[Research highlight] Enhancing p53 pathway can efficiently suppress colon cancer

 

Colorectal cancer is the third most diagnosed cancer and leads to the second mortality among cancers worldwide. The first-line chemotherapeutic drugs of colorectal cancer are mainly 5-fluorouracil(5-FU) based. However, these drugs exhibit compromised efficacy due to significant toxicity, drug resistance or patient inconvenience. Novel chemotherapeutic drugs for efficient treatment of colorectal cancer patients are urgently needed. Cabazitaxel (CBT), a microtuble inhibitor, can promotes polymerization of tubulin and stabilize microtubules. It shows antitumor activity in docetaxel-refractory metastatic prostate cancer and breast cancer.

However, it is not clear whether CBT is effective in inhibiting colorectal cancer and what the underlying mechanism is. In response to this question, The researchers from The Sixth Affiliated Hospital of Sun Yat-sen University, Wen Zhang et al. published an article named “Cabazitaxel suppresses colorectal cancer cell growth via enhancing the p53 anti-tumor pathway” in FEBS Open Bio. In this study, the researchers screened 160 FDA approved drugs with HCT116 cells, the commonly used colorectal cancer cell lines, and found that Cabazitaxel, by enhancing the P53 antitumor pathway, can efficiently inhibit the proliferation and migration of colorectal cancer cells via a series of in vitro assays. 

 

 

Cabazitaxel inhibits colorectal cancer cell growth via activating p53 signaling pathway

KEGG pathway analysis revealed that CBT treatment induced genes were enriched in the well-known anti-tumor p53 signaling pathway (Fig. 1). Further gene set enrichment analysis (GSEA) revealed a positive correlation between p53 pathway genes with CBT upregulated genes in HCT116 cells, indicating that CBT indeed enhances the expression of p53 pathway genes (Fig. 2). To examine whether activation of p53 pathway plays a key role for CBT efficacy, they used CRISPR/Cas9 system to knock out TP53, p53 encoding gene, in HCT116 cells by two different gRNAs and generate TP53 KO1 cells and TP53 KO2 cells with two hTP53 gRNA KO plasmids (YKO-RP003-hTP53, YKO-RP003-hTP53) which were provided by Ubigene. MTT assay revealed that IC50 of CBT to TP53 KO1 cells and TP53 KO2 cells were about at least three fold higher than IC50 of CBT in HCT116 cells. The enhanced resistance to CBT of TP53 KO cells indicates that the inhibitory effect of CBT to HCT116 cells relies on the TP53 pathway (Fig. 3). All these results substantiate that CBT inhibits HCT116 cells mainly via activating p53 pathway. 

In this study, Ubigene provided hTP53 gRNA KO plasmids for the construction of TP53 knockout cell line. It was demonstrated that CBT inhibited HCT116 cells mainly by activating p53 pathway. Ubigene now provides TP53 knockout cell lines, which can be delivered within one week for only $1980. Click to learn more > >  

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Fig. 1

 

Fig. 2

 

Fig. 3

 

In this study, they showed that CBT can efficiently inhibit colorectal cancer proliferation and migration.  It suppresses colorectal cancer via enhancing the expression of multiple p53 downstream effector genes and promoting cell cycle arrest, apoptosis and inhibition of angiogenesis. Hence, CBT may be served as an alternative option for colorectal cancer treatment in future. 

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[Research Highlight] miR-302a is involved in vascular smooth muscle cell proliferation and restenosis

 

Vascular stenosis in different parts of body can lead to diseases of organs or tissues dominated by blood vessels in corresponding areas, such as Acute coronary syndrome(ACS) which is led by coronary artery stenosis and other cardiovascular diseases. ACS is a common serious cardiovascular disease, a serious type of coronary heart disease, percutaneous coronary interventions (e.g., balloon angioplasty or stenting) are considered the first-choice treatment for it. However, patients face an increased risk of restenosis after surgery. Excessive smooth muscle cell (SMC) proliferation, migration and invasion are considered important contributors to restenosis, which together lead to neointimal hyperplasia. Therefore, inhibition of SMC hyperplasia is an important therapeutic approach for restenosis.

