Application of PCSK9 in diagnosis and treatment of esophageal squamous carcinoma

[0060] Example 1 Screening for gene markers associated with esophageal squamous cell carcinoma

[0061] 1. Sample collection

[0062] Paracancerous tissues and esophageal squamous cell carcinoma tissue samples were collected from 6 cases of esophageal squamous cell carcinoma. All the above specimens were obtained with the consent of the organizational ethics committee.

[0063] 2. RNA sample preparation (using E.Z.N.A. kit to operate)

[0064] The tissues obtained above were shredded, put into liquid nitrogen and ground into powder, and RNA was extracted and isolated according to the instructions in the kit. details as follows:

[0065] 1) Isolation of RNA:

[0066] A. Add RNA to tissue homogenate or cells- Reagent II 1ml;

[0067] B. Stand at room temperature for 3 minutes, add 0.2ml chloroform and shake vigorously for 15 seconds;

[0068] C. Place on ice for 10 minutes;

[0069] D. Centrifuge at 12000g for 15min at 4°C;

[0070] E. Transfer 80% of the aqueous phase

[0085] Example 2 QPCR sequencing to verify the differential expression of PCSK9 gene

[0086] 1. Large-sample QPCR verification of differential expression of PCSK9 gene. According to the sample collection method in Example 1, 50 cases of normal esophageal tissues and 50 cases of esophageal squamous cell carcinoma tissues were selected.

[0087] 2. The RNA extraction steps are as described in Example 1.

[0088] 3. Reverse transcription:

[0089] 1) Reaction system:

[0090] Reagent

volume

MgCl 2

2μl

10×RT Buffer

1μl

RNase-free water

3.75μl

dNTP mix

1μl

RNase inhibitor

0.25μl

AMV reverse transcriptase

0.5μl

Oligo-dT Adapter Primer

0.5μl

Experimental sample

1μl

[0091] 2) Reverse transcription reaction conditions

[0092] According to the reverse transcription reaction conditions in RNA PCR Kit (AMV) Ver.3.0.

[0093] 42°C~55°C for 60 minutes, 99°C for 2 minutes, 5°C

[0111] Overexpression of embodiment 3PCSK9 gene

[0112] 1. Cell culture

[0113] Human esophageal squamous carcinoma cell line KYSE 150 was incubated at 37°C and 5% CO in DMEM containing 10% fetal bovine serum and 1% P / S. 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.

[0114] 2. siRNA design

[0115] siRNA sequence against PCSK9 gene:

[0116] Negative control siRNA sequence (siRNA-NC):

[0117] The sense strand is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.7),

[0118] The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.8);

[0119] siRNA1-PCSK9:

[0120] The sense strand is 5'-UCAUUGAUGACAUCUUUGGCA-3' (SEQ ID NO.9),

[0121] The antisense strand is 5'-CCAAAGAUGUCAUCAAUGAGG-3' (SEQ ID NO.10);

[0122] siRNA2-PCSK9:

[0123] The sense strand is 5'-UGUUUGAAUGGUGAAAUGCCC-3' (SEQ ID NO.11),

[0124] The antisense strand is 5'-GCAUUUCACCAU