ShRNA targeting CPE and application thereof

Abstract

The invention discloses shRNA (short hairpin Ribonucleic Acid) targeting CPE (Chlorinated Polyethylene), and belongs to the technical field of biological medicines, and the shRNA has the following sequence: 5 '-AATC-GCTATCTTCCATAGCACCGTATAACTTATTTAGCCTTGCCAGAG-ACTGG-3'. The invention also provides a cell and a medicine comprising the shRNA targeting CPE, and an application of the shRNA targeting CPE in preparation of medicines for preventing and treating liver cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a shRNA targeting CPE and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the seventh most common malignant tumor in the world and the third most lethal. In China, it causes more than 440,000 deaths each year, accounting for about 45% of the total global deaths. Due to the complexity of the pathogenesis of liver cancer, it remains a disease with serious unmet medical needs in recent years. Therefore, effectively controlling the invasion and metastasis of HCC is the fundamental way to improve the treatment effect of HCC patients and obtain long-term survival. It is imperative to seek efficient and safe immunotherapy as soon as possible.

[0003] Studies have shown that carboxypeptidase E (CPE) is a multifunctional protein that plays a vital role in the endocrine and nervous systems. It promotes tumor growth and spread and is a predictive biomarker for endocrine and non-endocrine tumor metastasis. Increased levels of CPE gene and protein are associated with poor prognosis in patients with colorectal cancer and liver cancer. In addition, studies have found that glycolytic reprogramming and lipogenesis play an important role in HCC. Aerobic glycolysis (Warburg effect) controls liver cancer progression through rate-limiting enzymes, and enhanced aerobic glycolysis can stimulate the anabolism of liver cancer cells. Abnormal activation of fatty acid synthase and related de novo lipogenesis play a key role in the development of liver cancer. However, how CPE affects the progression of liver cancer and whether it is related to glycolytic reprogramming and lipogenesis are key to identifying specific target antigens and developing cancer immunotherapy. How to develop targeted drugs for liver cancer based on the above information has become a top priority. Summary of the invention

[0004] The present invention aims to provide a shRNA targeting CPE, which can knock down the expression level of CPE and is used for preventing and treating liver cancer.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a shRNA targeting CPE, wherein the shRNA has the following sequence:

[0007] 5'-AATC-GCTATCTCTCCATAGCACCGTATAACTCTATTTAGCCTTG

[0008] CCAGAGAG-ACTGG-3'.

[0009] In a second aspect, the present invention provides a cell comprising the above-mentioned shRNA.

[0010] In a third aspect, the present invention provides a drug comprising the above-mentioned shRNA.

[0011] In a fourth aspect, the present invention provides use of the above shRNA, the above cell or the above drug in the preparation of a method for preventing and treating liver cancer.

[0012] The beneficial effects of the present invention are as follows: the shRNA provided by the present invention can knock down the expression of CPE in vivo and in vitro, and can be directly used as the basis for shRNA drug design for preventing and treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 High-fat diet induces the expression of CPE in vivo. (A) Analysis of differential gene expression in the GSE151620 dataset; (B) Comparison of gene expression between tumor and normal groups; (C) Comparison of gene expression fold changes between ND-HCC and HFD-HCC groups (mice); (D) Comparison of gene expression fold changes between non-obese and obese HCC patient groups; (E) Western blot analysis of CPE between ND-HCC and HFD-HCC groups and between non-obese and obese HCC patient groups; (F) Immunohistochemistry (IHC) of CPE; (G) Western blot analysis of CPE in normal / tumor tissues.

[0015] Figure 2 The effect of CPE knockdown on high-fat-induced liver cancer in mice. (A) Tumor imaging; (B) Measurement of tumor volume; (C) Measurement of tumor weight; (D) Immunohistochemistry (IHC) of Ki67; (E) Survival analysis; (F) Determination of serum alanine aminotransferase (ALT) level; (G) Determination of serum aspartate aminotransferase (AST) level; (H) Measurement of fasting blood glucose (FPG) level; (I) Measurement of fasting insulin (FINS) level.

