SWELL1 Promotes The Proliferation And Migration Of Hepatocellular Carcinoma And Study On The Molecular Mechanism

Background and Objective Hepatocellular carcinoma(HCC)is a very common and severe malignancy,which is one of the leading causes of cancer-associated mortality worldwide.HCC is characterised by rapid metastasis and development,reducing the time for the treatment of patients.To date,the biological mechanism about the development of HCC has not been investigated.Recently,SWELL1 is reported to be an indispensable part of the volume-regulated anion channel(VRAC).This marked a significant breakthrough,revealing more possibilities for SWELL1.Beyond its pivotal role in cell volume regulation,VRAC is also involved in cell proliferation,apoptosis,and migration.However,the correlation between SWELL1 and HCC remains poorly-understood.Therefore,in this study,we tried to explore the role of SWELL1 in the proliferation,apoptosis,and metastasis of HCC,including the molecular mechanism.SWELL1 may be a potential intervention target for HCC.The results of our study might aid in better understanding the functional capacity of SWELL1.Methods(1)Immunohistochemistry(IHC)and quantitative real-time-PCR(qRT-PCR)was used to measure SWELL1 expression in HCC samples obtained from patients with HCC.(2)Transfection with the small interfering RNA(si RNA)and plasmid was performed to achieve the over-expression and suppression of SWELL1 in HCC cells.The effects of SWELL1 on HCC cell proliferation were analysed by the Cell Counting Kit-8(CCK8),5-ethynyl-2’-deoxyuridine(Ed U)and cell cycle analysis.Besides,quantitative real-time-PCR(q RT-PCR)and western blot was adopted to evaluate the expression of cell cycle-related proteins cyclin D1,cyclin E,CDK2,and CDK4.(3)The Hoechst 33258 staining,TUNEL assay,Annexin V and PI staining,cellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP),and intracellular ATP content were adopted to analyse the effects of SWELL1 on apoptosis in HCC cells.(4)Western blot was performed for the molecular mechanism of SWELL1-mediated cell proliferation in HCC cells.(5)The cell adhesion and epithelial-to-mesenchymal transition(EMT)assays were adopted to detect the role of SWELL1 on migration in HCC cells.(6)Western blot was performed to investigate the molecular mechanism of SWELL1-mediated cell metastasis of HCC.Result(1)According to the results of IHC and q RT-PCR,SWELL1 expression was significantly up-regulated in HCC samples.(2)According to the results of the CCK8 and Ed U assay,the over-expression of SWELL1 enhanced the proliferation in HCC cells.Conversely,suppressing SWELL1 had the opposite effects.In addition,the results of cell cycle analysis indicated that high expression of SWELL1 promoted G1/S transition in HCC cells.In contrast,inhibition of SWELL1 expression had the opposite effects.The q RT-PCR and western blot results were in accord with the cell cycle analysis.(3)The results of the Hoechst 33258 staining,TUNEL and Annexin V and PI staining indicated that cells with high SWELL1 expression had much fewer apoptotic cells.Besides,increasing SWELL1 expression had a significantly decreased ROS production,less MMP loss and elevated levels of ATP.In contrast,suppressing SWELL1 had the opposite effects.These results indicated that SWELL1 suppressed apoptosis in HCC cells.(4)Results of western blot indicated that SWELL1 could promote HCC cell growth by cyclin D1/CDK2 pathway via connecting with protein kinase C alpha(PKCa)at the signalling level.Besides,further studies indicated that Sphingosine kinase 1(SPHK1)was an upstream factor of SWELL1 and that SPHK1 promoted cell proliferation via the SWELL1-PKCa pathway in HCC.(5)According to the results of the cell adhesion and EMT assays,up-regulation of SWELL1 dramatically inhibited the cell adhesion and promoted the EMT of HCC cells,which in turn enhanced the cell migration.Conversely,the migration ability was reduced in cells suppressing SWELL1.(6)Western blot results indicated that SWELL1 could regulate NKCC1 expression and promote the migration of HCC cells via the JNK-NKCC1 pathway.Conclusion(1)SWELL1 was highly expressed in HCC tissues.(2)SWELL1 promotes cell proliferation and protects cells from apoptosis in HCC.(3)SWELL1 induced cell growth by activating the cyclin D1/CDK2 pathway via connecting with PKCa at the signalling level.Besides,SPHK1 acts as an upstream factor of SWELL1 and promotes cell proliferation via the SWELL1-PKCa pathway in HCC.(4)SWELL1 could enhance the EMT and suppresses cell adhesion of HCC cells,which promote the metastasis.(5)SWELL1 regulates HCC cell migration through the JNK-NKCC1 signalling pathway.In conclusion,SWELL1 acts as a promoter in the growth and metastasis of HCC cells and may be a potential intervention target for HCC.