| Background and objectiveNasopharyngeal carcinoma is a malignant tumor of the head and neck that originates from the epithelial cells of the nasal mucosa and is highly prevalent in the southern part of China,Southeast Asia and North Africa.According to the burden and scope of the tumor,patients with nasopharyngeal carcinoma are usually treated with a combination of radiation therapy primarily combined with surgical resection and chemotherapy.After receiving radiotherapy or chemotherapy,about 30%of nasopharyngeal cancer patients will relapse or metastasize,which is the main reason for the short overall survival of nasopharyngeal cancer patients.Although the research on the occurrence and metastasis of nasopharyngeal carcinoma has made great progress,the diagnosis and effective treatment of nasopharyngeal carcinoma remains one of the great clinical challenges at present.Long non-coding RNA(LncRNAs)are non-coding RNAs longer than 200 nucleotides,which can be used as competitive endogenous RNAs(ceRNAs)or RNA sponge targeting microRNAs(miRNAs)and competitively bind with them to affect the expression of target messenger RNA(mRNA)to regulate cell malignancy and tumor development,thus exerting a tumor-promoting or suppressive function.LncRNAs have unique expression patterns in tumors of different pathological types and advanced stages,and are expected to become tumor diagnostic markers and therapeutic targets.Recent studies have found that LncRNA HLA complex group 11(HCG11)may play different roles in different tumors,highlighting the role of HCG11 in cancer progression,which has attracted increasing attention in the field of oncology research.However,the molecular function and mechanism of LncRNA HCG11 related to nasopharyngeal carcinoma have not been reported so far.The expression of LncRNA HCG11 in nasopharyngeal carcinoma and its clinical correlation with nasopharyngeal carcinoma,whether LncRNA HCG11 expression has a regulatory role in the occurrence and development of nasopharyngeal carcinoma and its regulatory pathways are still unclear.Therefore,the molecular regulation mechanism of LncRNA HCG11 in nasopharyngeal carcinoma is a new research passsway worth exploring to reveal the pathogenesis of nasopharyngeal carcinoma,guide clinical diagnosis and treatment,and assist prognosis judgment.To this end,in this study,in the first part,the expression of LncRNA HCG11 was first detected using bioinformatics and nasopharyngeal carcinoma patient tissue samples and human nasopharyngeal carcinoma cell lines,and the correlation of this expression with patient tumor stage,cell differentiation,tumor load,metastasis and prognosis was analyzed and investigated.In the second part,by constructing nasopharyngeal carcinoma cells with knock down of LncRNA HCG11,observe the effect of LncRNA HCG11 expression on the biological behaviors of nasopharyngeal carcinoma cells such as proliferation,apoptosis and migration,while its effects on nasopharyngeal carcinoma progression in nude mice were analyzed using a xenograft tumor nude mouse model.The third part explores the specific molecular mechanism of LncRNA HCG11 regulating nasopharyngeal carcinogenesis and development through bioinformatics and in vitro experiments.The correlation between LncRNA HCG11 and nasopharyngeal carcinoma cell differentiation,tumor biological behavior and clinical stage grading as well as metastasis was revealed for the first time,providing an experimental basis for LncRNA HCG11 as a biomarker for the diagnosis of nasopharyngeal carcinoma and targeting LncRNA HCG11 for treatment,which has important clinical guidance.Part 1.The expression and clinical significance of LncRNA HCG11 in nasopharyngeal carcinomaObjectiveTo analyze the expression of LncRNA HCG11 in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cell lines,and to analyze the correlation between the expression of LncRNAHCG11 and the tumor burden,clinical stage,tumor metastasis and prognosis of patients with nasopharyngeal carcinoma.Methods(1)The pathological sections of 126 patients with nasopharyrigeal carcinoma were collected,and the FISH experiment was used to detect the expression of LncRNA HCG11 in nasopharyngeal carcinoma tissues and normal tissues.(2)qRT-PCR was performed to detect the expression of LncRNA HCG11 in 20 pairs of nasopharyngeal carcinoma tissue samples and 3 nasopharyngeal carcinoma cell lines.