[0016] Figure 3(A) Western blot analysis of CPE in LO2 / SNU449 and Huh7 cell lines; (B) CPE expression in Huh7 cells induced by palmitic acid (PA) to induce a high-fat state; (C) Survival analysis based on CPE levels. (A) Western blot analysis of CPE in LO2 / SNU449 and Huh7 cell lines; (B) CPE expression in Huh7 cells induced by palmitic acid (PA) to induce a high-fat state; (C) Survival analysis based on CPE levels.

[0017] Figure 4 CPE knockdown inhibits high-fat-induced cell proliferation in hepatoma cells. (A) Western blot of CPE protein levels; (B) Edu staining for cell proliferation.

[0018] Figure 5 CPE knockdown inhibits glycolysis in high-fat-induced hepatocellular carcinoma cells. (A) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements; (B) Glycolytic level and capacity measurements; (C) ATP generation and maximum respiration measurements; (D) Glucose and lactate production assays; (E) Western blot of glycolytic enzymes PDK1, PFKL, LDHA, and PKM2.

[0019] Figure 6 CPE knockdown inhibits lipogenesis in high-fat-induced hepatocellular carcinoma cells. (A) Cholesterol level measurement; (B) Triglyceride level measurement; (C) Western blot of lipogenic enzymes ACC1, ACLY, FASN, and SCD1. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Based on the Microarray data and RNA-Seq data from the TCGA database, the levels of CPE in genes differentially expressed in high-fat diet (HFD) and hepatocellular carcinoma (HCC) were analyzed. Specifically, Figure 1HFD induces CPE expression in HCC in vivo. Among them, A represents a selection array of 26 genes revealed by differential gene expression analysis based on the GSE151620 dataset, which changed between high-fat diet (HFD) liver tissue and normal diet (ND) liver tissue. It is worth noting that five genes (LY6D, LCN2, CPE, ELOVL7 and SPINK1) showed significant upregulation in both HFD liver tissue and HFD-HCC tissue. In contrast, 15 genes showed downregulation, while 6 genes showed different expression patterns under the two conditions. B shows that the five upregulated genes showed significant expression differences when compared between normal liver tissue and tumors, with tumor tissue showing higher expression levels. C shows that the differential expression of the five upregulated genes was more obvious in the HFD-HCC mouse model compared with the ND-HCC model. D indicates that the expression of these genes in obese HCC patients was significantly increased by two times compared with non-obese patients, further linking obesity to the severity of HCC. E is a Western blot analysis confirming that HFD-HCC mice and obese HCC patients showed higher levels of carboxypeptidase E (CPE) compared with HCC mice and normal-sized patients. F is the immunohistochemistry result, which found a higher proportion of CPE-positive cells in HFD-HCC tissues. G is a Western blot analysis comparing normal tissues and HCC tissues, highlighting the differential expression of CPE in HCC.

[0022] In summary, CPE may be involved in the occurrence of liver cancer, and obesity may further promote the progression of liver cancer through CPE.

[0023] According to the above results, the mRNA and protein expression of CPE in the HFD-HCC mouse model and obese HCC patient samples were significantly upregulated compared with the HCC mouse model and HCC patients, but how this upregulation regulates the progression of liver cancer is still unknown. Therefore, in the high-fat diet-induced liver cancer model, the key regulatory role of CPE on the occurrence and development of high-fat diet-induced liver cancer was analyzed:

[0024] C57BL / 6 mice (Shanghai Model Biological Center, China) were fed a high-fat diet to induce obesity, and their body weight was measured weekly. After 13 weeks, a small incision was made on the skin, and a Huh7 cell suspension (1×106 cells in 100 μl PBS) was injected subcutaneously using a 27-gauge needle, and the incision was then sutured with surgical sutures to construct the HFD-HCC mouse model. Figure 2It shows that CPE knockout can resist the effects of high-fat diet-induced liver cancer progression. A indicates that compared with the sh-NC group, the tumor growth in the sh-CPE group was significantly delayed. B shows that compared with the sh-NC group, the tumor weight in the sh-CPE group was significantly reduced. C is the result of immunohistochemistry, which confirms that the proliferation rate (Ki67 ratio) in the sh-CPE tumor was significantly reduced, and the above results are associated with the improved survival rate of the experimental animals (Figure D). Compared with the control group, the decrease in serum alanine aminotransferase (ALT) level (Figure E) and aspartate aminotransferase (AST) level (Figure F) indicates that the sh-CPE group has reduced liver damage and improved liver function; the fasting blood glucose (FPG) concentration (Figure G) in the sh-CPE group is significantly lower, indicating that glucose homeostasis is regulated; the fasting insulin (FINS) level is reduced (Figure H), indicating enhanced insulin sensitivity or reduced pancreatic β cell lineage.

