Effects Of Kallikrein-related Peptidase4(KLK4) Silencing On Proliferation,apoptosis,migration And Invasion Of Oral Squamous Cell Carcinoma(OSCC)

Oral squamous cell carcinoma(OSCC)is one of the most destructive and fatal malignancies.It has a large proportion of all the head and neck cancers and has become a great killer of human health.Because of the special anatomical location,OSCC often severely affects the patient's quality of life with an unfavourable prognosis.People's daily bad habits,such as smoking and drinking,are risk factors for cancer metastasis,and metastasis is a serious problem for cancer patients.Statistically,more than 90 percents of cancer-related deaths are due to metastasis.In OSCC,more than 50% of the patients had lymph node metastasis,and the 5-year survival rate of these patients decreased significantly.Kkallikreins(KLKs)are the largest serine protease gene cluster in human genes.The KLKs,which are encoded by 15 different genes,are located on the human chromosome 19q13.4,a highly conservative set of genetic structures and protein sequences.KLKs are expressed in multiple tissue and organs.And meanwhile,KLKs play an important role in many physiological processes.Recently,KLKs has been reported to play an important role in tumor growth and metastasis,and is considered a biomarker for various cancers.Many KLKs show abnormal expression in various malignancies,which are considered to be the prognostic markers of disease development.The current research indicates that KLKs is closely related to OSCC,which involves many aspects such as proliferation,apoptosis and metastasis of tumor,which seriously affect human life and health.KLK4,a member of the Kkallikreins family,has shown that KLK4 can cleave peptidebonds.And this feature may be crucial for its role in malignant tumors.The study suggests that KLK4 plays an important role in malignant tumors,and KLK4 may promote the development and progression of cancer by promoting cell proliferation,EMT and malignant transformation.In 1999,KLK4 was first discovered in the c DNA library of endometrial cancer by Stephenson.Ovarian cancer research has shown that KLK4 is associated with progression of ovarian cancer.In addition,KLK4 is also overexpressed in prostate cancer and plays an important role in the process of tumor cell epithelial mesenchymal transformation(EMT)and metastasis of prostate cancer.In addition,KLK4 plays an important role in other tumors such as kidney cancer and laryngeal cancer.The Wnt/?-catenin signaling pathway plays an important role in oral tissue development and related diseases.?-catenin is involved in the development of oral squamous cell carcinoma.The reduction of ?-catenin is associated with poor prognosis and is considered a biomarker for recurrent OSCC.Wnt signaling is associated with cell proliferation,migration,differentiation and survival.Deregulation of Wnt will affect the progression of cancer.In malignant tumors,similar regulation mechanisms are existed and PI3K/AKT signaling pathways are also included.This research is based on the technique of RNA interference.Aimed to interfere the gene expression in OSCC,and explore the influence and its mechanism of KLK4 silencing on OSCC proliferation,apoptosis,migration and invasion ability.This research is divided into the following parts:Part ?:Oral squamous carcinoma cell culture,transfection of KLK4 sh RNA and interference efficiency verification:Design and synthesis of the interfere fragment of KLK4(si RNA for KLK4:5 '-AATCCGTGTCCGAGTCTGAC-3').The above fragments were built into the plasmids of pgcsi-h1,and then the control plasmids of pgcsi-h1-KLK4 and the negative control plasmid of pgcsi-h1-nc were obtained.Then,transfect the OSCC(KB cells and Tca-8113 cells),select them by G418 to obtain the stably transfected cells.Gene silencing efficiency was verified by q RT-PCR method.The results showed thatthe interfering plasmid was transfected successfully and the cell lines and control cell lines of p GCsi-h1-klk4 were stabilized.Meanwhile,the silencing efficiency of KLK4 gene was verified by real-time quantitative PCR.Part ?: The effect of KLK4 silencing on proliferation of OSCC cells:The proliferation of cells in each group was detected by MTT assay(five days of continuous detection),and the cell proliferation ability of klk4-sh RNA group was significantly lower than that of the pure cell group and the negative control group.Cell cycle was verified by flow cytometry detection.And it showed that KLK4 gene disruption make the proportion of the OSCC cells in G1 phase increased significantly,inhibit OSCC cells mitosis,thus inhibiting tumor cell proliferation.By clone formation assay in vitro,it was found that KLK4 silence inhibits the proliferation and colony formation of OSCC.The expression of PCNA was detected by immunofluorescence method,and the ratio of proliferative capacity in all cells decreased significantly after the KLK4 silence.These results indicate that KLK4 silence significantly inhibits the proliferation of OSCC.Part ?: The effect of KLK4 silencing on apoptosis of OSCC cells:Apoptosis of