| Cervical cancer is one of the common malignant tumors in women.Among the common pathological types of cervical cancer,squamous cell carcinoma accounts for 85% and adenocarcinoma accounts for 15%.The main cause of the disease is the persistent infection of HPV with high risk,HPV16 is mainly related to squamous cell carcinoma,HPV18 is mainly related to adenocarcinoma,but the process of cervical cancer development and development is not the only cause of HPV persistent infection.This process is a complex process involving multiple genes,and multiple regulatory pathways work together.The specific molecular mechanism is still unclear.Thrombospondin 2(THBS2)is a glycoprotein that mediates cell-cell or cell-interstitial action and inhibits angiogenesis in a variety of tumors.Matrix metalloproteinases(MMPs)family is a family of proteases that can affect tumorinvasion and metastasis by degrading extracellular matrix.In our previous studies,it has been confirmed by bioinformatics that THBS2 is a target gene of miR-1246,the expression level of THBS2 in cervical squamous cell carcinoma SiHa cells transfected with miR-1246 was significantly decreased in vitro,and the characteristics of promoting cell invasion and migration were observed;The expression level of THBS2 in SiHa cells of cervical squamous cell carcinoma with miR-1246 was down-regulated,and the expression of MMP2 and MMP9 in nude mice was decreased in vitro.Promote invasion and migration of SiHa cells after overexpression of THBS2,and it is unclear whether there is a regulatory pathway between THBS2 and MMPs that affects the invasion and metastasis of cervical squamous cell carcinoma.This study was conducted to detect whether there is a difference in the expression of MMPs family after overexpressing THBS2,and selected types with a difference to over-express.It was confirmed whether there was a difference in the expression of THBS2 after overexpression of MMPs.This study provides a basis for miR-1246 to affect the expression of MMPs by regulating THBS2 and participate in the invasion and migration of cervical cancer,and propose a new idea for gene therapy of cervical cancer.,and propose a new idea for gene therapy of cervical cancer.Part I Differential expression of MMPs in cervical cancer SiHa cells after overexpression of THBS2Objective: After overexpression of thrombospondin 2(THBS2),the expression of matrix metalloproteinases(MMPs)in the cervical squamous cell carcinoma SiHa cells was differentially expressed.Methods: The groups were as follow.The cervical squamous cell carcinoma SiHa cells Transfected with THBS2 lentivirus were Transfected into the THBS2-LV group,and the cervical squamous cell carcinoma SiHa cells Transfected with the negative control virus were NC group,The cervical squamous cell carcinoma SiHa cells that were not Transfected with virus were blank group,and the cervical squamous cell carcinoma SiHa cells were Transfected with THBS2 overexpression lentivirus.The mRNA expression level of THBS2 was detected by real-time quantity PCR,and detected whether the mRNA expression levels of MMP1,MMP2,MMP7 and MMP9 were different after transfection;Western Blot was used to detect whether the expression of protein level was increased before and after THBS2 transfection overexpression of lentivirus,and whether the expression of MMP1,MMP2,MMP7 and MMP9 protein level was changed after transfection.Result:The experimental results shows that,the relative expression of THBS2 mRNA in the THBS2-LV group was significantly higher than that in the NC group and the blank group after transfection of THBS2 overexpressing lentivirus.There was no significant difference in the expression of MMP1,MMP2 and MMP7 mRNA expression.The mRNA level of MMP9 was significantly lower than that of NC group and blank group,and the difference was statistically significant.In the Western Blot experiment,the relative expression of THBS2 protein in the THBS2-LV group was significantly higher than that in the NC group and the blank group.The protein expression level of MMP1,MMP2 MMP7 and MMP9 in THBS2-LV group was significantly lowerthan that in NC group and blank group,and The difference was statistically significant.Conclusion: THBS2 may inhibit the expression of MMP1,MMP2,MMP7 and MMP9 in cervical squamous cell carcinoma SiHa cells.Part II Effect of overexpression of MMP1/MMP2 on invasion and migration of cervical squamous cell carcinoma Si Ha cellsObjectiv : Overexpression of MMP1 and MMP2 was overexpressed to investigate the effects of MMP1 and MMP2 on proliferation,apoptosis,invasion and migration of cervical squamous cell carcinoma Si Ha cells.Methods: The second part was grouped as follows.The cervical squamous cell carcinoma Si Ha cells Transfected with MMP1 overexpressed lentivirus were MMP1-LV group,and the cervical squamous cell carcinoma Si Ha cells Transfected with MMP2 overexpressing lentivirus were MMP2-LV group,The cervical squamous cell carcinoma Si Ha cells Transfected with the negative control virus were NC group,and the non-Transfected cervical squamous cell carcinoma Si Ha cells were blank group.Through Statistical analysis,two types of proteases,MMP1 and MMP2,were selected,and MMP1 and MMP2 were overexpressed in Si Ha cells by lentiviral transfection.Real-time quantity PCR and Western Blot were used to detect wether the expression of MMP1 and MMP2 m RNA and protein levels in Si Ha cells were increased after transfection of lentivirus.CCK8 method was detected the difference in the ability of cell proliferation of Si Ha cells after transfected by MMP1 and MMP2 overexpressing lentivirus.Flow cytometry was used to detect the apoptosis of Si Ha cells Transfected with MMP1 or MMP2 overexpressing lentivirus.Difference of invasion ability of Si Ha cells after transfection of MMP1 or MMP2 overexpressing lentivirus by Transwell method.Differences in migration ability of Si Ha cells after transfection of MMP1 and MMP2 overexpressing lentivirus by cell scratch assay.Result:The experimental results is as follows,after transfection of MMP1 overexpressing lentivirus,the relative expression of MMP1 m RNA in MMP1-LV group was significantly higher than that in NC group and blank group.The relative expression of MMP2 m RNA in MMP2-LV group was significantly higher than that in NC group and blank group.In the Western Blot experiment,the relative expression of MMP1 protein in MMP1-LV group was significantly higher than that in NC group and blank group.The relative expression of MMP2 protein in MMP2-LV group was significantly higher than that in NC group and blank group.But the m RNA and protien expression level of THBS2 in MMP1-LV group and MMP2-LV group has no significantly difference than that in NC group and blank group.The results of CCK8 showed that the cell proliferation ability of MMP1-LV group was significantly higher than that of NC group and blank group.The cell proliferation ability of MMP2-LV group was significantly higher than that of NC group and blank group.Flow cytometry was used to detect apoptosis.The apoptosis of MMP1-LV group was inhibited compared with NC group and blank group.The apoptosis of MMP2-LV group was inhibited compared with NC group and blank group.according to Transwell results,the cell invasion ability of MMP1-LV group was significantly higher than that of NC group and blank group.The cell invasion ability of MMP2-LV group was significantly higher than that of NC group and blank group.The results of cell scratch test showed that the cell migration ability of MMP1-LV group was significantly higher than that of NC group and blank group.The cell migration ability of MMP2-LV group was significantly higher than that of NC group and blank group.Conclusion: MMP1 and MMP2 enhance the proliferation,invasion and migration of cervical squamous cell carcinoma Si Ha cells and inhibit their apoptosis.Therefore,MMP1,MMP2,MMP7,and MMP9 may be target genes of THBS2 in cervical cancer,and MMP1 and MMP2 may be oncogenes of cervical cancer. |
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