The Expression And Clinical Significance Of MMP-14 In Laryngeal Squamous Cell Carcinoma And Overexpression Of MiR-133a Promotes Proliferation Of Laryngeal Carcer By Targeting MMP-14

Objective: Laryngeal cancer is one of the most common tumors in the world and has the highest mortality.The epidemiological studies have showed the incidence of laryngeal carcinoma was higher in men and people aged more than 40 years,and more than 90% of laryngeal carcinoma was laryngeal squamous cell carcinoma(laryngeal squamous cell carcinoma,LSCC).Although a great deal of progress on early diagnosis and treatment of laryngeal cancer has been made over the last several decades,the five-year survival rate remained the same,and no obvious early symptoms and lack of reliable early diagnosis biomarkers may be the important reasons.The laryngeal cancer patients would die of recurrence and metastasis due to the unsatisfactory surgical effects.The pathogenesis of laryngeal cancer is not clear yet,therefore,it is very important clinical significance to explore the pathogenesis,development and invasion of laryngeal carcinoma cell.Researchs have shown that matrix metalloproteinases(MMPs)might play a very important role in the invasion and metastasis of laryngeal carcinoma by taking part in the degradation process of extracellular matrix.The membrane of matrix metalloproteinase(membrane type-1matrix metalloproteinase,MT1-MMP),as an important member of MMPs,play a very important role in the process of development of laryngeal carcinoma.Therefore,the main purpose of this study is to understand the expression of MMP-14 in LSCC tissue and its clinical significance,and to further explore the relationships between the expression of MMP-14 protein and the occurrence,development and prognosis of LSCC.This study will provide a new biomarker that can make early diagnosis and evaluating prognosis of laryngeal cancer,as well as provide a scientific basis for early diagnosis,follow-up monitoring and targeted treatment of laryngeal cancer.Methods: We selected 40 LSCC patients following total / partial laryngectomy,and collected their carcinoma tissue and normal mucosa tissues after approved by the ethics committee of the First Affiliated Hospital of Medical University Of Anhui and the informed consents were obtained from all patients.We used self-administrated questionnaires to abstract the basic information,clinical data,pathological types of cancer tissues,treatment,and postoperative survival data.The expressions of MMP-14 protein in cancer tissue and adjacent mucosal tissues were detected by immunohistochemical staining,and the expressions of MMP-14 m RNA were detected by q RT-PCR method.The changes in MMP-14 protein expression in laryngeal squamous cell carcinoma and normal mucosa tissues were determined by Western blotting method.We used Microsoft Office Excel 2007 to store medical records and laboratory data and IBM SPSS Statistics 19 software to perform data analyses.The difference of MMP-14 gene expression between different groups were detected by two independent samples t-test or Kruskal-Wallis Test,while the positive expression rate of MMP-14 gene were detected by Chi-square continuity correction formula and Fisher exact probability analysis.The interactive effects were also analyzed by crossover analysis.Were also used Kplan-meier to analyse the prediction effect of MMP-14 protein expression on postoperative survival rate of LSCC patients and used Log Rank test to compare the difference of cumulative survival rate between different MMP-14 protein expression level.In the last we used the COX proportional hazard model to estimate the mortality risk of positive expression of MMP-14 protein compared with negative expression.Results: Of 40 LSCC patients,30 patients had significantly higher expression of MMP-14 in laryngeal carcinoma tissues than that in adjacent normal mucosa,4patients had significantly lower expression of MMP-14 in laryngeal carcinoma tissues than that in adjacent normal mucosa,and 6 patients had similar MMP-14 expression between laryngeal carcinoma tissues and adjacent normal mucosa.There were no difference on MMP-l4 gene expression between different age group,smoking group,alcohol drinking group,and clinical type group(P>0.05),while significant differences were found between dfferent clinical stages,different differentiated degree,and different lymph node metastasis status(P<0.05).The MMP-14 gene expression level was significantly weaker in low clinical stage(I or II)than that in high clinical stage(III or IV)(Z=2.153,P=0.031),higher in poorly differentiated LSCC patients than that in high differentiated LSCC patients(Z=4.364,P<0.001),and higher in LSCC patients with positive lymph node metastasis than that in negative lymph node metastasis(Z=3.544,P<0.001).The MMP-l4 protein expression was significantly higher in laryngeal carcinoma tissues than that in adjacent normal mucosal tissue,and there was consistent expression on MMP-l4 protein and MMP-l4 gene.The relationships between the expression rate of MMP-14 protein and clinical stages,lymph node metastasis and tumor differentiation were statistically signifant.The patients with higher clinical stages(? and ?)