cells in each group was detected by flow cytometry.And the results showed that KLK4 sh RNA elevated the percentages of apoptosis cells.By Hoechst staining of apoptotic cell nucleus changes,we can clearly observed KLK4 inhibit apoptosis rate increased significantly,and the nuclear chromatin gathered can be clearly observed from apoptotic cells.Through the Western blot method,we detected the protein expression levels of cleave caspase-3,cleave PARP,Bax and bcl-2,which are closely related to apoptosis.The expression levels of cleave caspase-3,cleave PARP and Bax were significantly increased in the KLK4 sh RNA group.And meanwhile,the expression of bcl-2 was significantly reduced.The results showed that the silencing of KLK4 could significantly induce apoptosis of OSCC cells.Part ?:The effectof KLK4 silencing on themigration andinvasion of OSCCcells:The ability of cells migration is detected through wound healing assay.And it showed that KLK4 silencing significantly inhibited the migration of KB cells andtca-8113 cells.And we evaluated the invasion ability of OSCC cell by transwell experiments,after the transfection of KLK4 sh RNA,the amount of cells which can go through the microporous membrane matrix significantly reduced.It indicates that the KLK4 silencing suppressed the invasion of OSCC cells.The expression levels of MMP-2 and MMP-9 were detected by the gelatin zymography and Western blot,and we found that the levels of MMP-2 and MMP-9 protein in the KLK4 sh RNA group were significantly reduced,suggesting that the KLK4 silencing inhibited the expression and activity of MMP-2 and MMP-9.These results suggest that the KLK4 silencing can significantly reduce the metastasis and invasion of OSCC cells.Part ?: The effect of KLK4 silencing on epithelial mesenchymal transformation(EMT)of OSCC cells:The protein expression levels of E-cadherin,Vimentin and N-cadherin were detected by western blot,and the results showed that the levels of E-cadherin in the KLK4 sh RNA group were significantly higher.The levels of Vimentin and N-cadherin were significantly reduced.Otherwise,the protein expression levels of u PA,TIMP-1and TIMP-2 of EMT were also detected by western blot.The results showed that the u PA protein level of the KLK4 sh RNA group was significantly reduced.However the protein levels of TIMP-1 and TIMP-2 were significantly higher.These results indicate that KLK4 can promote the transformation of epithelial mesenchymal transformation(EMT)in OSCC cells.Part ?:Theeffectof KLK4 onthe Wnt/?-cateninsignalingpathwayin OSCCcells:The levels of Wnt1 and ?-catenin protein in the KLK4 sh RNA group were significantly decreased by western blot detection.In the expression and distribution of?-catenin,immunofluorescence detection showed that ?-catenin in the KLK4 sh RNA group showed low expression,and that ?-catenin entered the nucleus and was inhibited by the KLK4 sh RNA.By western blot,we detected the expression of Wnt/?-catenin signaling pathway related products of GSK-3?,p-GSK-3?,cyclin D1 and c-myc.And we found that there were no significant differences of GSK-3?protein levels in each group.And the expression of p-GSK-3?,cyclin D1 and c-mycdecreased significantly.The above results provide further evidence for the suppression of the activation of the Wnt/?-catenin signaling pathway by the KLK4 silencing.However,after treatment with SB216763,which is the activator of Wnt/?-catenin signaling pathway.The inhibition effect of KLK4 sh RNA on the cell viabilities was rescued.These results demonstrated that SB216763 reversed the inhibitory effect of KLK4 silencing on the proliferation of OSCC cells and KLK4 can play a carcinogenic role through the Wnt/?-catenin signaling pathway.Part ?:The effectof KLK4 on the PI3K/AKTsignaling pathway in OSCC cells:The expression of P-PI3 K,PI3K,P-AKT and AKT was detected by western blot,indicating that the P-PI3 K and P-AKT protein levels of the KLK4 sh RNA group were significantly reduced,while the levels of PI3 K and AKT protein showed no difference.Similarly,the expression of P-AKT protein in the KLK4 sh RNA group was significantly reduced,while the expression of AKT protein showed no significant change,indicating that the KLK4 silencing inhibited the activation of the PI3K/AKT signaling pathway.After the treatment of the PI3K/AKT signaling pathway activator740 y-p,the effects of the KLK4 silencing before were partially reversed through the detection of wound healing assay,transwell experiments,gelatin zymography and western blot.The above results showed that KLK4 can be used to induce and promote malignant tumors through PI3K/AKT signaling pathway.Based on all the studies above,we can conclude that KLK4 silencing can significantly inhibit the proliferation,metastasis and invasion of OSCC,and promote apoptosis of OSCC.The mechanism is that KLK4 silencing can inhibit the activation of the Wnt/?-catenin signaling pathway and the PI3K/AKT signaling pathway.It also reveals the KLK4 as a kind of oncogene of the OSCC,the targeting therapy of KLK4 may provide us the new ideas and new weapons to conquer oral malignant tumors.