Investigation Of The Effect And Mechanism Of RNAi Silencing KDELR2 Synergizing TMZ To Promote Human Glioma Cell Apoptosis

Brain glioma originates from glial and neuronal cells of the nervous system,accounts for 80% of all malignant brain tumors.Glioblastoma Multiforme(GBM),which occupies more than 50%,is the highest malignant degree of glioma.Although with the deepening of understanding of tumor genes,immunity,and microenvironment and the development of new technologies,significant progress has been made in the development and related mechanisms of gliomas,making clinical diagnosis more accurate,pathological classification more standardized,and clinical prognosis evaluation more precise.However,due to the high heterogeneity,invasiveness of glioma,and special brain function of glioma,it is difficult to be resected thoroughly and easy to relapse.Surgery,radiotherapy,and chemotherapy are still the standard treatments for glioma.The malignant degree of glioma is increasing with the extension of its growth years and the increase of recurrences times.At present,the clinical efficacy of glioma is still not ideal,especially in GBM.Temozolomide(TMZ),which is the most commonly used drug for GBM treatment,prolongs survival by only 2-5 months,and its efficacy varies from person to person.Chemotherapy resistance to TMZ is a major cause of treatment failure of GBM.Therefore,enhancing the chemosensitivity of TMZ and developing new drugs are the hotspots in glioma research.Like other cancers,glioma cells are exposed to complex internal and external environmental stresses,including oncogene activation,chromosome number changes,genomic instability,oxidative stress,nutritional deficiencies,acidosis,and dysregulated calcium homeostasis.These stresses lead to the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum(ER)beyond the folding capacity of the ER,resulting in endoplasmic reticulum stress(ERS).In response to ERS,cells activate a series of adaptive responses which is called unfolded protein response(UPR)to enhance the recovery of misfolded or unfolded proteins and eventually make them survive in harsh conditions rather than apoptosis.When ERS is too strong or lasts too long in cells,UPR mediates apoptosis.The regulation of UPR has been considered a new direction in glioma treatment.KDEL receptor is a seven-transmembrane protein that consists of three members,KDEL1,KDELR2,and KDELR3.KDELR mediates protein recycling from the Golgi to the endoplasmic reticulum,which can effectively maintain the residence of endoplasmic reticulum luminal proteins and prevent peptide chain misfolding and misassembly.Current studies have confirmed that KDELR participates in ERS via its retrieval ability,inducing p38 phosphorylation which regulates the p38 MAPK signaling pathway,and activating PKA and Src kinases which regulate the membrane transport system of ER and Golgi.Therefore,KDELR is considered one of the UPR genes.KDELR is closely related to the occurrence and development of non-small cell lung cancer,malignant melanoma,breast cancer,and GBM.KDELR2 is thought to promote glioma progression through m TOR/HIF1α and CCND1,and may be used as a prognostic biomarker for glioma.However,its mechanism is still at a preliminary stage,and whether it can be used as a therapeutic target for glioma is still lake research.Therefore,exploring the effect of targeting KDELR2 on the biological function of glioma cells,and on the effect of TMZ chemotherapy,may provide new ideas for improving the prognosis of patients with glioma.This study is divided into the following three parts.Part 1.Correlation of KDELR2 expression and clinicopathology in glioma.Objective: To detect the expression of KDELR2 in glioma tissues and glioma cells,to explore the relationship between KDELR2 and glioma,and prepare for the follow-up study.Methods: Clinical specimens were collected from 42 patients(23 males,19females)diagnosed with glioma were obtained from the Department of Neurosurgery at the Affiliated Jiujiang Hospital of Nanchang University from January2016 to September 2019,aging 47.14±8.74 years,including 14 cases of astrocytomas and 28 cases of glioblastomas,and including 9 cases of low-grade gliomas(LGG,grade Ⅰ～Ⅱ)and 33 cases of high-grade gliomas(HGG,gradeⅢ～Ⅳ).All the specimens were from the first surgery,and no preoperative chemoradiotherapy was performed.Immunohistochemistry and western blot were applied to detect the expression of KDELR2 in these glioma tissues,and RT-q PCR was used to detect the m RNA level of KDELR2 in glioma cell lines.Results: In the cohort of 42 glioma patients detected by IHC,KDELR2 protein