CN104450703A - Kit and method for detecting serum of liver cancer patient by taking miR-146a as marker - Google Patents

Abstract

The invention discloses a kit and method for detecting the serum of a liver cancer patient by taking miR-146a as a marker, belonging to the technical field of molecular biomedicines. According to the method, a miR-146a specific primer is designed, the changes of miR-146a contents in serums of a normal person and the liver cancer patient are detected by using a quantitative PCR method, and the serum in which the miR-146a content is remarkably reduced is the serum of the liver cancer patient. The method disclosed by the invention is simple, rapid, sensitive and suitable for detecting whether patients suffer from liver cancer.

Description

A kind of utilize miR-146a to be mark liver cancer patient blood serum detection kit and method

Technical field

The invention belongs to molecular biosciences medicine technology field, be specifically related to a kind of utilize miRNA-146a to be mark liver cancer serum detection kit and method.

Background technology

Liver cancer is a kind of common malignant tumour, it is reported, in world wide, annual newly-increased patients with hepatocellular carcinoma reaches 700, more than 000.China is the country that global onset of liver cancer rate is the highest, and have statistics display, China's onset of liver cancer number accounts for the whole world 55%, and death toll accounts for the whole world 45%, the second of row cancer mortality, and sickness rate constantly rises.The early discovery of hepatocellular carcinoma, early diagnosis, early treatment to the prognosis of liver cancer patient and the survival time most important.Alpha-fetoprotein (alpha-fetoprotein, AFP) be first tumor markers with diagnostic value finding of the mankind so far, important role is play in primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) develops.But according to clinical data, about have 30% ~ 40% liver cancer patient blood serum AFP negative, and China is the country occurred frequently of primary hepatocellular carcinoma, the Serum AFP negative primary hepatocellular carcinoma reported in recent years has and increases trend.This part liver cancer patient lacks effective tumor markers for Index for diagnosis and therapeutic evaluation.Therefore, new liver cancer serum mark, especially the detection of the blood serum designated object of AFP negative hepatocellular carcinoma has important clinical meaning.

MicroRNA (miRNA) is the non-coding small RNA molecular that a class is about 22nt, and they, by being combined the target gene or suppress to translate of degrading with target gene 3 ' UTR, play a significant role in various physiological processes.The generation evolution of liver cancer is participated in by the regulatory pathway of Various Complex, and wherein miRNA take part in the post-transcriptional control of liver cancer pathogenic related gene.The pathogenesis of liver cancer has a lot, and as Inflammatory response, endothelial dysfunction etc., more and more study discovery in the recent period, miRNA can participate among the regulation and control of above-mentioned pathologic process.

The present invention confirms, miR-146a content in the liver cancer patient blood plasma that AFP is negative is starkly lower than normally, and this characteristic can as the marker molecule of this disease, for clinical diagnosis.Further research confirms that miR-146a can suppress propagation, the transporting action of human liver cancer cell in liver cancer cell.

Summary of the invention

The object of the present invention is to provide miR-146a as liver cancer serum molecular marker.

The object of the present invention is to provide a kind of primer detecting miR-146a expression amount.

The present invention also aims to provide a kind of liver cancer serum detection kit.

The present invention also aims to the liver cancer serum detection method that a kind of non-diagnostic object is provided.

Detect a primer for miR-146a expression amount, the nucleotide sequence of described primer is as shown in SEQ ID No.2, SEQ ID No.3 in sequence table.

A kind of liver cancer serum detection kit, described test kit comprises SEQ ID No.2 in sequence table, the primer shown in SEQ ID No.3, standard model, internal reference primer, ThermoScript II, dNTPs, buffer, RNA enzyme inhibitors and aqua sterilisa.

Described standard model is the cDNA of the RNA reverse transcription one-tenth in untreated human serum sample.

A liver cancer serum detection method for non-diagnostic object, carry out in accordance with the following steps:

(1) total serum IgE of measuring samples is extracted;

(2) be cDNA by the total serum IgE reverse transcription that step (1) obtains;

(3) utilize a kind of liver cancer serum detection kit described in claim 3 or 4, the cDNA obtained with step (2), for template, does quantitative PCR, detects the expression amount of miR-146a in sample;

(4) if the expression amount of measuring samples is significantly higher than the expression amount of standard model, then this measuring samples takes from the biological sample of liver cancer patient.

Beneficial effect of the present invention:

Accompanying drawing explanation

The result of miR-146a content in Fig. 1 to be quantitative PCR detection normal human serum and AFP be negative liver cancer patient blood serum.

Fig. 2 is the result of miR-146a content in quantitative PCR detection human liver cancer cell.

After Fig. 3 transfection NC, si-VASN, miR-146a mimics and miR-146a inhibitors, on the impact of VASN mRNA level in-site in HepG2 cell lines.