(3)The FISH experiment was used to further analyze the correlation between LncRNA HCG11 in the pathological sections of 126 patients with nasopharyngeal carcinoma and the clinical parameters such as tumor burden,clinical stage,tumor metastasis and prognosis of nasopharyngeal carcinoma patients.Results(1)According to FISH analysis,compared with normal tissues,LncRNA HCG11 is highly expressed in cancer tissues of patients with nasopharyngeal.carcinoma(P<0.01).(2)Using qRT-PCR to analyze the clinical nasopharyngeal carcinoma patients’cancer tissues and paraneoplastic tissues samples,the results showed that the expression of LncRNA HCG11 in the cancer tissues of nasopharyngeal cancer patients was significantly higher than that in the paraneoplastic tissues(P<0.01);meanwhile,the normal nasopharyngeal cell line NP-69 and three nasopharyngeal carcinoma cell lines(CNE-2,5-8F and CNE-1)was detected,and it was found that LncRNA HCG11 was in CNE-2,5-8F and CNE-1.The expression level in the.cell line was significantly higher than that in the NP-69 cell line(P<0.001).(3)Statistical analysis of the correlation between LncRNA HCG11 and tumor progression in patients with nasopharyngeal carcinoma showed that the expression of LncRNA HCG11 was higher in patients with advanced nasopharyngeal carcinoma(P<0.001);the expression of LncRNA HCG11 gradually increased with the increase of tumor stage,and there was a statistically significant difference(P<0.001);The expression of LncRNA HCG11 is higher in the cancer tissues of patients with metastatic nasopharyngeal carcinoma,and its expression level is positive with the high tumor burden and metastasis of nasopharyngeal carcinoma patients(P<0.01),and the prognosis of nasopharyngeal carcinoma patients with high expression of LncRNA HCG11 was worse(P<0.001).ConclusionLncRNA HCG11 was highly expressed in both cancer tissues of patients with nasopharyngeal carcinoma and nasopharyngeal carcinoma cell lines,and its expression is significantly related to the tumor burden,clinical stage and tumor metastasis of patients with nasopharyngeal carcinoma,and it was also related to the poor prognosis of nasopharyngeal carcinoma patients.The results suggest that LncRNA HCG11 has potential value as a prognostic biomarker for predicting the prognosis of patients with nasopharyngeal carcinoma.Part 2.The influence of the expression of LncRNA HCG11 on the biological function of nasopharyngeal carcinomaObjectiveTo clarify the effect of LncRNA HCG11 on the biological function of nasopharyngeal carcinoma,using in vitro cell function experiments and in vivo animal experiments,Methods(1)Clone formation assay and CCK8 proliferation assay were used to observe the effect of knockdown of LncRNA HCG11 on the proliferation ability of nasopharyngeal carcinoma cells;Flow cytometry was used to detect the effect of LncRNA HCG11 knockdown on the apoptosis of nasopharyngeal carcinoma cells;Transwell experiment analyzed the effect of LncRNA HCG11 knockdown Influence of the migration ability of nasopharyngeal carcinoma cells.(2)A subcutaneous transplantation tumor model of nasopharyngeal carcinoma in nude mice was constructed,and the effect of LncRNA HCG11 expression on the growth and progression of nasopharyngeal carcinoma xenograft tumor in nude mice was observed in vivo experiments;the expression of LncRNA HCG11 and Ki67 in the transplanted tumor tissue of nasopharyngeal carcinoma were analyzed by FISH and immunohistochemistry,respectively.Results(1)The results of the clonogenic assay revealed that when LncRNA HCG11 was knocked down,the proliferation ability of the two nasopharyngeal carcinoma cell lines CNE-2 and 5-8F were reduced(P<0.01);flow cytometry analysis showed that LncRNA HCG11 was inhibited the apoptosis of these two nasopharyngeal carcinoma cell lines increased;and after the addition of the chemotherapy drug cisplatin,the apoptosis of CNE-2 and 5-8F cells further increased(P<0.05);The results of transwell experiment showed,the migration ability of CNE-2 and 5-8F cells were reduced after LncRNA HCG11 knockdown(P<0.01).