Effect Of RNA M6A Demethylase ALKBH5 In Pancreatic Stellate Cells On The Progression Of Pancreatic Cance

Background and Purpose:Pancreatic cancer is a highly aggressive malignancy holding extremely poor prognosis,which is thought to be one of the most lethal cancers with a life expectancy of 10%at 5 years.Due to the long term of asymptomatic phase and rapid progression,majority of patients with pancreatic cancer are incapable of getting timely diagnosis and treatment.And surgical excision is only possible in 10%-15%of patients.Additionally,the chemotherapy and radiotherapy-resistant characteristics of pancreatic cancer further increase the therapeutic difficulty.Therefore,it is urgent to find novel molecular mechanism of pancreatic cancer progression.Ninety-five percent of pancreatic cancers are pancreatic ductal adenocarcinoma(PD AC),which owns 80%-90%of fibrotic stroma in whole tumor.It is acknowledged that there are various cells and extracellular matrix constitute tumor microenvironment(TME),which is closely related to poor prognosis.Pancreatic stellate cells(PSCs)are the most abundant cells in stroma and maintain intimately complex interaction with pancreatic cancer cells(PCCs),which play key role in pancreatic cancer progression.Hence,PSCs are one of the potential targets for precision medicine in the future.N6-methyladenosine(m6A)methylation is the most common form of mRNA modification in mammals.m6A participates in the progression of multiple disease through regulating RNA splicing,transport,degradation,and translation etc.Previous studies have found that m6A taking parts in molecular mechanisms and providing new direction for clinical diagnosis,molecular therapy,and prognosis in various cancers,such as glioma,lung cancer,liver cancer,colorectal cancer,pancreatic cancer etc.However,whether m6A regulates functions of PSC remains unknown.Therefore,this study intended to explore the m6Amediated biological function of PSC and its influence on PSC-PCC interaction.Methods:1.Active primary PSCs were acquired from surgically removed tumor tissues by outgrowth method and deactivated through all-trans retinoic acid(ATRA)treatment.2.Characteristics of quiescent PSCs were validated by morphological observation,Oil Red O staining and immunofluorescence analysis.3.Western blot analysis was utilized to screen differentially expressed m6A regulators between active and quiescent PSCs.4.Subcellular localization of m6A regulator ALKBH5 was observed by immunofluorescence analysis.5.siRNA transfection was used to suppress the ALKBH5 expression in PSCs and colony formation assay was performed to detect proliferation of PSCs regulated by ALKBH5.6.Western blot was applied for detecting influence of ALKBH5 on autophagy in PSCs.7.PSC-PCC co-culture Transwell system and cell-counting-8(CCK8)assay were constructed and performed for probing migration,invasion,and proliferation of PCCs affected by ALKBH5 in PSCs.8.Conditioned-medium from PSCs with or without ALKBH5 knockdown was applied for PCC incubation and western blot was performed to detect epithelial-mesenchymal transition(EMT)markers in PCCs.9.To differentiate whether small molecule proteins are critical in PSC-PCC interaction,boiled PSC conditioned medium was used to perform Transwell assay to investigate whether protein-inactivated medium also affects migration and invasion of PCCs.10.Cytokine array was applied to sift differential cytokines secreted by PSCs after ALKBH5 knockdown.11.ELISA analysis was used to demonstrate intracellular and extracellular cytokine concentration between PSCs with or without ALKBH5 knockdown.Recombinant proteins were used to rescue the inhibition on migration and invasion of PCCs,which was caused by ALKBH5 knockdown in PSCs.Results:1.RNA m6A demethylase ALKBH5 was highly expressed in active PSCs.2.Knockdown of ALKBH5 could inhibit proliferation and autophagic flux in PSCs.3.ALKBH5 knockdown in PSCs could not significantly suppress PCCs’ proliferation but could decrease the migration and invasion ability of PCCs.4.ALKBH5 knockdown in PSCs suppressed EMT in PCCs.5.ALKBH5 could modulate cytokine secretion such as LIF,VEGFA,CXCL12 and TARC in PSCs.VEGFA may be the most critical factor in PSC-PCC interaction regulated by ALKBH5 in PSCs.Conclusion:These findings implicate that ALKBH5 is critical in proliferation and autophagy in PSCs,and it can also mediate cytokine secretion of PSCs and thereby regulate EMT in PCCs,which ultimately affects migration and invasion of PCCs and PDAC metastasis.Therefore,ALKBH5 may be the potential molecular target in PSCs for PDAC precision medicine in the future.