| Oral squamous cell carcinoma(OSCC)is the most common oral cancer in the world.Currently,the use of surgery-based combination of radiation therapy,chemotherapy and other therapies to treat OSCC.Although some achievements have been made,the prognosis of patients with advanced OSCC is still not ideal.If it can be detected early,the long-term prognosis of patients can be greatly improved.Therefore,in-depth study of the molecular mechanism of OSCC and finding new and highly effective biomarkers are currently urgent problems to be solved.In recent years,studies have shown that long non-coding RNA(LncRNA)plays an important role in the occurrence,development and metastasis of tumors.LncRNA,more than 200 nucleotides in length,does not code for protein,and performs biological functions through competitive endogenous RNA(ce RNA)hypothesis and miRNA competitive combination.Studies have found that LncRNA HCG11 affects and participates in the proliferation,infiltration and metastasis of a variety of tumor cells,and its expression level and mechanism of action in OSCC are still unclear,and we need to further study.In this study,human OSCC tissue,oral squamous cell carcinoma cell line and subcutaneous heterotopic xenograft in nude mice were used as the research objects.We used quantitative real-time PCR(qRT-PCR),Western Blot,MTT,flow cytometry,immunofluorescence,double luciferase assay,immunohistochemistry,xenotopic tumor transplantation in nude mice and other experimental methods respectively.Firstly,the expression differences of LncRNA HCG11 and PTPRS in OSCC and adjacent normal tissues were studied,and then the effects of inhibition or overexpression of LncRNA HCG11 on proliferation of three OSCC cell lines(TSCCA,CAL-27 and Fa Du)were studied in vitro.In addition,dual luciferase assay was used to study the potential mechanism of LncRNA HCG11 affecting the proliferation ability of OSCC cell lines.We explored whether LncRNA HCG11/miR-455-5p molecular axis could participate in the development of OSCC through the targeted regulation of PTPRS.Finally,TSCCA cell lines with stable inhibition of LncRNA HCG11 were successfully transfected to construct subcutaneous heterotopic xenograft tumor in nude mice,and its effect on the proliferation of xenograft tumor in vivo was observed.By exploring the role of LncRNA HCG11 in the proliferation of oral squamous cell carcinoma,it is helpful to reveal the pathogenesis of OSCC,and also provide the basis and direction for the search of new biomarkers for OSCC.Part One The expression of LncRNA HCG11 and PTPRS in Oral Squa-mous Cell CarcinomaObjective:Oral squamous cell carcinoma is a malignant tumor of the oral cavity,which has a strong aggressiveness,a poor prognosis,and a low survival rate.Studies have shown that LncRNA HCG11 and PTPRS play an important role in tumor development and regulation.In this study,cancer tissues and normal adjacent tissues of patients with OSCC were collected,and the expression levels of LncRNA HCG11 gene and PTPRS were detected respectively,and the relationship between them and oral squamous cell carcinoma was analyzed.Methods:1.The qRT-PCR method was used to detect the expression levels of LncRNA HCG11 and PTPRS mRNA in cancer tissues and normal tissues adjacent to cancer.2.Western Blot was used to detect the expression level of PTPRS protein in cancer tissues and normal tissues adjacent to cancer.Results:1.The expression levels of LncRNA HCG11 in OSCC and adjacent normal tissues detected by qRT-PCR were 0.034±0.008 and 0.019±0.005,respectively.After statistical analysis,compared with adjacent tissues,the expression level of LncRNA HCG11 in OSCC tissues was significantly lower,and the difference was statistically significant(P<0.01).2.qRT-PCR detection of PTPRS mRNA expression levels in OSCC and normal tissues adjacent to cancer.The expression levels of PTPRS mRNA in normal tissues adjacent to oral cancer and OSCC tissues were 0.100±0.008and 0.022±0.002,respectively,and the difference was statistically significant.