Target molecules for the development of therapeutic products for cervical diseases
A technique for cervical squamous cell carcinoma and drug candidates, applied in the field of biomedicine
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Embodiment 1
[0066] Example 1 Screening for Gene Markers Related to Cervical Squamous Cell Carcinoma
[0067] 1. Sample collection
[0068] 32 cases of cervical squamous cell carcinoma (CESC) tissues, 18 normal tissues (N), and 24 cases of cervical intraepithelial neoplasia (CIN) tissues were collected. All cases did not receive any immunosuppressant treatment, radiotherapy and chemotherapy before operation. All included All the subjects signed the informed consent before collecting the specimens. All the above specimens were obtained with the consent of the organizational ethics committee. Four samples were taken from each group for detection and analysis of gene expression profiles, screening of differentially expressed genes, and verification experiments were carried out in all case samples of each group.
[0069] 2. Preparation of RNA samples
[0070] Use QIAGEN tissue RNA extraction kit to extract total RNA in each group of tissues, please refer to the instruction manual for specif...
Embodiment 2
[0081] Example 2 QPCR sequencing to verify differential expression of FRMD4B gene
[0082] 1. Large-sample QPCR verification of differential expression of FRMD4B gene.
[0083] 2. RNA extraction
[0084] Use QIAGEN tissue RNA extraction kit to extract total RNA in each group of tissues, please refer to the instruction manual for specific steps.
[0085] 3. QPCR
[0086] 1) Reverse transcription reaction
[0087] Use FastQμant cDNA First Strand Synthesis Kit (Product No.: KR106) for lncRNA reverse transcription, first remove the genomic DNA reaction, add 5×gDNA Bμffer 2.0μl, total RNA 1μg, add RNase Free ddH 2 O to bring the total volume to 10 μl, and heat in a water bath at 42°C for 3 min.
[0088] 10× Fast RT Bμffer 2.0μl, RT Enzyme Mix 1.0μl, FQ-RT Primer Mix 2.0μl, RNase Free ddH 2 O 5.0 μl, after mixing, add to the above test tube and mix together a total of 20 μl, heat in a water bath at 42°C for 15min, then at 95°C for 3min.
[0089] 2) Primer design
[0090] QPCR a...
Embodiment 3
[0108] Example 3 Overexpression of FRMD4B gene
[0109] According to the FRMD4B gene sequence information in NCBI, specific primers were designed and amplified to obtain the corresponding genes. After the gene fragments were recovered by gel, they were connected with the expression vector pEGFP-C1 and transformed into DH5α competent cells. Positive clones were picked and subjected to PCR and After sequencing and identification, the pEGFP-C1-FRMD4B recombinant plasmid was obtained.
[0110] 1. Cell culture
[0111] Cervical squamous cell carcinoma cells (Hela) were cultured in RPIM-1640 medium containing 10% fetal bovine serum and 1% P / S at 37°C, 5% CO 2 When the cells grew to 80%-90%, they were routinely digested and passaged with 0.25% EDTA-containing trypsin.
[0112] 2. Transfection
[0113] The experiment was divided into 3 groups, namely the control group (Hela), the blank control group (transfected with pEGFP-C1), and the experimental group (transfected with pEGFP-C1-...
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