| Objective:Oral squamous cell carcinomas(OSCC),one of the most common malignancy of oral and maxillofacial,is the eighth of common systemic malignancy worldwide and.the incidence of oral and maxillofacial malignant tumors accounted for more than 80%.Oral squamous cell carcinoma often develops rapidly,transfers to the regional lymph node in the early stages,occurs distant metastasis in the late stages,affects the quality of life of patients and even life-threatening seriously.Although we made a certain degree of breakthrough with the introduction of a multidisciplinary sequence of treatment,the survival rates remained at 50%for 5-year after surgery.Therefore,study of the molecular mechanism of the development of oral squamous carcinoma and to explore effective biomarkers and therapeutic targets,has important clinical significance and practical value.for us to prevention,control and treatment of oral squamous cell carcinoma.BCL2/adenovirus E1B19kd-interacting protein 3(BNIP3)is a kind of apoptosis protein which can combine with adenovirus ElB19kD protein each other and plays an important role in the development and progression of various tumor,Sexual cell death or apoptosis.BNIP3 gene can induce cell programmed death or apoptosis under hypoxia condition.Recent studies have found that BNIP3 can also cause autophagy.MicroRNA(miRNA)is a small molecular non-coding RNA of about 17?25nt,which can degrade or inhibit mRNA translation by binding to target mRNA to achieve negative regulation of target genes.Several studies have confirmed that the abnormal expression of miRNAs is associated with many kinds of cancer that regulate cancer-related genes,involved in tumor cell proliferation,apoptosis,invasion or angiogenesis,and play a role similar to oncogenes or tumor suppressor genes.Therefore,miRNAs become a new research direction for tumor diagnosis and treatment.MiR-150 is an important member of miRNA family.It is found that miR-150 is abnormal in various tumors,and the abnormal expression of miR-150 affects the proliferation,apoptosis and migration of tumor cells.We found that miR-150 and BNIP3 were negatively correlated in oral squamous cell carcinoma by bioinformatics methods,suggesting that there may be a targeted regulatory relationship between the two,but still need relevant experimental evidence to confirm.The aim of this study was to investigate the expression of BNIP3 in oral squamous cell carcinoma,the possible molecular regulation mechanism and the biological function in oral squamous cell carcinoma,and provide a new idea and basis for clinical treatment.This paper is divided into the following three parts.Part 1:The expression levels of BNIP3 and miR-150 in oral squamous cell carcinoma were detected at the cellular and tissue levels by Western Blot and PCR,then the relationship between miR-150 expression and BNIP3 was analyzed.Part 2:We built stable overexpression and silencing of miR-150 cell lines,then observe the effect of miR-150 on the biological behavior of CAL-27 cells to reveal the role of miR-150 in the development of oral squamous cell carcinomas,and provide a reference with early diagnosis,targeted therapy and improve the prognosis of oral squamous cell carcinomas.Part 3:Western Blot analysis of miR-150 expression changes in the regulation of BNIP3 protein,and research the effect of miR-150 on the autophagy level of oral squamous cell carcinoma in vitro.To provide a new target.of oral squamous cell carcinomas about the molecular therapy,we preliminary investigate miR-150 regulation the molecular mechanism of autophagy in oral squamous cell carcinomas.Methods:1.Expression of BNIP3 protein levels in oral squamous cancer cells CAL-27 exposed to normal oxygen(20%O2)and hypoxia(1%O2)and matching of eight cases of oral squamous carcinoma tissues and adjacent normal tissue samples(n = 8)were detected by Western Blot method.2.Expression of miR-150 and BNIP3 mRNA levels in oral squamous cancer cells CAL-27 exposed to normal oxygen(20%O2)and hypoxia(1%O2)and matching of eight cases of oral squamous carcinoma tissues and adjacent normal tissue samples(n = 8)were detected by real-time fluorescent quantitative PCR and the expression of miR-150 was correlated with BNIP3 were analyzed by Spearman analysis.3.The different concentrations of miR-150 mimics and miR-150 inhibitors were transfected into CAL-27 cells by riboFECTTMCP Reagent transfection reagent and establish stable overexpression and silencing of miR-150 cell lines.After 48h,the transfection efficiency of miR-150 was detected by qRT-PCR,and the best treatment concentration was identified and screened for later experimental study.4.The effects of miR-150 on the invasion and migration of oral squamous cell carcinoma were detected by cell scratch test,Transwell invasion assay and Transwell migration assay.5.To imitate tumor hypoxic microenvironment,we establish a model of oral squamous cell carcinoma under hypoxic culture in vitro.After CAL-27 cells were exposed to 1%O2 for 4h,8h,16h,24h,autophagosome fluorescent particles aggregation were observed by immunofluorescence tests,the number of autophagy bodies were observed by transmission electron microscopy,and the expression of autophagy protein BNIP3,LC3 ?