Application of gene HES2 to auxiliary diagnosis, prognosis and treatment of esophageal squamous carcinoma
A technology for esophageal squamous cell carcinoma and auxiliary diagnosis, which is applied in the fields of molecular biology and tumor drugs, and can solve the problems of limited tools for guiding treatment and immature markers of esophageal cancer
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Embodiment 1
[0074] Example 1: HES2 is up-regulated in esophageal squamous cell carcinoma
[0075] (1) TCGA database analysis
[0076] Methods: In order to explore the expression of HES2 in esophageal cancer, the biological analysis method was used to analyze the mRNA expression data of Normal and Tumor in related esophageal cancer in The Cancer Genome Atlas (TCGA) database, and the prognosis analysis was carried out.
[0077] result( figure 1 A): The HES2 gene RNA level in esophageal squamous cell carcinoma tissue was higher than that in normal esophageal tissue and esophageal adenocarcinoma tissue.
[0078] (2) qRT-PCR detection of the mRNA level of HES2 gene in esophageal squamous cell carcinoma tissues and paired paracancerous tissues
[0079] Through the above analysis, it is known that the mRNA level of HES2 is highly expressed in esophageal squamous cell carcinoma, and the present invention further performs qRT-PCR detection and verification on HES2 in esophageal squamous cell car...
Embodiment 2
[0114] Example 2: Using small hairpin RNA (shRNA) to stably silence HES2 can suppress the malignant phenotype of esophageal cancer.
[0115] (1) Cell culture and stable strain construction
[0116] Methods: Esophageal cancer cells Eca109 and Kyse520 were treated with negative control shGFP and shRNA that can silence HES2 (including shHES2#1 and shHES2#2). WB verification was carried out by harvesting the corresponding protein. The targeting sequence of shHES2#1 is: 5'-CTGGAGCACCTGTGGCGGAG-3' (SEQ ID NO: 5), and the targeting sequence of shHES2#2 is: 5'-GCCTTGCGACAGCTACCGCG-3' (SEQ ID NO: 6).
[0117] Results: Control cells Eca109 shGFP, Kyse520 shGFP and HES2-silenced cells Eca109 shHES2#1, Eca109 shHES2#2, Kyse520 shHES2#1, Kyse520 shHES2#2 were obtained respectively.
[0118] (2) Cell growth experiment
[0119] Method: The culture medium containing 10% fetal calf serum was used to prepare a single cell suspension, and 1000-10000 cells per well were inoculated into a 96-we...
Embodiment 3
[0130] Example 3: Stable silencing of HES2 using antisense oligonucleotides (AON) targeting HES2 can inhibit the malignant phenotype of esophageal cancer
[0131] (1) Cell culture and stable strain construction
[0132] Methods: Esophageal cancer cells Eca109 were treated with negative control Scramble and AONs that could silence HES2 (HES2-AON#1, HES2-AON#2, HES2-AON#3, HES2-AON#4, HES2-AON#5), respectively, The working concentration is 5 nM.
[0133] HES2-AON #1: 5'-GCTCACTAGTCCACTA-3' (SEQ ID NO: 7);
[0134] HES2-AON#2: 5'-GCTGGTTAGATCTTAC-3' (SEQ ID NO: 8);
[0135] HES2-AON#3: 5'-GTGAAATTGATGCTCG-3' (SEQ ID NO: 9);
[0136] HES2-AON#4: 5'-AAGCAAGAGGTGGCAA-3' (SEQ ID NO: 10);
[0137] HES2-AON #5: 5'-TCCAGAGTCAAGCAAG-3' (SEQ ID NO: 11).
[0138] 1) qRT-PCR detection of HES2 mRNA expression level in esophageal squamous cell carcinoma cell lines after antisense oligonucleotide chain silenced HES2 gene.
[0139] Methods Six groups of cells were collected from the negat...
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