MicroRNAs (miRNAs) are a family of endogenous noncoding small RNAs consisting of 18–22 nucleotides that can regulate gene expression at the post-transcriptional level by pairing with imperfect complementary target sites in the 3’UTR of their target mRNAs. Previous studies have suggested that miRNAs are involved in regulating SMC proliferation and migration. The miR-302a functions as a tumor suppressor by inhibiting Akt-dependent cell proliferation in different cancer cells, indicating that miR-302a may negatively regulate Akt activation. And the lack of Akt has been shown to inhibit VSMC proliferation and migration.

Given the importance of Akt in regulating cell proliferation, Ying-ying Liu et al from SUN YAT-SEN UNIVERSITY speculated that miR-302a is involved in Akt-mediated vascular SMC (VSMC) proliferation and restenosis. They published an article “MicroRNA-302a promotes neointimal formation following carotid artery injury in mice by targeting PHLPP2 thus increasing Akt signaling” in Acta Pharmacologica Sinica. In the article, they used MiR-302 heterozygous mice and MiR-302 conditional KO mice building the carotid artery injury model, to investigated whether and how MicroRNAs regulated vascular SMC proliferation and vascular remodeling following carotid artery injury in mice. The study demonstrate that carotid artery injury-induced neointimal formation was remarkably ameliorated in miR-302 heterozygous mice and SMC-specific miR-302 knockout mice. In contrast, delivery of miR-302a adenovirus to the injured carotid artery enhanced neointimal formation. Upregulation of miR-302a enhanced the proliferation and migration of mouse aorta SMC (MASMC) in vitro by promoting cell cycle transition, whereas miR-302a inhibition caused the opposite results.

miR-302a potentiates MASMC proliferation by increasing Akt activity

miR-302a promoted Akt activation by corporately decreasing Akt expression and increasing Akt phosphorylation in MASMCs. To further confirm whether Akt activity is required for the regulatory function of miR-302a in VSMC proliferation, cells were treated with miR-302a mimics and the Akt inhibitor GSK690693 in the presence of PDGF-BB. Compared with miR-302a mimics-treated MASMCs, treatment with GSK690693 inhibited cell proliferation and migration, which was potentiated by miR-302a upregulation. These results suggest that miR-302a regulates VSMC proliferation dependent on Akt signaling.

Figure 1

PHLPP2 is essential for the function of miR-302 in neointimal formation

They further revealed that miR-302a directly targeted at the 3′ untranslated region of PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) and negatively regulated PHLPP2 expression. Restoration of PHLPP2 abrogated the effects of miR-302a on Akt activation and MASMC motility. To further evaluate the requirement of PHLPP2 during miR-302- regulated restenosis in vivo, recombinant adenovirus harboring leucine rich repeat protein phosphatase 2 shRNA (Ad-shPHLPP2) (provided by Ubigene) was administered to miR-302^+/− mice. The result showed that the inhibitory effects of miR-302 knockdown on neointimal formation are largely dependent on the elevation of PHLPP2 expression(Fig.2).

The shRNA adenovirus provided by Ubigene in this study was used to further study the effect of PHLPP2 on the formation of mir-302 in neointima. The purity and titer of adenovirus determine its quality and way of use. The adenovirus particles provided by Ubigene are ultra-centrifuged and filtered, and the titer is determined by hexonimmunoassay to ensure the purity and accurate titer. At present, Ubigene provides adenovirus with titer of 10 ^{^ 10} ~ 10 ^{^ 12} PFU/ml. Click here to learn >>

Figure 2

In conclusion, the study demonstrated that miR-302a potentiates VSMC hyperplasia and subsequent neointimal formation by Akt activation via targeting its phosphatase PHLPP2, suggesting that inhibition of miR-302a may be a novel strategy for restenosis treatment, and this study demonstrated for the first time that PHLPP2 is a novel target of miR-302a. It provided a new idea for vascular restenosis treatment in future.

Ubigene is focusing on cell genome editing. We provide high-quality gene editing cell lines, stable cell lines, virus packaging and other related services. Our KO cell bank contains nearly 2000 KO cell lines, involving in 8 popular signal pathways, such as key genes of Akt signal pathway. 

