MMP1\7 Mediate Proliferation Metastasis And Invasion Of Tongue Squamous Cell Carcinoma

ObjectiveTo investigate whether MMP1/7 was elevated expression in tongue spuamous cell carcinoma and whether it could promote proliferation,metastasis and invasion of tongue carcinoma cells,we had this research.MethodsPart I Five tongue cancer samples and their respective adjacent non-tumor tissues after surgical resection operation were collected to explore the oncogenes by RNA-seq measure.To make sure the RNA-seq measure result that MMP1/7 were two of the oncogenes of tongue cancer,another 53 paired samples were utilized to quantify the mRNA expression of MMP1/7 by RT-PCR trials.Futhermore,IHC trials was also for the oncogene role of MMP1\7.PartII The siRNA-MMP1\7 targeting MMP1\7 gene sequence were designed and synthesized in vitro and the overexpression plasmid of MMP1\7(pCDH-CMVMCS-EF1�Puro-MMP1\7)were testified having the right gene sequences.They were transfected into CAL-27 or SCC9 cells line.The expression level of MMP1\7 mRNA and protein were testified by Real-time PCR and Western Blot.CCK8 and plate cloning assays were used to detect the proliferation ability of CAL-27 or SCC9 cells;transwell and wound healing assays were utilized to check cells metastasis and invasion ability after silencing or overexpressing MMP1/7 in vitro.PartIII Our researching team have foud that NOTCH and WNT signal pathway could mediate proliferation,metastasis and invasion of tongue squamous cell carcinoma.And the papers before reported that MMPs could act as the targeting gene of NOTCH and WNT signal pathway to regulate cancer cells.To investigate this connection in tongue cancer,we detected MMP1 protein expression after silencing NOTCH and WNT signal pathway by Western Blot.Also,as some papers held the message that MMP7 interaced with TIMPs/MMPs/CDH1 in many tumors,we investigated this by RT-PCR.At the same time,We explored the possible relations between MMP1/7 and TIMP1 in tongue cancer.Part IV As the results of the trails in vitro,to investigate the oncogene role of MMP7 in vivo,we explored the metastasis and invasion ability of the high metastatic tongue cancer cell line(LN4)from our researching team before by shRNA targeting MMP7.ResultsPart I RNA-seq measure revealed MMP1/7 were overexpressed in tongue squamous cell carcinoma compared to their respective adjacent non-tumor tissues,which was in line with the IHC results.Real-time PCR trials showed that overexpression of MMP7 was detected in 45 of 53(84.9%)TSCC sample compared to their adjacent tissues;MMP1 was 50 of 53(94.33%).Part II The siRNA-MMP1\7 knocked down MMP1\7 and PCDH-MMP1\7 overexpressed MMP1\7 successfully both at mRNA and protein level.CCK8 and plate cloning assays showed the growth of CAL-27 cells transfected with siRNA-MMP1 and the growth of CAL-27 and SCC9 cells transfected with siRNA-MMP7 was significantly suppressed compared to the negative control group.And aslo,the metastasis and invasion ability of CAL-27 or SCC9 cells were limited after silencing MMP1\7,Which were testified by transwell and wound healing assays.The PCDHMMP1\7 groups got the opposite results.Part III when knocked down the NOTCH and WNT signal pathway in CAL27 cells,MMP1 protein expression was decreased obviously.The RT-PCR illustrated a lower expression of MMP9,TIMP2.3.4 and a higher expression of MMP1/3/10/13,TIMP1,CDH1 at mRNA level after silencing MMP7 in CAL27 cell;a lower expression of MMP9 and a higher expression of MMP3.10.13,TIMP1,CDH1 at mRNA level after silencing MMP7 in SCC9 cell.Of all the 15 paired samples,13 tumors TIMP1 mRNA expressed higher than compared normol tissue and 11 tumors had an enhanced expression of MMP1/7 mRNA and a lower level of TIMPs mRNA at the same time.Part IV In vivo,the lymph node metastasis rate of the nude mice with high metastatic tongue cancer cell transplantation tumor was lower in the MMP7 silencing group.Conclusions1.MMP1\7 were elevated expression in the tongue cancer compared with in the normal tissue.2.The ability of the proliferation,migration and invasion of tongue cancer cells could reduce or enhance while silencing or overexpressing MMP7.3.MMP1 mediated tongue cancer proliferation,migration and invasion by acting as the targeting gene of NOTCH and WNT signal pathway or by degrating TIMP1.MMP7 might cooperate with other genes(CDH1,TIMPs,and individual MMP1.3.9.10.13)in regulating tongue cancer progression.4.MMP7 promotes the migration and invasion of tongue cancer cells in nude mouse high metastasis model.Herein,MMP1\7 may be regarded as two targeted therapy biomarkers to cure tongue cancer patients.