(OR=5.44,95%CI=1.14-25.95,lower tumor differentiation(OR=9.33,95%CI=1.65-52.92),and positive lymph node metastasis(OR=15.55,95%CI=1.73-139.65)were more likely to have a positive MMP-l4 protein expression.The results of crossover analysis showed that there were significant interaction effects between any two of these three variates,i.e.,clinical stages,lymph node metastasis,and differentiated degree.There significant interaction effect between clinical stages and lymph node metastasis on positive MMP-l4 protein expression(c 2=8.801,P=0.032),and the positive expression rate was 32(95%CI= 2.76-370.81)times higher in patients with high clinical stages and positive lymph node metastasis compared to with patients with low clinical stages and negative lymph node metastasis.There significant interaction effect between clinical stages and differentiated degree on positive MMP-l4 protein expression(c2=9.322,P=0.002),and the positive expression rate was 24(95%CI = 2.21-260.29)times higher in patients with high clinical stages and poor differentiated degree compared to with patients with low clinical stages and high differentiated degree.There significant interaction effect between lymph node metastasis and differentiated degree on positive MMP-l4 protein expression(c2=9.322,P=0.002).Interestingly,the interaction effects were observed only in non-glottic LSCC,but but not in glottic LSCC.The results of survival analysis showed that the average survival time of 40 LSCC patients was 3.84(SE=0.43)years,and the cumulative survival rate of 1,3 and 5 years was 82.50%,55% and 32.51% respectively.The results of Kplan-meier analysis showed that the patients with strong positive expression of MMP-14 protein had poorer survival rate comparing with patient with weak positive or negative expression,and the results of Log Rank test showed that the difference on survival rate between strongly positive group and weakly positive(c2=21.17,P<0.001),strongly positive group and negative group(c2=20.843,P<0.001)were siginifant;while between weak positive group and negative group was no significant(c2=1.468,P=0.226).The results of COX proportional hazards model showed that the patient with strongly positive MMP-14 protein expression had signifantly higher mortality risk comparing with patients with negative expression(HR=13.43,95%CI=4.63-38.94),as well as with weakly positive expression(HR=8.20,95%CI=3.21-20.94).However,there was no significant difference in the mortality risk between the weakly positive and the negative patients(HR=1.64,95%CI=0.70-3.81).Conclusion: The study found that the MMP-14 protein was commonly observed in the carcinoma cell cytoplasm and tumor interstitial fibroblast cells,but not in normal mucosa cells.The expression of MMP-14 gene and protein was significantly higher in laryngeal carcinoma tissues than that in adjacent normal mucosa.There were siginiant relationships between the expression of MMP-14 gene and protein and three important clinical pathological indexes,i.e.,clinical stage,tumor differentiation,lymph node metastasis,and the three clinical pathological indexes had signifant interaction effect between each others.Whatmore,the patients with strong positive expression of MMP-14 protein had poorer survival rate comparing with patient with weak positive or negative expression,while there was no difference in the cumulative survival rate between the patients with weak positive expression and those with negative expression.Our results have great clinical significance and may provide scientific guiding for early diagnosis and prognosis of laryngeal cancer.Objective:Laryngeal cancer is one of the common malignant tumors in the head and neck,accounting for 5.7%-7.6% of the total malignancy.The incidence rate was increasing year by year.Laryngeal cancer patients had a poor prognosis due to the fact that most patients were diagnosed in the middle and late stage.However,the molecular pathogenesis was not clear yet,it is of great significance to explore the molecular mechanism of the laryngeal carcinoma.Micro RNAs(mi Rs)are a large family of non-coding RNAs,which play an important role in regulating gene expression.Clinical evidences have showed that mi Rs played an important role in the development of tumors as a biological marker.This study was to investigate the expression of mi R-133 a and MMP-14 in LSCC and to analyze the relationship between the expression of mi R-133 a and the overexpression of MMP-14 