Preliminary Study On The Mechanisms Of NRP1 Regulating Autophagy In Human Colon Cancer Cell Line HT29 By AKR1B10

Colorectal cancer is one of the most common digestive tract cancers.The major methods for the treatment of colorectal cancer are surgery,supplemented by radiotherapy and chemotherapy.The effect of these methods for patients with advanced colorectal cancer is still not significant.At present,the developed mechanism of colorectal cancer needs to be further explored.Therefore,it is necessary to study the mechanism of its development and provide a new theoretical basis for the treatment of colorectal cancer.Autophagy is an important process in eukaryotes which is evolutionarily conserved and transfers intracellular substances.In this process,the damaged,deformed,aged,misfolded,non-function proteins and damaged organelles are encapsulated by autophagosomes and then degraded by lysosomal hydrolases after combining to them.Afterwards,amino acids and other nutrients are produced.The present study shows that autophagy plays a different role in the development of tumor.On the one hand,autophagy in the early stage of tumor can inhibit the development of tumor;on the other hand,in the late stage of tumor,it can provide the mechanisms of anti-nutrition deficiency and anti-hypoxia and promote the survival and development of tumors.Autophagy can be a potential target for the treatment of tumors.NRP1(Neuropilin-1)is highly expressed in a variety of tumor cells and plays an important role in tumor growth,proliferation,migration and angiogenesis.AKR1B10(Aldo-keto reductase family 1 member B10)is Aldehydes and ketones reductase family 1 member 10 and is currently unclear about the relationship and molecular mechanism of NRP1/AKR1B10 with autophagy.The purpose of this study is to explore the molecular mechanism of NRP1/AKR1B10 regulation of autophagy in cells to provide a favorable basis for autophagy treatment of colorectal cancer.In this study,shRNA and molecular cloning techniques were used to knock down and replenish NRP1 and AKR1B10 in human colon cancer cell line HT29.Dosing inhibited AKR1B10 enzyme activity and constructed HT29 cell line of AKR1B10 enzyme active site point mutation.Western blotting and immunofluorescence assay were used to detect autophagy in human colon cancer cell line HT29.Proteins interacting with the NRP1/AKR1B10 pathway during autophagy were detected by immunoprecipitation and mass spectrometry.The effects of AKR1B10 on the biological function of colon cancer cells were investigated by cell counting,CCK8 and cell colony experiments.The results showed that:1.The knock down and replenish of NRP1 in human colon cancer cell line HT29,the knock down and replenish of AKR1B10 and enzyme active site point mutation of AKR1B10 ceIl line were constructed successfully.2.With the prolongation of induction time,autophagy increased gradually,and the expression of NRP1 gradually increased.3.Knockdown of NRP1 attenuated autophagy,and overexpression of NRP1 increased autophagy.4.The expression of AKR1B10 was up-regulated after knocking down NRP1 in HT29 cells.5.Knockdown of AKR1B10 cells increased autophagy,and overexpression of AKR1B10 cells attenuated autophagy.6.Enhanced autophagy after inhibition of AKR1B10 enzyme activity.7.Co-IP showed that AKR1B10 interacted with GAPDH during autophagy.8.The knockdown of AKR1B10 increased the entry into the nucleus of GAPDH.The replenish of AKR1B10 inhibited the entry into the nucleus of GAPDH,and the entry into the nucleus of GAPDH was not inhibited after the replenish of AKR1B10 enzyme activity site point mutation.9.Autophagy induced by glucose starvation did not affect the expression of AKR1B10,GNAI3 and Synectin.10.NRP1,AKR1B10,Synectin and GNAI3 interacted with each other in human colon cancer cell line HT29.11.Autophagy induced by glucose starvation in HT29 cells was independent on the mTOR pathway,while rapamycin-induced autophagy in HT29 cells xwas dependent on the mTOR pathway.12.Other proteins which might interact with AKR1B10 were selected out by Co-IP combined with mass spectrometry.13.The ability of proliferation and colony forming of human colon cancer cell line HT29 were affected by AKR1B10.Therefore,we can draw conclusions as follows:1.NRP1 promotes autophagy in HT29 cells by inhibiting the expression of AKR1B10.2.AKR1B10 inhibits autophagy of HT29 cells by inhibiting the entry of GAPDH into the nucleus.3.Autophagy regulated by NRP1/AKR1B10 is closely related to GNAI3 and RGS19 signaling pathway.4.The autophagy of NRP1/AKR1B10 induced by glucose starvation is independent of the mTOR pathway,and the autophagy of NRP1/AKR1B10 induced by rapamycin is dependent on the mTOR pathway.5.AKR1B10 interacts with BIP,G6PD,CALM1,SODC,CD59 and EPCAM during autophagy...6.AKR1B10 promotes the proliferation and colony formation of human colon cancer cell line HT29.This study first discovered that NRP1/AKR1B10 regulated autophagy,and its preliminary mechanism had been explored.It will provide a new idea and a theoretical basis for the treatment of colorectal cancer.