/LC3 ?,Beclin-1 and P62 in the cells were detected by Western Blot.6.After miR-150 mimic and miR-150 inhibitor were transiently transfected to oral squamous cell carcinoma cell line CAL-27 under hypoxic,Western blot,cell immunofluorescence and electron microscopy were used to verify the effect of miR-150 on autophagy in oral squamous cell carcinoma.7.MiR-150 mimic and miR-150 inhibitor were transfected into CAL-27 cells.After 48h,the expression of BNIP3 protein was detected by Western Blot which verified that miR-150 had negative regulatory effect on BNIP3 in CAL-27cells under hypoxia.Results:1.Compared with normal oxygen(20%O2),the expression of BNIP3 mRNA in hypoxia(1%O2)was significantly up-regulated(P<0.05),and the expression level of BNIP3 mRNA in cancer tissue was also significantly higher than that in adjacent normal tissue samples(P<0.05).The expression level of BNIP3 mRNA in CAL-27 cells was significantly higher than that in 8 cases of adjacent normal tissue samples average expression(P<0.05).Western Blot results show that the protein expression of BNIP3 was the same as that of mRNA expression.2.The expression level of miR-150 in oral squamous carcinoma tissues was significantly lower than that in adjacent normal tissue samples(P<0.05),and the expression of miR-150 in CAL-27 cells exposed to hypoxia was significantly lower than that in normal oxygen(20%O2)(P<0.05).Compared with adjacent to carcinoma normal oral tissues,the expression of miR-150 in CAL-27 cells was significantly lower than that in 8 cases of normal tissue average expression(P<0.05).3.Spearman correlation analysis showed that the expression level of miR-150 was negatively correlated with the expression of BNIP3 in matched oral squamous cell carcinoma(r =-0.482,P<0.01).4.The results of real-time fluorescent quantitative PCR showed that the expression of miR-150 in CAL-27 cells with the final concentration of 100 nmol/L miR-150 mimic was highest(P<0.01).Therefore,100 nmol/L miR-150 mimic was selected as as the treatment concentration of miR-150 mimic in this experiment.The lowest expression of miR-150 was expressed in the 50 nmol/L miR-150 inhibitor group in CAL-27 cells(P<0.01).Therefore,50 nmol/L miR-150 inhibitor was selected as the treatment concentration of miR-150 inhibitor in this experiment and used for subsequent experimental study.5.Wound healing assay results show that the migration rate of CAL-27 cells transfected with miR-150 mimic was significantly slower than that of the control group,and the 24-hour mobility was respectively(14.28 ± 2.46)%and(42.86 ±8.31)%,the results have significant difference(P<0.05);The migration rate of CAL-27 cells transfected with miR-150 inhibitor was significantly faster than that of the control group,and the 24-hour migration rate was respectively(64.28 ± 9.73)%and(35.71 ± 2.59)%,the results have significant difference(P<0.05),indicating that overexpression of miR-150 can inhibit the migration of oral squamous cell carcinoma cell line CAL-27 in vitro.6.After transfection with miR-150 mimic CAL-27 cells for 24h,the number of cells crossing the Transwell membrane(32 ±12)was significantly lower than that of the control group(81 ± 13)cells,the difference was statistically significant(P<0.05),while transfection with miR-150 inhibitor CAL-27 cells for 24 h,compared with the control group(95± 11),the number of CAL-27 cells through Transwell cell membrane was increased significantly(173±15),the difference was statistically significant(P<0.05),indicating that overexpression of miR-150 can inhibit the migration of oral squamous cell carcinoma cell line CAL-27 in vitro.7.After transfection with miR-150 mimic for 24h,the number of cells penetrating the matrix gel(35 ±09)in CAL-27 cells was significantly lower than that in the control group(97± 32),and the difference was statistically significant(P<0.05),while transfection with miR-150 inhibitor for 24h,compared with the control group(99 ± 25),the number of CAL-27 cells penetrating matrix adhesive was increased significantly(195 ± 29),and the difference was statistically significant(P<0.05),indicating that overexpression of miR-150 can inhibit the invasive ability of oral squamous cell carcinoma cell line CAL-27 in vitro.8.After oral squamous carcinoma cells CAL-27 exposed to hypoxia(1%02)environment for 4h,8h,16h and 24h,autophagy in cells CAL-27 began to rise.With the extension of hypoxic exposure time,the expressions of LC3-?,BNIP3 and Beclin-1 were up-regulated and the expression of p62 protein was decreased.in autophagy model,the aggregation of LC3 punctate dots were visualized by fluorescence microscopy,the number of autophagic bubbles and autophagic bodies were significantly increased.by transmission electron microscopy.9.After transfection with miR-150 inhibitor CAL-27 cells exposed to hypoxia for 24h,compared with the normal controls,the number of aggregation of LC3 punctate dots,autophagic bubbles and autophagic bodies were significantly increased.by fluorescence microscope and transmission electron microscopy(P<0.05),suggesting that autophagy levels enhanced;while transfection with miR-150 inhibitor CAL-27 cells for 24h,compared with the control group,the number of aggregation of LC3 punctate dots,autophagic bubbles and autophagic bodies were significantly reduced by fluorescence microscope and transmission electron microscopy(P<0.05),suggesting that autophagy levels weakened.10.The expression of BNIP3 protein in CAL-27 cells transfected with miR-150 mimic for 48h was significantly lower than that in control group(P<0.05).In contrast,the expression of BNIP3 protein in CAL-27 cells transfected with miR-150 inhibitor was significantly higher than that in the control group(P<0.05),suggesting that miR-150 had negative regulatory effect on BNIP3 in the CAL-27 cells under hypoxia condition..Conclusion:The expression of miR-150 was low in both oral squamous cell carcinoma and hypoxia-exposed CAL-27 cells,while BNIP3 was highly expressed in oral squamous cell carcinoma and hypoxia-exposed CAL-27 cells.There was a negative correlation between miR-150 and BNIP3 expression.The overexpression of miR-150 can significantly inhibit the migration and invasion of oral squamous cell carcinoma.miR-150 inhibits the autophagy of oral squamous cell carcinoma by negatively regulating the expression of BNIP3 protein. |
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