(P<0.01).3.Western Blot detection of PTPRS protein expression in OSCC and adjacent normal tissues.The relative expression levels of PTPRS protein in adjacent normal tissues and cancer tissues were 0.974±0.096 and 0.631±0.153,respectively.The expression of PTPRS in OSCC tissues was significantly reduced,and the difference was significant(P<0.01).Summary:1.The expression of LncRNA HCG11 gene in OSCC tissues was significantly lower than that in normal tissues adjacent to the cancer.2.The expression of PTPRS mRNA and protein in OSCC tissues was significantly lower than that in normal tissues adjacent to the cancer.3.LncRNA HCG11 and PTPRS may become new targets for the diagnosis of OSCC.Part Two The proliferation mechanism research of LncRNA HCG11/miR-455-5p regulation of the PTPRS on oral squamous cell carcinomaObjective:In vitro,multiple co-transfection systems were established to explore the regulatory effect of HCG11 on OSCC cells,and to verify the targeted binding relationship of miR-455-5p with HCG11 and PTPRS.We wanted to investigate the effect and mechanism of LncRNA HCG11/miR-455-5p/PTPRS molecular axis on the proliferation of OSCC.Methods:1.qRT-PCR was used to detect the expression level of LncRNA HCG11in three OSCC cell lines.LncRNA HCG11 interference experiment was conducted on the two cells with high expression level,and LncRNA HCG11overexpression experiment was conducted on the cells with low expression level.2.After inhibiting or overexpressing LncRNA HCG11,the effects of LncRNA HCG11 on the proliferation of OSCC cell lines were observed by MTT,qRT-PCR,Western Blot,flow cytometry,and immunofluorescence detection.3.qRT-PCR was used to detect the expression level of miR-455-5p in TSCCA and CAL-27 cells in the LncRNA HCG11 interference experiment.RNA binding protein immunoprecipitation(RIP)experiment was used to detect the enrichment of LncRNA HCG11 and miR-455-5p.Dual luciferase experiment verified the targeted binding relationship between LncRNA HCG11 and miR-455-5p.4.To analyze whether LncRNA HCG11 affects the proliferation of OSCC cells through miR-455-5p.In TSCCA and CAL-27 cell lines,miR-455-5p was subjected to functional rescue experiments to observe the reversal of miR-455-5p inhibitors on cell proliferation mediated by LncRNA HCG11interference.5.To analyze the effect of miR-455-5p on the proliferation of OSCC cell lines and its regulatory effect on PTPRS.qRT-PCR was used to detect the expression level of miR-455-5p in three OSCC cell lines TSCCA,CAL-27and Fa Du.After transfection of miR-455-5p inhibitor or miR-455-5p mimics,the effects of miR-455-5p on OSCC cell line proliferation,PTPRS mRNA and protein expression were observed by qRT-PCR,Western Blot technology,and MTT.The dual luciferase experiment verifies the targeted binding relationship between them.6.To analyze whether miR-455-5p affects the proliferation of OSCC cell lines through the downstream signaling pathway of PTPRS,namely epidermal growth factor receptor/phosphoinositide 3-kinase/protein kinase B(EGFR/PI3K/AKT).Performing functional rescue experiments on PTPRS to observe the reversal of cell proliferation mediated by miR-455-5p inhibitors by inhibiting PTPRS,and observe the changes in the levels of PTPRS and related proteins in downstream signaling pathways.7.To analyze the influence of LncRNA HCG11/miR-455-5p molecular axis on the downstream signaling pathway of PTPRS EGFR/PI3K/AKT.HCG11 si RNA and miR-455-5p inhibitor were co-transfected in TSCCA and CAL-27 cells.qRT-PCR detected the expression level of PTPRS mRNA in the cells after transfection.Western Blot detected the changes in the levels of PTPRS and related proteins in the downstream signaling pathways in the cells.Results:1.The expression level of LncRNA HCG11 in three cell lines LncRNA HCG11 had the highest expression level in TSCCA(1.30±0.13),followed by CAL-27 cells(1.00±0.00),and the lowest expression level in Fa Du cells,with a relative expression level of 0.74±0.08.Therefore,TSCCA and CAL-27 were selected for HCG11 interference