Folic Acid Deficiency Regulates Colorectal Cancer Cells Proliferation And Apoptosis Via Mir-379-p Mediated Downregulation Of HSPA5

Colorectal cancer(CRC)is the most common malignant tumor in the digestive system,and its morbidity and mortality are increasing year by year.Dietary micronutrients have various effects on the occurrence and development of CRC.Folate is a kind of micronutrients involving in DNA synthesis and repair,DNA and protein methylation and other biochemical reactions in one-carbon metabolism.The risk of CRC increases for improper intake and metabolic disorder of folate.Both studies of in vivo and in vitro suggested that folate has a dual regulatory role in the development of CRC.mi RNA,which is the non-coding small RNA responding to internal and external environmental changes,participate in multiple biological processes of CRC.It is a scientific issue to be further explored and clarified whether mi RNA responds the folate concentration changes to regulate the biological process of CRC cells and the involved molecular mechanisms.In this study,we focused on screening mi RNAs that both respond to the concentration changes of oxidized folic acid(FA)and target the CRC regulation and their mechanisms.Human CRC cell lines HCT116 and SW620 and normal colonic epithelial cells CCD-841-CON were treated with the deficient concentration(22.6nmol/L),according to the definition FA concentration of maintaining the the stability of human cell genome and sufficient concentration(2260 nmol/L)for 28 days.We discussed the effects of colorectal cells in different pathophysiological states respond to FA deficiency through TUNEL assay,Annexin V-FITC/PI,Transwell and other methods.A series of techniques including m RNA/mi RNA transcriptomic sequencing,bioinformatics analysis,RT-QPCR,Western Blot and Luciferase reporter assay were used to identify and verify the mi RNAs and their targeted genes that responded to FA deficiency and regulated the biological process of CRC cells.Based on above results,a chain of evidence for folate-mi RNA-CRC cell biological process changes was established.The results showed that:1.In the 7th day after continuously exposed to FA deficiency,the cell proliferation and migration ability of normal colonic epithelial cells CCD-841-CON decreased significantly while the apoptosis rate increased significantly(p<0.05 ～ 0.01).On the contrary,the migration ability of the two CRC cells(HCT116 and SW620)decreased significantly and the apoptosis rate increased significantly in response to the continuous intervention by deficient FA for 28 days(p<0.05 ～ 0.001),meanwhile the proliferation rate of HCT116 cells decreased significantly(p<0.01),and cell cycle of HCT116 was arrested in G0/G1 phase(p<0.05).These results suggest that CRC cells in different pathophysiological states respond to FA deficiency with different biological outcomes.2.The signaling pathway of HSPA5-IRE1α-XBP1,which is associated with endoplasmic reticulum stress(ERS),was enriched to be influenced by FA concentration after the analysis of the transcriptome sequencing results of HCT116 cells under FA deficiency/sufficient concentration.Then,mi RNAs and their targeted important genes in response to FA concentration and regulating the biological processes of CRC were also uncovered by a mi RNA regulated m RNA network associated with ERS.The expression of HSPA5,as an ERS marker gene,had significantly differences affected by FA deficiency in CRC cells with different pathophysiological states.3.Luciferase reporter assay confirmed mi R-379-5p negatively regulated HSPA5,and play the role of tumor suppressor in HCT116 validated by Ed U assay,TUNEL assay and other experiments.The HCT116 cells with HSPA5 knock-down,responded to FA deficiency for 28 days by downregulating the expression of IRE1α and XBP1-s(p<0.05),upregulating the expression of CASP4 to inhibit BCL-2(p<0.01～0.001)and initiate ERS apoptosis signaling pathway mediated by caspase-4 to induce a large-scale apoptosis.However,the role of overexpressed HSPA5 protein on promoting cell proliferation and reducing cell apoptosis was antagonized after FA deficiency for 28 days by down-regulating p53 and CHOP expressions(p<0.05～0.01)in HCT116 to reverse high proliferation and low apoptosis rate.In conclusion,in HCT116 cells,the expression of mi R-379-5p was significantly up-regulated after 28 days of FA deficiency,which downregulating its target HSPA5,inducing ERS-mediated apoptosis and inhibiting the proliferation by inhibiting the proteins in HSPA5-IRE1α-XBP1 axon of the ERS.We demonstrated that mi R-379-5p respond to FA deficiency inducing various HSPA5 expressions in colorectal cells with different pathophysiological states.To our knowledge,our study showed for the first time that,mi R-379-5p plays the role of "tumor suppressor gene" in CRC cells in response to FA deficiency in vitro.Our study may reveal the new mechanism that inhibits the proliferation and promotes the apoptosis of CRC cell from the perspective of micronutrients change to mediate the expression of core gene in ERS pathway.