(2)In the nude mouse model of nasopharyngeal carcinoma xenograft tumor,when LncRNA HCG11 was stably knocked down,the tumor growth rate of nude mice becomes slower(P<0.001),and the tumor volume and weight of nude mice in the LncRNA HCG11 knockdown group were obvious smaller than the control group(P<0.01);Immunohistochemical analysis showed that the expression of Ki67 in the transplanted tumor tissue of nude mice in the LncRNA HCG11 knockdown group was significantly lower than that in the control group(P<0.05);FISH results showed that the expression level of LncRNA HCG11 was positively correlated with the weight of the transplanted tumor(P<0.05).ConclusionThe LncRNA HCG11 gene positively regulates the proliferation and migration of nasopharyngeal carcinoma cells and inhibits their apoptosis;the nude mouse xenograft model further confirms that the expression of LncRNA HCG11affects the growth and progression of nasopharyngeal carcinoma.Part 3.LncRNA HCG11 targeting miR-490-3p regulates the effect of MAP3K9/MAPK/ERK pathway on the progression of nasopharyngeal carcinomaObjectiveTo explore the main role of LncRNA HCG11 microRNA and its downstream molecules and signaling pathways,and to reveal the pathways of action of LncRNA HCG11 affecting the development of nasopharyngeal carcinoma and its key molecules.Methods(1)The Starbase database was used to predict the miRNA targeted by LncRNA HCG11,and the dual luciferase reporter gene experiment and qRT-PCR assay were used to further validate the regulatory role of LncRNA HCG11 on the possible target gene miRNAs.(2)Clone formation experiment and CCK8 experiment were used to observe the effect of LncRNA HCG11 targeting miR-490-3p on the proliferation of nasopharyngeal carcinoma cells;Transwell experiment was used to observe the effect of LncRNA HCG11 on the migration ability of nasopharyngeal carcinoma cells through miR-490-3p.(3)Prediction of miR-490-3p-regulated target genes by combining miRDB,TargetScan and miRTarBase databases,qRT-PCR observation of the expression levels of miR-490-3p possible target genes in nasopharyngeal carcinoma cells,application of dual luciferase reporter gene assay used to fuyrther to verify the targeting relationship between miR-490-3p and the possible target gene MAP3K9.(4)qRT-PCR and Western Blot were used to observe the effect of LncRNA HCG11 on the expression of MAP3K9/MAPK/ERK signaling pathway molecules through miR-490-3p.Results(1)Prediction from the Starbase database found that LncRNA HCG11 might regulate the expression of miR-490-3p,miR-1297 and miR-455-5p.Further qRT-PCR analysis showed that when LncRNA HCG11 was knocked down,the expression of miR-490-3p in nasopharyngeal carcinoma cells was up-regulated(P<0.01),while the expression of miR-1297 and miR-455-5p showed no significant changes,,suggesting that miR-490-3p may be the target gene of LncRNA HCG11;the dual luciferase reporter gene experiment further confirmed that LncRNA HCG11 had a targeted regulatory effect on miR-490-3p(P<0.05).(2)The results of clone formation experiment and CCK8 experiment showed that when LncRNA HCG11 was knocked down,the proliferation ability of nasopharyngeal carcinoma cell 5-8F decreased,and the number of cell migration was less.However,after the addition of miR-490-3p inhibitor,the proliferation of 5-8F cell ability was partially restored(P<0.01),and the migration ability of 5-8F cells was reversed(P<0.001);(3)Using TargetScan,miRDB and miRTarbase databases to predict target genes,the results showed that MAP3K9,NUFIP2,RBPJ,CLCC1,SMARCD1,and PAPPA may be the target genes of miR-490-3p;qRT-PCR analysis found that when miR-490-3p was knocked down,the expression of MAP3K9 in nasopharyngeal carcinoma cells 5-8F was significantly reduced,while the expression of NUFIP2,RBPJ,CLCC1,SMARCD1 and PAPPA did not show significant changes.Further dual luciferase reporter gene experiments confirmed that miR-490-3p targeted regulate the expression of MAP3K9 gene(P<0.05).(4)Analysis of the cancer tissues of clinical nasopharyngeal carcinoma patients showed that the expression of LncRNA HCG11 and miRNA-490-3p was negatively correlated(P<0.05),and the expression of LncRNA HCG11 was positively correlated with MAP3K9(P<0.05),and miRNA-490-3p was negatively correlated with MAP3K9(P<0.05).Further analysis by qRT-PCR and Western Blot confirmed that simultaneous inhibition of LncRNA HCG11 and miR-490-3p expression significantly increased the transcript level and protein expression level of MAP3K9 gene(P<0.01),and when simultaneously inhibiting the expression of LncRNA HCG11 and miR-490-3p,the phosphorylation level of ERK was significantly up-regulated(P<0.01).ConclusionLncRNA HCG11 regulates the expression of MAP3K9/MAPK/ERK signaling pathway molecules by targeting miR-490-3p,which affects the biological functions(proliferation,apoptosis and migration)of nasopharyngeal carcinoma cells,thereby promotes the progression of nasopharyngeal carcinoma. |