protein in LSCC.We then aimed to explore the effects of Mi R-133 a targeted-regulating MMP-14 on the laryngeal cancer cell proliferation and its potential mechanism.Methods:We selected 40 LSCC patients following total / partial laryngectomy,and collected their carcinoma tissue,normal mucosa tissues,and Hep-2 laryngeal carcinoma cell lines.Mi R-133 a mimics and inhibitors were transfected into Hep-2 laryngeal carcinoma cells by Lipofectamine TM 2000 reagent,and evaluated all related indexes after 36-48 hours.The histopathological changes of LSCC cells were observed using HE staining and immunohistochemical staining.The expression of mi R-133 a and MMP-14 in LSCC and Hep-2 laryngeal carcinoma cells was determined by q RT-PCR.The expression of MMP-14 protein in LSCC and Hep-2 laryngeal carcinoma cells was detected by Western blotting.MTT assay was used to detect the effect of mi R-133 a mimics on the proliferation of Hep-2 laryngeal carcinoma cells.Results:Our results indicated that the MMP-l4 gene expression was significantly higher in laryngeal carcinoma tissues than that in adjacent normal mucosal tissue(t=12.19, P<0.001);The MMP-l4 protein expression was very distinct in the cytoplasm and mesenchyme of LSCC carcinoma cells.The relationships between the expression rate of MMP-14 protein and clinical stages,lymph node metastasis and tumor differentiation,and survival rate were statistically signifant,which indicated that the over-expression of MMP-14 in LSCC carcinoma tissue might be significantly associated with the development,invasion and metastasis of carcinoma cell.The expression of mi R-133 a was significantly decreased in the laryngeal carcinoma tissues comparing with the adjacent normal mucosal tissue(t=25.68,P<0.001).The high expression of mi R-133 a was observed in the Hep-2 laryngeal carcinoma cells after transiently transfected by mi R-133 a mimics,and mi R-133 a expression level was sigifantly higher in mi R-133 a mimics group comparing with negative control group(t=63.83,P<0.001)and blank control group(t=62.00,P<0.001).The high expression of mi R-133 a could inhibit the proliferation activity of Hep-2 laryngeal carcinoma cells,and the proliferation activity of Hep-2 laryngeal carcinoma cells was sigifantly lower in mi R-133 a mimics group comparing with negative control group(t=7.56,P<0.001)and blank control group(t=8.11,P<0.001).The expression of MMP-14 m RNA and protein in Hep-2 laryngeal carcinoma cells might be inhibited by the high expression of mi R-133 a.1)The expression of MMP-14 m RNA was sigifantly lower in mi R-133 a mimics group comparing with negative control group(t=16.23,P<0.001)and blank control group(t=14.82,P<0.001),while no difference on the expression of MMP-14 m RNA was observed between negative control group and blank control group(t=0.21,P=0.833);2)The expression of MMP-14 protein was sigifantly lower in mi R-133 a mimics group comparing with negative control group(t=19.43,P<0.001)and blank control group(t=15.20,P<0.001),while no difference on the expression of MMP-14 protein was observed between negative control group and blank control group(t=1.23,P=0.211).On the contrary,the expression of MMP-14 m RNA and protein in Hep-2 laryngeal carcinoma cells might be increased by inhibiting the expression of mi R-133 a.1)The expression of MMP-14 m RNA was sigifantly higher in mi R-133 a inhibitors group comparing with negative control group(t=26.63,P<0.001)and blank control group(t=25.46,P<0.001),while no difference on the expression of MMP-14 m RNA was observed between negative control group and blank control group(t=1.32,P=0.189);2)The expression of MMP-14 protein was sigifantly lower in mi R-133 a inhibitors group comparing with negative control group(t=41.273,P<0.001)and blank control group(t=42.85,P<0.001),while no difference on the expression of MMP-14 protein was observed between negative control group and blank control group(t=1.18,P=0.243).The results of Kplan-meier analysis showed that the patients with low expression of mi R-133 a had poorer survival rate comparing with patient with high expression,and the results of Log Rank test showed that the difference were siginifant(?2=14.658,P<0.001).The mortality risk of patients with low expression of mi R-133 a was signifantly increased(HR=3.949,95%CI=1.815-8.589)comparing with those with high expression.Conclusion:In summary,our study suggests that the expression of mi R-133 a is significantly decreased in the laryngeal carcinoma tissues,which negatively targeted-regulating of MMP-1 protein expression and dramatically increase the MMP-1 protein expression in laryngeal cancer cell.Mi R-133 a and MMP-14 may paly a important role in the development,infiltration,and metastasis of laryngeal cancer cell,they together paly a important role in the prediction and assessment of the prognosis.