experiments,and Fa Du was selected for HCG11 overexpression experiments.2.LncRNA HCG11 was involved in regulating the proliferation of OSCC cell lines.After inhibiting LncRNA HCG11 in TSCCA and CAL-27 cell lines,the cell proliferation ability was significantly enhanced;after overexpression of LncRNA HCG11 in Fa Du cells,the cell proliferation ability was signif-icantly weakened(P<0.05).3.Regulation of the influence of HCG11 on the expression level of miR455-5p and their targeted binding verification.After inhibited the LncRNA HCG11 of TSCCA and CAL-27,the expression level of miR-455-5p was significantly increased(P<0.01),and the RIP experiment showed that LncRNA HCG11 and miR-455-5p were in a highly enriched state.Dual luciferase experiments showed that there was a targeted binding relationship between them.4.LncRNA HCG11 affected the proliferation of OSCC through miR-455-5p.The expression level of miR-455-5p was negatively regulated by LncRNA HCG11.miR-455-5p inhibitor could partially reverse the cell viability enhancement,Ki67 increase,Cyclin D1 and E2F1 increase,and p21decrease caused by LncRNA HCG11 inhibition.5.The effect of miR-455-5p on the proliferation of OSCC and its regulatory effect on PTPRS.miR-455-5p inhibitor could significantly increase the expression levels of PTPRS mRNA and protein in Fa Du and CAL-27 cells,and significantly reduce cell viability.Transfection of miR-455-5p mimics in TSCCA cells significantly reduced the expression levels of PTPRS mRNA and protein,and significantly enhanced cell viability.Dual luciferase assay showed a targeted binding relationship between them.6.miR-455-5p affected the proliferation of OSCC cell lines through the downstream signaling pathway of PTPRS,namely epidermal growth factor receptor/phosphoinositide 3-kinase/protein kinase B(EGFR/PI3K/AKT).The inhibition of miR-455-5p expression could significantly increase the expression level of PTPRS in Fadu and cal-27 cell lines,decrease the cell proliferation ability,and decrease the expression levels of p-EGFR and p-Akt,but at the same time,the inhibition of PTPRS gene could partially reverse the above cell activities induced by miR-455-5p inhibition.7.The effect of LncRNA HCG11/miR-455-5p molecular axis on the downstream signaling pathway of PTPRS EGFR/PI3K/AKT.After the transfection of LncRNA HCG11 si RNA,the expression level of PTPRS in TSCCA and CAL-27 cell lines was significantly decreased,however,the expression level of PTPRS was significantly increased by inhibiting the expression of miR-455-5p while inhibiting HCG11 si RNA(P<0.05).The expression results of EGFR/PI3K/Akt pathway related proteins showed that the expression levels of p-EGFR and p-Akt were higher in HCG11 gene suppressed cells than in NC si RNA transfected cells,but there was no significant change in EGFR and Akt.When the expression levels of miR-455-5p and HCG11 were inhibited at the same time,the expression levels of p-EGFR and p-Akt were significantly decreased(P<0.05).Summary:1.LncRNA HCG11 participated in regulating the proliferation of OSCC cell lines.The cell proliferation ability enhanced after inhibition HCG11.The proliferation ability was significantly weakened by overexpression HCG11.2.LncRNA HCG11 and miR-455-5p had a targeted binding relationship.The expression of miR-455-5p is negatively regulated by LncRNA HCG11.3.PTPRS and miR-455-5p had a targeted binding relationship.The expression of PTPRS is negatively regulated by miR-455-5p.4.The expression of PTPRS was positively regulated by LncRNA HCG11,which acted as a tumor suppressor gene,and the tumor suppressor mechanism may be as follows:LncRNA HCG11 could adsorb miR-455-5p,weaken the expression level of miR-455-5p,increase the expression of downstream target gene PTPRS,inhibit the activation of downstream signaling pathway EGFR/PI3K/Akt,and thus inhibit tumor growth.Part Three The study of the effect of LncRNA HCG11 on oral squam-ous cell carcinoma transplantation in nude miceObjective:Vivo experiments were conducted to further verify