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Application of PD-L1 (Programmed Death-Ligand 1) spliceosome c as target for predicting tumor prognosis

A splice body and prognostic target technology, applied in the fields of biomedicine and tumor immunotherapy, can solve the problems that no research has shown the role of cancer, no research has shown the role of three splice bodies and their mechanism, etc.

Active Publication Date: 2018-11-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] It has been disclosed in the prior art that there are three alternative splicing bodies (isoforms) of PD-L1, namely Isoform a, Isoform b, and Isoform c, but only the existence of the three splicing bodies is disclosed, and no research has shown that the three splicing bodies exist in In vivo action and mechanism, and no studies have shown its role in a specific cancer

Method used

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  • Application of PD-L1 (Programmed Death-Ligand 1) spliceosome c as target for predicting tumor prognosis
  • Application of PD-L1 (Programmed Death-Ligand 1) spliceosome c as target for predicting tumor prognosis
  • Application of PD-L1 (Programmed Death-Ligand 1) spliceosome c as target for predicting tumor prognosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1 Analysis of PD-L1 expression in colorectal cancer cell lines

[0141] Collect all colorectal cancer cell lines in the laboratory for PD-L1western bolt test, the results are as follows Figure 3a Shown. The PD-L1 antibody is Abcam: Anti-PD-L1 antibody [28-8] (ab205921). It can be seen from the figure that among the common cell lines of colorectal cancer, only RKO and RKO-MUT cell lines express PD-L1. Among them, RKO-MUT cells are mutant cells of RKO cells and are preserved by the Pathology Laboratory of Zhejiang University School of Medicine. The inventor promises that the cells can provide the cell line to the public for free within 20 years from the patent application date.

Embodiment 2

[0142] Example 2 RNAi interferes with the expression of PD-L1 of RKO / RKO-MUT

[0143] RNAi RKO / RKO-MUT cell line PD-L1 expression detection, the siRNA sequence used in it is shown in the following table:

[0144]

[0145]

[0146] The result is Figure 3b Shown. On the way, it can be seen that after RNAi, the expression of PD-L1 in RKO / RKO-MUT cells has decreased, and the expression of PD-L1 in RKO cell line has decreased more significantly than that in RKO-MUT cell line. In addition, surprisingly, the inventors observed that the band position of PD-L1 in the RKO-MUT cell line is different from that in the RKO cell line, suggesting that PD-L1 may have multiple alternative spliceosomes. After searching, the PD-L1 protein of Homo sapiens has three natural alternative splicing bodies, namely isoforma / b / c. The comparative relationship between the encoded nucleotide sequences can be seen in Figure 1. The encoded polypeptides For the sequence, please refer to NCBI Accession Number NCBI...

Embodiment 3

[0147] Example 3 RT-qPCR to detect the expression of three spliceosomes in different colorectal cancer cell lines

[0148] Primers are designed in the common sequence region of the three variants, which can detect all three variants; the primers are designed for the three variants, and only one variant can be specifically detected. The mRNA expression of the three variants was detected by qPCR, and the primer sequences are shown in the following table:

[0149] Primer name

[0150] The result is Figure 4 As shown, similar to the results of western-blot, except for PD-L1 expression in RKO and RKO-MUT cell lines, only trace amounts of PD-L1 mRNA in other colorectal cancer cell lines were transcribed and detected. In addition, the expression of isoform b was not detected in RKO and RKO-MUT cell lines.

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Abstract

The invention discloses application of a PD-L1 (Programmed Death-Ligand 1) spliceosome c as a target for predicting tumor prognosis. Expression of three different variable spliceosomes of PD-L1 can beobserved in normal and mutant colorectal cancer cell systems, and eukaryotic expression vectors of the three spliceosomes are established and transfected into tumor cells without or with low PD-L1 expression; tests show that proliferation, migration and invasion of tumor cells are promoted by isoform c, occurrence and development of tumor can be promoted by isoform c, and isoform c is a potentialtumor diagnosis and treatment target and a monitoring target for poor prognosis of tumor; meanwhile, results further show that in co-culture of a tumor cell system with isoform b over-expression andimmune cells, apoptosis of the immune cells is promoted, secretion of immune cell factors is inhibited, inhibition of immunoreactions in bodies of patients can be reminded, and isoform c is a monitoring target of poor prognosis of tumor.

Description

Technical field [0001] The invention relates to the application of a PD-L1 spliceosome in guiding the medication of tumor immunotherapy, and belongs to the field of biomedicine, specifically the field of tumor immunotherapy. Background technique [0002] In recent years, immune checkpoints against programmed death-1 (PD-1 / CD279) / programmed death ligand-1 (PD-L1 / CD274) pathway have been blocked. Anti-tumor therapy is a research hotspot in antitumor therapy. At present, reagents such as Atezolizumab, nivolumab, and pembrolizumab have been used in clinical trials of melanoma, non-small cell lung cancer, colorectal cancer (CRC) and other malignant tumors, and have achieved certain therapeutic effects [1]. Among them, colorectal cancer is the world's third most common cancer, and its fatality rate ranks fourth among malignant tumors, and research on its application in diagnosis and treatment is imminent. [0003] PD-L1 is a potential indicator of CRC diagnosis and treatment, and there...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 张红河王超彦翁梦涵来茂德
Owner ZHEJIANG UNIV
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