The Inhibition Of MMP-10 And MMP-13 Effects On Cell Function Of Human Tongue Squamous Cell Carcinoma Cell Line

In the world,oral cancer accounts for about 3% of the world's malignant tumors,tongue cancer is the most common oral cancer.Although the rapid development of science and technology of the medical ladder,and treatment of tongue cancer also get continuous improvement,the five-year survival of patients is still low,postoperative quality of life is also unsatisfactory.As a result,it is a hot research topic to study the molecular mechanism of tongue carcinogenesis,invasion and metastasis in order to reach the goal of molecular targeted therapy of tongue cancer and improve patients' quality of life and prognosis.Our group previously found that MMP-10,MMP-13 are both expressed at high levels in tongue cancer tissue by RNA-seq,and they are colosely related to tongue cancer.Therefore,we plan to investigate the changes of the proliferation,invasion and migration abilities after inhibition of MMP-10 and MMP-13,hoping to further clarify the role of MMP-10/MMP-13 in tongue cancer cells(CAL-27),exploring their possible mechanisms and providing new thoughts for the treatment of tongue cancer.Objective To study the effect of inhibiting the expression of MMP10 and MMP13 on the proliferation,invasion and migration of tongue squamous cell carcinoma CAL27.Methods Part I:RNA-seq based on NGS of five pairs tissue samples preliminary found differentially regulated genes-MMPs;The tissue specimens were quantified using realtime PCR to further test the expression of MMPs.Part II:Inhibiting MMP-10,MMP-13 gene and studying the impact on the biological behavior of CAL-27 cell line.The corresponding si RNAs were designed and synthesized according to the sequence of MMP-10 and MMP-13 respectively.CAL-27 cells were transfected.The expression of two genes at RNA level was detected by Real-time PCR and two si RNAs with good interference effect were screened.Western blot was used to verify the changes of the two genes at the protein level.The CCK8 and plate cloning assays were used to investigate the changes of CAL-27 cell proliferation after interference of MMP-10 and MMP-13.The migration and invasion changes of CAL-27 cells were investigated by wound healing assay and transwell.Part III:An animal model was established to observe the ability of cancer cells to metastasize in nude mice after inhibiting the expression of MMP-10 of highly metastatic cell line of tongue cancer(LN-4).The lentiviral vector targeting MMP-10 gene was constructed,and the highly metastatic tongue cancer cell line was infected.Establishing the tongue carcinoma in situ in nude mice and calculating the lymph node metastasis rate.Results Part I:RNA-seq Sequence analysis showed MMP-10,MMP-13 expression is relatively high in five pairs carcinoma tissues,q RT-PCR results also found two genes expressed relatively high in most of the tongue cancer tissues compared to their paired normal tissues.Part II:Transfected CAL-27 cells with si RNA-MMP10 / si RNA-MMP13,Real-time PCR and Western blot revealed that the corresponding MMP-10 / MMP-13 expression was decreased both at RNA and protein levels.The results of CCK8 and plate cloning assays showed that the growth of MMP-13 inhibited CAL27 cells was obviously suppressed than that of the NC group.Wound healing assay and transwell showed that the migration and invasion ability of CAL-27 cells decreased after inhibiting the expression of MMP-10 / MMP-13.Part III:Lentivirus inhibited stable transformation of MMP-10 protein in the 1336 group compared with the NC group.The migration ability was lower than that in the NC group.We selected 1336 stably transfected cells and NC stably transfected cells for animal experiment and found lymph node metastasis rate decreased in 1336 nude mice compared with NC group.Conclusions The expression of MMP-10 and MMP-13 in cancerous tissues was relatively high.CAL-27 cells?proliferation?migration and invasion abilities were decreased after inhibiting the expression of MMP-13.The migration and invasion abilities of CAL-27 cells were decreased after inhibiting the expression of MMP-10.MMP-10 and MMP-13 may play a key role in diagnosis,prognosis and gene therapy of tongue cancer.