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[Research highlight] New target for NAFLD treatment--Metformin activates TFEB to induce autophagy

 

Nonalcoholic fatty liver disease (NAFLD) is a major liver disease subtype , which is characterized by an excessive hepatic lipid accumulation, covers a wide range of liver disorders, including steatosis, steatohepatitis, fibrosis, and cirrhosis. Metformin is commonly used to treat type 2 diabetes mellitus and, in recent years, it was found to play a potential role in the amelioration of NAFLD. Transcription factor EB (TFEB) is a master transcriptional regulator of lysosomal biogenesis and autophagy and, when activated, is effective against disorders of lipid metabolism.

TFEB plays a key role in lipid metabolism. However, whether TFEB is involved in the protective effect of metformin against NAFLD has not yet been reported. In response to this question, Dan Zhan et al. from first Affiliated Hospital of Kunming Medical University published an article “Metformin Alleviates Hepatic Steatosis and Insulin Resistance in a Mouse Model of High-Fat DietInduced Nonalcoholic Fatty Liver Disease by Promoting Transcription Factor EB-Dependent Autophagy” in Frontiers in Pharmacology.

In this report, mice were divided into five groups: 1) the control group, 2) high fat diet (HFD)-fed group, 3) HFD + Metformin (Met) group, 4) HFD + Met + Scramble control group, and 5) HFD + Met + TFEB shRNA group. The result demonstrates that the activity of TFEB is reduced in the liver of mice fed a high-fat diet. Metformin treatment significantly reverses the activity of TFEB, and the protective effect of metformin against hepatic steatosis and insulin resistance is dependent on TFEB. They found that metformin-induced autophagy is regulated by TFEB, and the findings reveal that TFEB acts as a mediator, linking metformin with autophagy to reverse NAFLD, and highlight that TFEB may be a promising molecular target for the treatment of NAFLD. 

 

 

Metformin Induces Autophagy in the Liver of NAFLD Mice

A metformin treatment can signifificantly improve an excessive lipid accumulation, hepatic steatosis, and IR in the mice fed an HFD. And an autophagy dysregulation may contribute to the pathogenesis of NAFLD. So the researchers hypothesized that metformin reduces obesity, hepatic steatosis, and IR, probably via the activation of autophagy. To verify the activation of autophagy in the liver, they evaluated the levels of autophagy proteins Atg7, LC3B-II, as well as autophagic substrate p62/SQSTM1 by using Western blotting. The results demonstrate that metformin promotes autophagy in the liver of HFD-induced NAFLD mice (Figure 1).

 

Figure 1

 

Metformin Induces Autophagy Through the Activation of TFEB in the Liver of NAFLD Mice

TFEB is a major regulator of autophagy and lysosomal biogenesis. The activity of TFEB mainly depends on its phosphorylation status and cytoplasm-nucleus shuttling , and the nuclear translocation is a hallmark of TFEB activation. Firstly, the researchers used Western blotting to examined whether metformin activated TFEB, the result showed that metformin treatment resulted in an increased nuclear accumulation of TFEB (Figure 2A) and reduced phosphorylation of TEFB. Then, to further test whether TFEB is involved in metformininduced autophagy, the researchers performed the knockdown of TFEB via tail vein injection with an AAV8 virus expressing an shRNA (provided by Ubigene) that targets TFEB (shTFEB). Compared with the injections of a scrambled control shRNA, the injections of recombinant AAV8-shRNA-TFEB reduced the expression of TFEB, as well as that of its target genes. Western blotting revealed that the knockdown of TFEB downregulated the expression of LC3B-II and upregulated the protein expression of p62/SQSTM1 (Figure 2C). Likewise, a downregulated TFEB level did not affect the protein level of Atg7. Taken together, these results confirm that metformin induces autophagy through the activation of TFEB.

In this study, Ubigene provided the recombinant AAV8 shRNA TFEB and the control AAV to study whether TFEB was involved in metformin induced autophagy. The AAV particles provided by Ubigene are ultra-purified, and the virus titer is 10^{^12}~10^{^13} GC/ml, so it is highly adaptable to in vivo experiments. Click here to learn about our AAV production > >

 

Figure 2

 

Metformin improves excessive lipid accumulation and IR in HFD-fed mice via TFEB

To test whether the activation of autophagy mediated by TFEB was involved in the alleviation of an excessive lipid accumulation and IR in HFD-fed mice, they measured the hepatic steatosis level using H&E and Oil red O staining after the knockdown of TFEB(Fig.3). The result showed that the protective effects of metformin on excessive lipid accumulation and IR are critically dependent on the TFEB in HFD-fed induced NAFLD mice.

