Research On The Molecular Mechanism By Which SiRNA-mediated LATS1 Knockdown Induces Pyroptosis In Bladder Cancer Cells

Objective: Bladder Cancer(BLCA)is the ninth most common malignant tumour globally,with an estimated 573,000 new cases and 213,000 deaths which is a comparatively high mortality rate by 2020.In China,BLCA accounts for the first incidence of genitourinary tumors.At present,although BLCA can be treated with conventional surgical resection,chemotherapy and immunotherapy,the treatment effect of BLCA is not ideal,and its recurrence rate and mortality have not been significantly improved.At present,there is no clear target factor for the treatment of BLCA,so it is urgent to find the molecular markers and therapeutic targets of BLCA.Large Tumor Suppressor 1(LATS1),a key component of Hippo signaling pathway,is a conserved serine/threonine protein kinase that inhibits cell proliferation and organ size.Previous studies have shown that LATS1 mainly plays an inhibitory role in tumors,but recent studies have found that LATS1 has an opposite effect in different tumors.Pyroptosis is a type of programmed inflammatory cell death activated by inflammasomes.Inflammasome is a polymer complex formed by pattern recognition receptor(PRR)indirectly linked to Pro-Caspase-1 via speckle-like protein(ASC).PRR components of inflammasome mainly include NLRP1,NLRP3,NLRC4 and melanoma deficiency factor 2(AIM2),which are members of the NLR protein family.The inflammasome activates caspase-1,cleaves Pro-IL-1β and Pro-IL-18 into mature forms,and cleaves Gasdermin D to form C-terminal(GSDMD-C)and N-terminal(GSDMD-N)fragments to induce cell pyroptosis.Cell pyroptosis is closely related to the occurrence of many kinds of tumors and plays different functions in different tumors.Studies have shown that cell pyroptosis can promote the proliferation of lung adenocarcinoma cell A549,but can inhibit the growth of liver cancer cells.Therefore,cell pyroptosis may be a "double-edged sword" in the process of tumor occurrence and development.As a key factor in the regulation of apoptosis,inflammation and immunity,TNF-α plays an important role in local and systemic inflammation.It has been shown that the combined action of TNF-α and IFN-γ can induce cell pyroptosis of bone marrow-derived macrophages.TNF-α is one of the known activators of the NF-κB pathway.Activation of the NF-κB pathway can enhance the expression of NLRP3 and induce apoptosis of cervical cancer,melanoma,colorectal cancer and nasopharyngeal cancer cells.In cervical cancer cells,SIRT1 deficiency activates NF-κB/p65 to upregulate AIM2 expression and induce cell pyroapoptosis,thereby inhibiting cell proliferation.At present,TNF-α/NF-κB pathway and cell apoptosis have not been reported in the development of bladder cancer.At present,the role of LATS1 in the occurrence and development of BLCA is still unclear.Through TCGA database analysis,we found that BLCA patients with high expression of LATS1 had poor prognosis.Therefore,we took LATS1 as the entry point to study the possible role and mechanism of LATS1 in BLCA and provide a new molecular target for the treatment of BLCA.Method:1.We used siRNA to silence the expression of LATS1 in BLCA 5637 and Biu-87 cells(si-LATS1).2.RT-qPCR and Western Blot methods were used to verify the effect of si-LATS1 on the expression of LATS1 in 5637 and Biu-87 cells.3.CCK-8 assay and clone formation assay were used to detect the cell viability and proliferation of 5637 and BIU-87 cells by si-LATS1.4.Flow cytometry to detect the effect of si-LATS1 on apoptosis and cell cycle of 5637 and Biu-87 cells.5.Bulk RNA-Seq analysis of gene expression differences and gene function enrichment of si-LATS1 in BLCA.6.Western Blot detects the expression of TNF-α/NFκB signaling pathway-related proteins and inflammation-related proteins in the si-LATS1 group of5637 and Biu-87 cells,such as Caspase-1,TNF-α,IL-1β,IL-18,p-NF-κB/p65,NF-κB,GSDMD,NLRP3,NLRC4,AIM2,etc.7.Immunofluorescence method was used to detect the influence of si-LATS1 on the intracellular localization of NF-κB.8.The kit detects the activity of Caspase-1 and Caspase-4 and the release of lactate dehydrogenase(LDH)in the 5637 and Biu-87 cells of the si-LATS1 group.9.DNA ladder kit was used to detect DNA damage in si-LATS1 group.10.CCK-8 method detects the effect of NF-κB inhibitor PS-341 on cell viability of si-LATS1 group.11.Western Blot method detects the effect of PS-341 on the expression of inflammation-related proteins in si-LATS1 group.12.Western Blot method detects the expression of TNF-α/NFκB signaling pathway related proteins and inflammation-related proteins in 5637 and Biu-87 cells with high expression of LATS1(OE-LATS1),such as Caspase-1,TNF-α,IL-1β,IL-18,p-NF-κB/p65,NF-κB1,NF-κB,GSDMD,NLRP3,NLRC4,AIM2,etc.13.Subcutaneous tumor formation experiments in nude mice verified the effect of si-LATS1 on tumor cell growth.Result: Compared with the control group(NC),the cell viability of 5637 and Biu-87 cells in si-LATS1 group was significantly decreased.The apoptosis and cell cycle of5637 and Biu-87 cells in si-LATS1 group were not significantly changed.The expressions of TNF-α,p-NF-κB /p65,NF-κB1,NLRP3,NLRC4 and AIM2 in 5637 and Biu-87 cells of si-LATS1 group were significantly increased.NF-κB entry into the nucleus was significantly enhanced in the si-LATS1 group.The activity of caspase-1 and the expression of IL-1β,IL-18 and GSDMD were increased in the si-LATS1 group.OE-LATS1 inhibited the expression of TNF-α,p-NF-κB/p65,NLRP3,NLRC4 and AIM2 in 5637 and Biu-87 cells,and significantly decreased the expression of IL-1β,IL-18 and GSDMD splice body.Conclusion: In 5637 and BIU-87 cells,LATS1 deficiency promotes the formation of NLRP3,NLRC4,and AIM2 inflammasomes by activating the TNF-α/NF-κB signaling pathway,which in turn activates caspase-1 cleavage of GSDMD and induces cell pyroptosis.Therefore,LATS1 can be a potential target for BLCA therapy.