Application of LINC01876 as molecular marker for diagnosing liver carcinoma
A liver cancer and auxiliary diagnosis technology, applied in the field of biomedicine, can solve the problems of liver cancer insensitivity and delay the disease, and achieve the effect of improving the survival rate
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Embodiment 1
[0053] Example 1 Screening of differentially expressed non-long coding RNA
[0054] 1. Materials:
[0055] Surgically resected cancer tissues and paracancerous tissues of 5 patients with primary liver cancer were used as experimental samples. All cancer tissues were pathologically confirmed as liver cancer. All patients with primary liver cancer had not received radiotherapy and chemotherapy before operation, and the clinical data of all cases were complete. All patients signed the informed consent.
[0056] 2. Steps:
[0057] (1) Use TRIzol reagent to extract total RNA from tissue samples, and use RNasey Mini Kit to purify the extracted RNA;
[0058] (2) Use QuickAmp Labeling Kit, One-Color (Agilent p / n 5190-0442) to synthesize and label double-stranded cDNA;
[0059] (3) After the labeled double-stranded cDNA is purified, it is hybridized to the Human 8x60K LncRNA expression array information chip of Arraystar;
[0060] (4) After hybridization and rinsing, scan and ana...
Embodiment 2
[0064] Example 2 Large sample verification screened differentially expressed LncRNA
[0065] Based on the results of previous chip sequencing and according to the size of P value, we selected LINC01876 for verification.
[0066] 1. Sample collection
[0067] According to the method of Example 1, 30 liver cancer tissues and 30 corresponding paracancerous tissues were collected.
[0068] 2. Validation at the mRNA level
[0069] Reagents: Reverse transcription kit (DDR037A) was purchased from Treasure Bioengineering (Dalian) Co., Ltd. SYBR Premix Ex Taq for real-time quantitative PCR (polymerase chain reaction) TM (Tli RNaseHPlus) kit was produced by Japan Takara Company.
[0070] 2.1 Extract tissue RNA
[0071] Step is with embodiment 1.
[0072] 2.2 Reverse transcription
[0073] Using the extracted total RNA (1 μg) as a template, add the following reaction system, specifically: Buffer4μL, RT Enzyme Mix 1 μL, Oligo dT Primer (50 μM) 1 μL, Random 6mers (100 μM) 1 μL, ...
Embodiment 3
[0083] Example 3 Effect of LINC01876 on the function of liver cancer cells
[0084] The human liver cancer cell line SMMC-7721 was purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured with DMEM medium plus 10% fetal bovine serum at 37°C, 5% CO 2 Cultured in an incubator, and the cells in the logarithmic growth phase were taken for experiments.
[0085]The INTERFERin transfection reagent was used to transfect the LINC01876 interference sequence (siRNA-LINC01876) and the universal negative control sequence (siRNA-NC) according to the reagent instructions. Both the universal negative control sequence and the LINC01876 interference sequence were provided by Shanghai Gemma Pharmaceutical Technology Co., Ltd. The LINC01876 interference sequence is 5'-AGCUUUUCAAGGUCAAAUCUCTT-3' (sense strand, (SEQ ID NO.7)) and 5'-GAUUUGACCUUGAAAAGCUUATT-3' (antisense strand, (SEQ ID NO.8)).
[0086] 1 day before transfection, according to 1×10 6 Cells were planted...
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