 

Figure 3

 

Overall, this study reveals a novel molecular mechanism for improving the NAFLD by using metformin, and highlight the potential beneficial effects of TFEB for the treatment of NAFLD.

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[Research highlight]A new target for human endometrial cancer treatment |Ubigene

 

Human endometrial cancer (EC) is one of the malignant tumors of the female reproductive tract . With the increase of aging populations, obesity, and other metabolic diseases, as well as the delay of marriage and the increasing number of infertile patients, the incidence of EC is increasing and shows a younger trend. Early diagnosis and treatment can improve the prognosis of patients with EC and reduce their clinical mortality. However, for some early asymptomatic people, the diagnosis is in the late stage of EC and the etiology of EC has not been fully clarified. Therefore, it is still an urgent issue to elucidate the mechanisms contributing to EC occurrence. 

 

In response to this question, Xingmei Wu et al from Lishui People's Hospital of Zhejiang province published an article “RNA Binding Protein RNPC1 Suppresses the Stemness of Human Endometrial Cancer Cells via Stabilizing MST1/2 mRNA” on MEDICAL SCIENCE MONITOR. RNA binding protein RNPC1 has been shown to play suppressive roles in various tumors, RNPC1 attenuates non-small cell lung cancer (NSCLC) progression by enhancing CASC mRNA through suppressing miR-181a binding to CASC mRNA. However, its roles in EC progression have never been demonstrated. This research shows that RNPC1 overexpression activated the Hippo pathway, a tumor-suppressive signaling, through directly binding to MST1/2 and inhibition of MST1/2 rescued RNPC1-mediated effects on EC sphere stemness.

 

RNPC1 was up or down regulated by overexpression lentivirus, knockdown lentivirus. All lentiviruses in this article were constructed by Ubigene (Guangzhou, China). Western blot, quantitative polymerase chain reaction and sphere forming analysis were performed to evaluate the stem-like traits of cells and RNPC1-induced effects on EC cell stemness. RNA immunoprecipitation (RIP) was constructed to investigate the underlying mechanisms. It was found that the spheres formed by EC cells, named EC spheres, exhibited a remarkably higher stemness than the parental cells, which is characterized as the increase of sphere forming ability, ALDH1 activity, stemness marker expression and migration ability. Notably, RNPC1 expression was decreased in poorly differentiated EC cells than that in EC cells with moderately differentiated. Additionally, RNPC1 expression was significantly decreased in EC spheres and RNPC1 overexpression attenuated the stemness of EC spheres. Moreover, RNPC1 overexpression decreased the migration ability of EC spheres. 

Further study showed that RNPC1 was identified as the upstream regulator of MST1/2, indicateing a possibility that targeting RNPC1 might be a good strategy to activating the Hippo pathway during EC progression, which means RNPC1 could be a potential target for EC treatment.

In this study, Ubigene provided the RNPC1 overexpressed lentivirus, knockdown (shRNA) lentivirus and empty vector to study the mechanism of inhibition of EC cell stemness by RNPC1 and MST1/2 mRNA binding. Ubigene has a mature virus packaging system and hundreds of cell transfection experience. We provide high titer viruses for in vitro cell infection and in vivo use. Now it is only $599, visit us to learn more>>

RNPC1 directly bound to MST1/2 and enhanced their mRNA stability

The Hippo pathway, a tumor-suppressive signaling, the main members of this pathway are MST1/2 kinase, LATS1/2 kinase, and their junction proteins Sav and MOBI1 in mammals. The main function of the Hippo signaling pathway is to regulate the activity of transcriptional co-activators YAP and TAZ. When the Hippo signaling pathway is activated, MST1/2 kinase binds to and activates its ligand protein Sav. Then phosphorylated LATS1/2 kinase and MOB1 protein increase the formation of LATS/MOB1 complex and activate LATS1/2 kinase. Activated LATS1/2 kinase inactivates YAP and its byproduct TAZ. As the RNPC1 always binds to mRNAs and enhances their stability, the researchers wondered whether RNPC1 could activate the Hippo pathway. It was found that the expression of MST1/2 and LATS1/2 was decreased in EC spheres compared with EC cells (Figure 1A,1B). However, RNPC1 overexpression increased MST1/2 but not LAST1/2 mRNA levels in EC spheres (Figure 1C). Thus, we speculated that RNPC1 could directly bind to MST1/2 mRNA and thus activate the Hippo pathway. As expected, RIP analysis showed that MST1/2 was enriched in RNA pulled down by anti-RNPC1 but not the control IgG in EC spheres, but LATS1/2 was not enriched (Figure 1D). Additionally, MST1/2 mRNA stability was enhanced by RNPC1 overexpression in EC spheres (Figure 1E, 1F).

