length <3000,excluding group-Treatment(raw)and groupNormal(raw)data of <200)further screened,67 lnc RNAs that met the screening requirements for upregulation,which contained HOXA11-AS1;HOXA11-AS1 expression in cancer tissues was 1.55-fold higher than that in paraneoplastic tissues(p=0.0023);PD-L1 expression in hypopharyngeal cancer was 1.57-fold higher than that in paraneoplastic tissues(p=0.0006),HOXA11-AS1 expression was negatively correlated with overall survival time(p=0.0282),PD-L1 and hypopharyngeal cancer overall survival time(p=0.0301);Pearson analysis showed that HOXA11-AS1 expression was positively correlated with PD-L1(p=0.0015,correlation coefficient=0.2363);HOXA11-AS1 expression was correlated with tumor size(p=0.0011),lymph node metastasis(p=0.0245),distant metastasis(p=0.0225),T-stage(p=0.0230)were significantly correlated with age(p=0.1167);Gepia’s database showed high expression of FOSL1 in head and neck tumors with negative prognosis,and Jaspar predicted a binding site between FOSL1 and PD-L1promoter;Starbase data predicted that HOXA11-AS1,FOSL1 and polypyrimidine binding protein 1(PTBP1),respectively.(2)qRT-PCR experiments showed that the relative expression of HOXA11-AS1 in Fa Du cells was 4.95 times higher than in NP69(p=0.0006)and in Detroit562 cells was 4.66 times higher than in NP69(p=0.0008);Western blot experiments showed that the relative PD-L1(p=0.0017)in Fa Du cells and 1.90-fold in Detroit562 cells(p=0.0344);the relative expression of FOSL1 in Fa Du cells was 3.10-fold in NP69(p=0.0009)and 1.83-fold in Detroit562 cells(p=0.0344);the relative expression of FOSL1 in Fa Du cells was 3.10-fold in NP69(p=0.0009)and 1.83-fold in Detroit562 cells(p=0.0008).1.83-fold in Detroit562cells(p=0.0179);immunohistochemistry showed high expression of PDL1 in hypopharyngeal cancer tissues;silencing HOXA11-AS1 or antiPD-L1 treatment increased the percentage of CD8+ T cells and interferon concentration in PBMC and decreased the percentage of CD4+ T cells in PBMC;silencing HOXA11-AS1 inhibited cell viability,attenuated cell The silencing of HOXA11-AS1 inhibited cell viability and diminished cell proliferation and migration ability;in Fa Du cells,HOXA11-AS1 was mainly localized in the cytoplasm.(3)qRT-PCR experiments showed that FOSL1 was highly expressed in hypopharyngeal cancer tissues,and the relative expression in cancer tissues was 1.45-fold higher than that in the paracancer(p=0.0036);FOSL1’s was negatively correlated with overall survival time,consistent with Gepia’s database analysis;silencing FOSL1 decreased PD-L1expression;chromatin immunoprecipitation(Chi P)and dual luciferase reporter assay confirmed FOSL1 binding to PD-L1 promoter;silencing HOXA11-AS1 reduced FOSL1 m RNA stability;RNA pulldown assay confirmed HOXA11-AS1 binding to FOSL1;RNA binding immunoprecipitation(RIP)results showed PTBP1 interacting with HOXA11-AS1;dual luciferase reporter assay showed PTBP1 interacted with FOSL1;qRT-PCR assay showed that silencing PTBP1 decreased the expression of HOXA11-AS1 and FOSL1 and shortened the m RNA decay rate of FOSL1 with HOXA11-AS1;(4)Overexpression of HOXA11-AS1 and PTBP1 enhanced the expression of FOSL1,PTBP1,HOXA11-AS1 and PD-L1;silencing PTBP1 partially reversed the upregulation of FOSL1 and PD-L1 caused by HOXA11-AS1 overexpression;silencing PD-L1 reversed the upregulation of HOXA11-AS1 caused by PD-L1 upregulation caused by HOXA11-AS1 overexpression,while PTBP1 did not change significantly with FOSL1;since HOXA11-AS1 increased PD-L1 levels in HSCC cells and promoted proliferation,metastasis and immune escape,we investigated the mechanism of PD-L1 regulation in HOXA11-AS1 overexpression cells.HOXA11-AS1 overexpression decreased the percentage of CD8+ T cells and increased the percentage of CD4+ T cells.In contrast,downregulation of PD-L1 gene led to the opposite result and partially reversed the HOXA11-AS1 overexpression-induced increase in T lymphocytes.Furthermore,PD-L1 knockdown increased the reduced interferon-γ concentration due to HOXA11-AS1 overexpression,suggesting that HOXA11-AS1 promoted immune escape of HSCC cells through regulation of PD-L1.Similarly,HOXA11-AS1 enhancement promoted cell proliferation,migration and invasion,and these effects were reversed by PD-L1 knockdown.Thus,we confirmed the role of HOXA11-AS1/PD-L1/PTBP1/FOSL1 axis in promoting HSCC progression in vitro.(5)In vivo experiments showed that the degree of tumor pathology and proliferation as well as the expression of PD-L1 and Ki-67 were reduced in the silenced HOXA11-AS1 group or in the mice injected with PBMCs,which were further enhanced by the downregulation of HOXA11-AS1 and the combined effect of PBMCs,indicating that HOXA11-AS1 deletion has an effective anti-tumor in vivo effects.Tumor volume and weight decreased after tumor silencing with HOXA11-AS1,and levels of PTBP1,FOSL1 and PD-L1 decreased in vivo.deletion of HOXA11-AS1 reduced the ability of tumors to metastasize to the lung.CONCLUSION: HOXA11-AS1 can increase the stability of FOSL1 by binding to PTBP1,thereby upregulating PD-L1 expression,resulting in enhanced tumor immune escape and promoting immune escape and metastasis in hypopharyngeal carcinoma. |
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