the effect of LncRNA HCG11 on tumor-bearing in nude mice with oral squamous cell line.Methods:1.p RNAH1.1 vectors carrying HCG11 sh RNA or NC-sh RNA sequences were transfected into TSCCA cells by Lipofectamine 2000.After screening,stable cell lines with HCG11 gene inhibition were obtained.2.TSCCA cells(1×10~7)transfected with HCG11 sh RNA(or NC sh RNA)were subcutaneously injected into nude mice,which were divided into three groups:TSCCA group,NC sh RNA group and HCG11 sh RNA group.The tumor size was measured every 4 days as the tumor began to form.Mice were sacrificed at 27 days.Tumors were collected and weighed.3.The expression levels of LncRNA HCG11,miR-455-5p and PTPRS mRNA in tumor tissues of each group were detected by qRT-PCR.4.Western Blot was used to detect the expression levels of cyclin D1,E2F1,p21,PTPRS,P-EGFR,EGFR,P-Akt and AKT proteins in tumor tissues of each group.5.Immunohistochemistry was used to detect the expression of Ki67protein in each group.Results:1.HCG11 gene inhibition promoted tumor growth in nude mice.Compared with NC sh RNA group,the HCG11 inhibition group showed faster tumor growth and greater tumor weight(P<0.01).2.The expression of HCG11,PTPRS and miR-455-5p in nude mice transplanted tumors.Compared with NC sh RNA group,the expression levels of HCG11 and PTPRS in HCG11 inhibition group were significantly lower,while miR-455-5p showed higher expression in HCG11 inhibition tumor tissues(P<0.01).3.The expression of proliferation-related proteins and signaling pathway-related proteins in transplanted tumors in nude mice.Western Blot results showed that compared with the control group,HCG11 down-regulation resulted in a significant decrease in p21 and an increase in cyclin D1 and E2F1expression in tumor tissues.At the same time,the expression of p-EGFR and p-Akt in HCG11 inhibition tumor tissues was increased while the level of PTPRS was decreased.4.The expression of Ki67 protein in transplanted tumors in nude mice.Immunohistochemical results showed that the positive Ki67 expression rate was significantly increased in HCG11 inhibition group.Summary:1.TSCCA oral squamous cell carcinoma cell line with stable inhibition of LncRNA HCG11 was successfully constructed,and subcutaneous xenograft tumor model was successfully constructed in nude mice.2.The inhibition of LncRNA HCG11 could accelerate the growth and increase the weight of Transplantation tumor in nude mice.3.After inhibiting LncRNA HCG11,the positive expression of Ki67 in transplanted tumors increased significantly,indicating that the proliferation activity of OSCC cells was enhanced.4.After the inhibition of LncRNA HCG11,the expression levels of HCG11 and PTPRS were decreased in the transplanted tumors,while the expression of miR-455-5p was higher,and the expression of p-EGFR and p-Akt in its downstream signaling pathway was enhanced instead.These results indicated that LncRNA HCG11/miR-455-5p axis targeting regulation of PTPRS affected the progress of OSCC.Conclusions:1.The expressions of LncRNA HCG11 and PTPRS in OSCC tissues were significantly lower than those in adjacent normal tissues.2.LncRNA HCG11 was involved in the regulation of the proliferation of Os CC cell lines,and the cell proliferation ability was enhanced when LncRNA HCG11 was inhibited.Overexpression of LncRNA HCG11significantly reduced cell proliferation.3.miR-455-5p had a targeted binding relationship with LncRNA HCG11and PTPRS,respectively.The expression of miR-455-5p was negatively regulated by LncRNA HCG11.The expression of PTPRS was negatively regulated by miR-455-5p.4.LncRNA HCG11 was low expressed in OSCC,as a tumor suppressor gene,LncRNA HCG11 may adsorb miR-455-5p,increase the expression of downstream target gene PTPRS,inhibit the activation of downstream signaling pathway EGFR/PI3K/Akt,and inhibit tumor growth.5.Inhibition of LncRNA HCG11 significantly accelerated the growth of subcutaneous heterotopic xenograft of human OSCC cell line in nude mice. |
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