Figure 1

 

Inhibition of MST1/2 rescued the inhibition of RNPC1 overexpression on the stemness of EC spheres

Finally, the researchers explored whether RNPC1 attenuated the stemness of EC spheres dependent on MST1/2. XMU-MP-1, an inhibitor of MST1/2, was added to EC spheres with RNPC1 overexpression. The expression of the downstream effectors of MST1/2, phosphorylated LATS1/2 (p-LATS1/2), was determined to evaluate the inhibitory efficiency of XMU-MP-1 (Figure 2A). The results showed that XMU-MP-1 partially reversed the decreased ALDH1 activity (Figure 2B), the reduced sphere forming ability (Figure 2C, 2D),reduction of stemness marker expression (Figure 2E, 2F), which were mediated by RNPC1 overexpression. These results suggest that RNPC1 attenuates the stemness of EC spheres through binding to MST1/2 and thus activating the Hippo pathway.

Figure 2

 

In this study, RNPC1 was identified as the upstream regulator of MST1/2. However, the detailed binding regions or sites were not elucidated in this study, and therefore should be investigated in future work. Although the main conclusion should be confirmed by further in vivo experiments, targeting RNPC1 might be a good strategy to activating the Hippo pathway during EC progression.

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U-87 MG with CRISPR/Cas9 technology can facilitates brain tumor research!

 

Background

In 1996, U-87 MG cell line was isolated from the malignant glioma of a 44-year-old female patient. This human primary glioblastoma cell line has epithelial morphology and can produce malignant tumors consistent with glioblastoma in nude mice. It is the most common brain tumor research model. In addition to being commonly used in brain cancer research, U-87 MG cell line is also widely used in biomedical industry, such as pathophysiology research, antibiotic screening, cancer drug research, etc. Ubigene provides wild-type U-87 MG cell line with low passage and good viability. STR authentication report is also provided. It is suitable for the construction of various gene-editing cell models.

 

Applications of CRISPR/Cas9 technology in U-87 MG cell line

CRISPR/Cas9 technology, as a popular gene-editing technology at present, can be used for the precise construction of various disease models, so as to facilitate research of cancer treatment, pathology, physiology and high-throughput drug screening. This technology has been well applied to U-87 MG cell line.

 

Case Study 1: Gene knockout U-87 MG help to study on growth inhibition of brain tumor cells

Glioblastoma multiforme (GBM), take up 70% of all gliomas, is the most common brain tumor in adults, but there is no curative treatment at present. All patients will basically relapse even after the maximum surgical resection, radiotherapy and chemotherapy. And the median survival time is only about 15 months. Therefore, we should deeply understand the molecular mechanism of glioma occurrence and development. And looking for new targets of treatment has become one of the research hotspots in recent years.

Some studies have found that the deletion of ACSVL3 in U-87 MG will reduce its tumorigenic properties and affect signal transduction through receptor tyrosine kinase. Therefore, Kolar et al knocked out ACSVL3 from U-87 MG cell line, and conducted multiple analyses to explore the effects of ACSVL3 deletion on the carcinogenicity of U-87 MG cell line and GBM cells. Proteomic analysis showed that the content of enzymes involved in ceramide synthesis was low, the level of TCA enzyme was low and the level of glycolytic enzyme was high in ACSVL3 KO U-87 MG cell line. It was found that the knockout of ACSVL3 would affect the synthesis of lipid raft and ganglioside by fluorescence microscope and thin-layer chromatography (TLC). At the same time, the antibody detecting the mitochondrial outer membrane marker Tom20 was used for immunofluorescence. It was observed that the mitochondrial morphology of ACSVL3 KO U-87 MG was different from that of wild type. In conclusion, Kolar et al. believe that ACSVL3 contributes to the GBM cell growth and proliferation, the synthesis of signal sphingolipids, and this enzyme may contribute to the carbohydrate metabolism involved in mitochondria ^[1]. This conclusion has certain research significance on how to inhibit the occurrence and expansion of GBM.