was highly expressed in HGG specimens.The immunohistochemical score of HGG(2.52±0.71)was higher than that of LGG tumor tissues(1.89±0.78),P<0.05.KDELR2 protein was not related to patient age and sex.The expression level of KDELR2 protein in HGG glioma specimens(6 cases)was higher than that in the LGG specimens(6 cases)detected by western blot.The expression level of KDELR2 protein in glioma tissues(8 cases)was higher than that in paired paratumor tissues detected by western blot.The m RNA levels of different subtypes of KDELR in the same cell line were different detected by RT-q PCR.The m RNA trend of KDELR2 was consistent in U251,U373,and U118 cells detected by RT-q PCR.Conclusion: The expression level of KDELR2 protein is higher in the more malignant glioma,and related to the pathological grade of glioma.Part 2.RNAi silencing KDELR2 promotes glioma cell apoptosis and associated molecular mechanisms.Objective: To investigate the effects of KDELR2 silencing on the proliferation and apoptosis of glioma cells and study the related molecular mechanisms by using the constructed high-efficiency KDELR2 si RNA and overexpressed plasmid were transfected into U373 cells.Methods: Three pairs of KDELR2 si RNA used in RNA interference(RNAi)technology and negative control si RNA were designed.In addition,the KDELR2 overexpression plasmid PLeno-K2 vector was constructed and transfected into U373 cells.Western blot and RT-q PCR were used to detect the KDELR2 in U373 cells to assess the effects of si RNA and plasmid PLeno-K2 vector.The proliferation capacity and clonogenic ability of U373 cells interfered with si RNA or plasmid PLeno-K2 vector were detected by CCK-8 assay and colony formation assay,respectively.The apoptosis of U373 cells interfered with si RNA or plasmid PLeno-K2 vector were detected by flow cytometry and hochest33258.The m RNA of genes(ATF4,ATF6,PERK,e IF2α,GRP78,and CHOP)and proteins(cleaved caspase 3,caspase 3,cleaved PARP,PARP,Bax,Bcl-2,JNK,p-JNK,p38,p-p38,ATF4,ATF6,XBP-1s,PERK,p-PERK,e IF2α,p-e IF2α,GRP78,and CHOP)were evaluated by RT-q PCR and western blot,respectively.Result: The high-efficiency KDELR2 si RNA that deduced the level of m RNA and protein in U373 cells by RNAi technology,and negative control si RNA were constructed successfully.The level of KDELR2 m RNA in U373 cells interfered with si RNA1 was lowest compared with the control group(17.03%±2.04% vs 100%),p<0.001.The m RNA level of KDELR2 in the transfected U373 cells with the p Len O‐K2 vector was 1.33±0.05 times of the control group,p<0.001.KDELR2 silencing significantly inhibited the proliferation and clonal formation of U373 cells.However,KDELR2 overexpression promoted cell proliferation and clonal formation.KDELR2 silencing increased the apoptosis rate of U373 cells.The proteins of cleaved PARP,cleaved caspase 3,and Bax were up-regulated in the KDELR2 silencing U373 cells.The m RNA levels of genes(ATF6,e IF2α,ATF4,PERK,GRP78,and CHOP)were significantly increased in the KDELR2 silencing U373 cells.The proteins of ATF6,ATF4,XBP1,GRP78,and CHOP were increased in the KDELR2 silencing U373 cells.KDELR2 silencing activated PERK and e IF2α,and JNK/p38 pathway in U373 cells compared with the control.Conclusion: RNAi silencing KDELR2 promotes apoptosis of glioma U373 cells through ERS dependent CHOP pathway and activation of the JNK/p38 pathway.Part 3.RNAi silencing KDELR2 synergizes TMZ to promote glioma cell apoptosis through CHOP and JNK/p38 pathways.Objective: To investigate KDELR2 silencing promotes the anti-glioma activity of TMZ through the CHOP pathway and JNK/p38 pathway.Methods: IC50 of temozolomide was determined in the U373 cells by CCK-8assay.The proliferation capacity of U373 cells intervened with TMZ and the KDELR2 silencing/TMZ was detected by CCK-8 assay.Cell apoptosis of U373 cells interfered with TMZ and the KDELR2 silencing/TMZ was detected by flow cytometry and hochest33258.The expression of proteins(KDELR2,JNK,p-JNK,p38,p-p38,GRP78,CHOP,and GAPDH)was detected to study the molecular mechanism by western blot.Results: The IC50 of TMZ was 301.3μmol/L(95% confidence interval: 281.8 to322.1 μmol/L)in U373 cells.Compared with the TMZ group,KDELR2silencing/TMZ significantly inhibited the proliferation of U373 cells,while it increased the apoptosis rate of U373 cells.KDELR2 silencing/TMZ significantly inhibited KDELR2 protein expression,activated the JNK/p38 pathway,and upregulated CHOP expression in U373 cells compared with the other control groups.Conclusion: RNAi silencing KDELR2 synergizing TMZ to promote human glioma cell apoptosis through CHOP and JNK/p38 pathways.