[0025] In summary, CPE knockout can improve HFD-induced adverse reactions. It is further demonstrated that CPE accelerates HCC development in the HFD-HCC mouse model, which provides a therapeutic target.

[0026] In order to better understand the relationship between CPE and the development of liver cancer, an in vitro HCC model induced by palmitic acid (PA) at a concentration of 200μM or 400μM was established to induce a high-fat state. The experiment included three cell lines: LO2, SNU449, and Huh7 cells. A shows that CPE was significantly expressed in liver cancer cell lines compared with normal cells by Western blot analysis of LO2, SNU449, and Huh7 cells. Figures B and C show that a high-fat state was induced by PA treatment in Huh7 cells, resulting in a dose-dependent upregulation of CPE gene and protein levels. Finally, survival analysis showed that increased CPE expression was associated with decreased survival (Figure D).

[0027] Based on the above in vitro experimental results, it was further verified that obesity may further promote the progression of liver cancer through CPE.

[0028] In order to further study how CPE regulates the development of liver cancer and whether it is related to PA-induced glycolysis and lipogenesis in liver cancer cells, the following experiments were conducted:

[0029] The efficacy of CPE knockdown was verified by Western blotting, which showed that CPE protein levels were significantly reduced in cells treated with PA+sh-CPE compared with those in the PA and PA+sh-NC groups ( Figure 4 A). The reduction of Edu-positive cells in the PA+sh-CPE group indicated that CPE knockdown attenuated PA-induced cell proliferation ( Figure 4 B) ECAR measurements showed that sh-CPE-mediated knockdown reduced the level of glycolysis increased by PA treatment ( Figure 5A). The elevated OCR in PA-treated cells was reduced to near normal control levels in the PA+sh-CPE group ( Figure 5 A). The glycolysis level and capacity enhanced by PA treatment were significantly reduced after CPE knockdown ( Figure 5 B). ATP production and maximum respiratory rate, which were upregulated after PA treatment, were significantly reduced after sh-CPE expression ( Figure 5 C). The production of glucose and lactate, two key end products of glycolysis, was reduced after CPE knockdown ( Figure 5 D). In the PA+sh CPE group, the expression of glycolytic enzymes PDK1, PFKL, LDHA, and PKM2 was downregulated ( Figure 5 E), which play a central role in the glycolytic pathway and are upregulated by PA. Cholesterol levels, a marker of lipogenesis increased by PA, were significantly reduced after sh-CPE expression, indicating that lipogenic activity was inhibited ( Figure 6 A). In addition, the triglyceride level in the PA+sh-CPE group was significantly lower than that in the PA and PA+sh-NC groups ( Figure 6 B). The data showed that knockdown of CPE alleviated the effects of a high-fat diet on glycolysis and lipogenesis in HCC cells, as evidenced by the inhibition of key lipogenic enzymes ACC1, ACLY, FASN, and SCD1 ( Figure 6 C). In conclusion, CPE is a potent regulator of metabolic pathways in HCC cells, and its knockout can reverse the effects of a high-fat environment.

[0030] In summary, obesity may regulate glycolysis and lipogenesis in liver cancer cells through CPE, further affecting the development of liver cancer.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A shRNA targeting CPE, characterized in that: The shRNA has the sequence as follows: 5'-AATC-GCTATCTCTCCATAGCACCGTATAACTCTATTTAGCCTTGCCAGAGAG-ACTGG-3'.

2. A cell comprising the shRNA according to claim 1. A drug comprising the shRNA according to claim 1.

4. Use of the shRNA according to claim 1, the cell according to claim 2, or the drug according to claim 3 in the preparation of a method for preventing and treating liver cancer.

Images