KO cell line plays an important role in studying the occurrence and expansion of tumors. Ubigene exclusive KO cell bank has nearly 2000 kinds of KO cell lines in stock, covering thousands of genes and hundreds of tumor dominated diseases, as low as $1980. Click here to access our KO cell bank>>

 

Case study 2: U-87 MG point mutant cell line contributes to the pathophysiology of Gaucher's disease (GD)

Gaucher's disease (GD) is a disease caused by acid β-Autosomal recessive lysosomal deposition caused by glucosidase gene (GBA1) mutation. Pavan et al. Used CRISPR/Cas9 to edit GBA1 in human monocyte THP-1 cell line and glioblastoma U-87 MG cell line to construct isogenic GD model (GBA1 mutant). It was found that the two edited cell lines showed low levels of mutant acidity β-Glucosidase expression, less than 1% residual activity, and a large amount of substrate accumulation. In addition, GBA1 mutant U87 MG showed that the mutant enzyme was retained in the endoplasmic reticulum and degraded by proteasome, triggering the unfolded protein reaction (UPR). Compared with wild-type cells, the accumulation of α-syn in GBA1 mutant U87 MG could increase interleukin-1β, and the mutant cell line has higher cell mortality ^[2]. These results provide a theoretical basis for the study of the pathophysiological mechanism of GD.

The construction of point mutation cell model is helpful to study the pathological mechanism of single gene mutation diseases, but the successful construction of the model not only needs to select appropriate gene-editing method, but also needs rich experience of cell line construction. Ubigene specialized in point mutation cell disease model generation, as low as $8480, exclusive CRISPR-U™, higher success rate! For more promotion details, please click here>>

 

Case study 3: Multiple U-87 MG point mutation cell lines contribute to cancer therapy development

Antibody drug conjugates can target cells and genes with high specificity. Kwon et al constructed multiple variants of TP53 gene in U-87 MG using CRISPR/Cas9 system, and determined that the main transcriptional difference was due to the loss of p53 function. Using transcriptome data, they predicted which mutant clones had less phenotypic difference from the wild-type, so as to screen the best candidate genes that can be used as a drug delivery test platform. Further experiments on cell morphology, proliferation rate and target antigen mediated uptake also confirmed their hypothesis. According to the results of comprehensive analysis, they had successfully screened the most suitable mutant clone. This study provides a great idea for exploring cancer therapeutic reagents ^[3].

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[Research highlight] A new idea of prevention and cure of COVID-19! |Ubigene

 

SARS-CoV-2 has caused a worldwide pandemic since 2019. Some studies have found that SARS-CoV-2 increases the number of autophagosomes, which seems to be a good breakthrough in prevention and treatment. However, a study on non-human primates showed that the FAD approved potential drug candidate chloroquine (CQ)/hydrochloroquine, as an autophagy regulator, could not inhibit SARS-CoV-2 infection, and it was found that it had no beneficial effect on the mortality of COVID-19 patients.

In view of this situation, Chao Shang, etc. (Changchun Veterinary Research Institute, Chinese Academy of Agriculture Sciences) believed that the mechanism of SARS-CoV-2 regulating autophagy in the host needs to be fully studied in different animal models. Recently, they published in the Journal of Virology (2020 IF: 5.1027) "Inhibition of autophagy suppresses SARS-CoV-2 replication and ameliorates pneumonia in hACE2 transgenic mice and xenografted human lung tissues" The results of SARS-CoV-2 infection on the established non-human primate Macaca fascilaris model and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and constructs the original SARS-CoV-2 infection model based on human lung xenotransplantation mice have been shown in the article. It reveals the new mechanism of SARS-CoV-2 manipulating autophagosome formation, and found that autophagy inhibitors may be potential drugs for the treatment of SARS-CoV-2 virus infection, which provides a new idea for the study of the pathogenic mechanism of SARS-CoV-2 and its prevention and treatment.

