Effects Of Interference Receptor Gene Notch1and Notch4Expression On Salivary Adenoid Cystic Carcinoma Cell Proliferation, Invasion And Metastasis

Salivary adenoid cystic carcinoma is one of the common malignant tumors inhead and neck. It’s characterized by perineural invasion and distant metastasis, whichare the main reasons of postoperative recurrence. Numerous researches have showedthat Notch pathway was associated with the development of many kinds of tumors.Our previous experiments had veritified that the receptor gene Notch1, Notch4hadmore highly expressed in SACC-M than SACC-2. The purpose of this study was toexplore the effect of Notch1and Notch4on the proliferation, invasion and metastasisof salivary adenoid cystic carcinoma cells. This research was divided into two partswhich are as followed.1. Using siRNA to verify the effect of Notch1, Notch4in salivary adenoid cysticcarcinoma cell.Aim: To verify the effect of receptor genes Notch1, Notch4in the proliferation,metastasis and invasion of salivary adenoid cystic carcinoma cell.Methods: First, siRNA was synthesized according to the sequence of Notch1,Notch4, and they were transfected into SACC-M, then Real-time PCR was used toexamin the expression level of Notch1, Notch4. Then the proliferation of SACC-Mwas tested by CCK8; The metastasis and invasion were evaluated by using transwellassay.Results: The results of Real-time PCR revealed that the mRNA level of targetgene was decreased. CCK8assay showed that the growth of SACC-M transfectedwith siRNA was significantly suppressed compared with negtive control aftertransfect for72h. And transwell assay revealed that the metastasis and invasionability of SACC-M were suppressed with the decreasing the expression of Notch1,Notch4. Conclusion: The Notch1and Notch4can infecte the proliferation, invasion andmetastasis of SACC-M.2. Construction of Recombinant Notch1, Notch4shRNA AdenovirusAim: To construct the recombinant adenovirus encoding Notch1and Notch4shRNA.Methods:We designed and synthetised shRNA fragment with restriction enzymesites of HindⅢ、XhoⅠaccording to the reference of the Notch1and Notch4siRNAsequence. Then the fragment was inserted into the shuttle vector pRNAT-H1.1/Adeno,which was digested by HindⅢ、XhoⅠ. The mixture then was used to transform intoE.coliDH5α, the positive plasmid was named pRNAT-H1.1-Notch4-1, pRNAT-H1.1-Notch4-2, pRNAT-H1.1-Notch1-1, pRNAT-H1.1-Notch1-2. The plasmid was digestedby endonuclease Pme I and transformed into Bj5183to homologously recombine,then identified by PacⅠdigestion, the positive recombinant adenovirus vector namedpAd-Notch1-1, pAd-Notch1-2, pAd-Notch4-1, pAd-Notch4-2. We used PacⅠtolinearize the adenovirus vector, and transfected them into AD-293cells. Theadenovirus vector would be packaged in AD-293cells. After packaging, supernatantand the cells were collected, obtained primary virus liquid. The primary virus liquidwas amplified to the fifth generation. At last, we used Real-time PCR to test the resultof interference.Results: The results of Real-time PCR revealed that the interference effect ofNotch1recombinant adenovirus vector was more than50%, but the effect of Notch4recombinant adenovirus vector was poor.Conclusion: The Notch1gene shRNA recombinant adenovirus vector wassuccessfullly constructed can be used for subsequent experiments; The Notch4geneshRNA recombinant adenovirus vector was successfully constructed, but theinterference effect was poor.