 

SARS-CoV-2  infection was found to trigger autophagy in the Macaca fascularis infection model

Researchers analyzed autophagy by collecting SARS-CoV-2 infection in the lungs of the Macaca fascularis and the corresponding areas of the infected lung. It was found that the expression level of Atg5-Atg12 and LC3-I /LC3-I was significantly increased after SARS-CoV-2 infection (Autophagy activation).

 

There are two main ways for SARS-CoV-2 to increase autophagy

LC3-I, which is mainly located in the cytoplasm, is transformed into LC3-II after autophagy activation. LC3-II is integrated into the extended phagocyte membrane and controlled to extend into a complete autophagosome. Therefore, LC3-II can be used as a reliable autophagosome marker. Western blot (WB) showed that the expression level of LC3-II in Vero E6 and Huh-7 cell lines increased significantly after SARS-CoV-2 infection. In order to visually observe the accumulation of autophagosomes, researchers constructed Vero E6 and Huh-7 cell lines transformed with GFP-LC3 plasmid (constructed by Ubigene), and found that the level of GFP in cells increased significantly after SARS-CoV-2 infection for 24 hours (hpi), indicating that SARS-CoV-2 infection induced the formation and accumulation of autophagosomes. In addition, EGFP-mCherry-LC3 transient Vero E6 cell line (constructed by Ubigene) was constructed to visualize the transfer of LC3 from autophagosome to lysosome. It was found that SARS-CoV-2 increased the number of autophagosomes in two ways: (1) the biogenic events of autophagosome increased; (2) the fusion of autophagosome and lysosome decreased.

Figure 1 SARS-CoV-2 induces autophagosome formation in cell lines and blocks autophagosome-lysosome fusion

 

Autophagy related cascade reactions were analyzed to determine the relationship between autophagosome formation and virus replication

In order to further clarify the mechanism of SARS-CoV-2-induced autophagosome formation, researchers analyzed a series of signal cascades involved in autophagosome formation in the Vero E6 cell line infected with SARS-CoV-2. mTORC1 signaling pathway is the most common upstream autophagy inhibitory regulator. Researchers choose to monitor its activity by phosphorylation of its downstream substrate. It was found that SARS-CoV-2 not only inhibited Akt-mTOR signaling pathway, but also activated ULK-1-Atg13 and VPS34-VPS15-Beclin1 complexes. Moreover, in order to determine the relationship between autophagosome formation and virus replication and the key factors of virus replication, researchers used broad-spectrum autophagy inhibitor 3-MA and VPS34 inhibitor SAR405. It was found that the pretreatment of 3-MA and SAR405 significantly reduced the virus load in the Vero E6 cell line, indicating the importance of autophagy formation and VPS34 complex activation for SARS-CoV-2 virus replication.

Figure 2 Autophagy formation and VPS34 complex activation promote the replication of the SARS-CoV-2 virus

Inhibition of autophagy can reduce SARS-CoV-2 replication and improve pneumonia in mice

Researchers used two different animal models to simulate the infection process of the SARS-CoV-2 in vivo. In the hACE2 transgenic mouse model, inhibition of autophagy can inhibit the replication of SARS-CoV-2 virus and improve pneumonia. On the contrary, rapamycin induced autophagy can promote viral autophagy and aggravate the pathological process of pneumonia.

 

In the innovative mouse model (transplanted with human lung tissue), they proved that SARS-CoV-2 could replicate itself in human lung tissue and spontaneously produce pneumonia.

Figure 3 Inhibition of autophagy can inhibit SARS-CoV-2 replication and improve pneumonia in the mouse model (transplanted with human lung tissue)

In conclusion, researchers confirmed that autophagy promotes the replication of SARS-CoV-2 and promotes the process of pneumonia, and proposed that autophagy inhibitors may be potential drugs for the treatment of SARS-CoV-2 virus infection. Through the mutual verification of in vivo and in vitro experiments. This study lays a theoretical foundation for the in-depth study of virus cell interaction in the future, and provides a useful idea for the development of autophagy related antiviral drugs.

Ubigene focuses on cell line engineering. In this study, engineered Vero E6 and Huh-7 cell lines were provided to explore the relationship between SARS-CoV-2 virus replication and autophagy induction mechanism.

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