After Fig. 4 transfection NC, si-VASN, miR-146a mimics and miR-146a inhibitors, on the impact of VASN protein level in HepG2 cell lines.

Fig. 5 is after MTT experiment detects transfection miR-146amimics and miR-146a inhibitor, on the impact of HepG2 cell lines propagation.

Fig. 6 is after scratch experiment detects transfection miR-146a mimics and miR-146a inhibitor, on the impact of HepG2 cell lines migration.

Fig. 7 is after Transwell experiment detects transfection miR-146a mimics and miR-146a inhibitor, on the impact of HepG2 cell lines migration.

embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described.

The unaccounted experimental procedure of following examples is with reference to " Molecular Cloning: A Laboratory guide " third edition, or the specification sheets of reference corresponding reagent box.

The extracting of RNA in embodiment 1 human serum sample

(1) sample (normal human serum, liver cancer patient blood serum) room temperature places 5 minutes, and the sample room temperature of taking out from-80 DEG C of refrigerators places 15 minutes to thawing.

(2) every 250 μ l samples add 750 microlitre Trizol and mix, then add 250 μ l trichloromethanes, acutely rock 15 seconds, and room temperature leaves standstill 3 minutes.

(3) 4 DEG C, centrifugal 15 minutes of 12000g, upper strata is RNA

(4) the 500 μ l that makeed an appointment by upper water transfer in new EP pipe, add equal-volume Virahol, and mixing 10 seconds of turning upside down is placed in-20 DEG C of refrigerators and precipitates two hours or spend the night.

(5) take out sample, centrifugal 15 minutes of 4 DEG C of 12000g, outwell supernatant and add 1ml80% ethanol (configuration of DEPC water) washing precipitation, spin upside down ten times, and white RNA precipitation is floated, and 4 DEG C, centrifugal 5 minutes of 7500g, carry out twice altogether.

(6) abandon supernatant, exhaust liquid in pipe, make ethanol volatilize clean (about 2-5 minute)

(7) when RNA precipitation edge starts to become dry transparent, DEPC water (20 μ about l) is added immediately.Get 1 μ l for quantitatively, other RNA saves backup with-80 DEG C.Can add 1 μ l RNAase inhibiter prevents RNA from degrading.

Embodiment 2 reverse transcription

M-MLV Reverse Transcriptase [Promega#9PIM170] Reverse Transcription box is adopted to carry out reverse transcription.

Each sample get 1 μ gRNA respectively with the special reverse primer mixing of miRNA-146a and U6,70 degree water-baths 10 minutes, placement on ice two minutes.Then each sample adds buffer5 μ l respectively, dNTP4 μ l, ThermoScript II 1 μ l, RNAase inhibitor0.2 μ l.The cDNA reversed preserves at-20 degree, avoids multigelation.

Quantitative-the PCR of embodiment 3

According to experiment grouping, the content of each sample detection internal reference and miRNA-146a.PCR forward primer and reverse primer are as shown in SEQ ID No.2 in sequence table and SEQ ID No.3, and the multiple hole of each experimental group three, system is 20 μ l, and instrument is Stratagene MxPro3000, SYBR Green mix is Promega

Products.Experimental system is: 10 μ l2 × SYBR Green mix, 8 μ l water, 1 μ l primer, 1 μ l cDNA.PCR reaction conditions be 95 degree 5 minutes, 95 degree 20 seconds, 60 degree 20 seconds, 72 degree 20 seconds, 50 circulations, solubility curve is 60 degree to 95 degree, and temperature interval is 0.4 degree.

As shown in Figure 1-2, in the liver cancer patient that AFP is negative, the copy number of miRNA-146a is starkly lower than the copy number (Fig. 1) of miRNA-146a in normal human serum to PCR result; In HepG2 cell lines, the copy number of miRNA-146a is starkly lower than the copy number (Fig. 2) of miRNA-146a in normal cell lines of human liver strain L02.

Embodiment 4MTT tests

With 2 × 10 ³individual cells/well is inoculated in 96 orifice plates; After overnight incubation, when cell density reaches 40%, carry out transfection.Cultivate 24h-72h for 37 DEG C, detect cell count; Every hole adds the mixed solution of 100 μ l substratum and 20 μ l MTS, hatches 1h for 37 DEG C, and enzyme-linked immunosorbent assay instrument 490nm reads absorbance, draws growth curve.

Embodiment 5 cell scratch experiment

With 5 × 10 ⁵individual cells/well is inoculated in 6 orifice plates; When cell density reaches 40%, transfection is carried out after overnight incubation.Cultivate 24h, make cell concn reach about 90% for 37 DEG C; To row dry in culture hole 2-3 bar cut with yellow rifle head, change fresh culture and continue to cultivate, observe cut healing state every 6h.

Embodiment 6 Cell migration assay

With 5 × 10 ⁵individual cells/well is inoculated in 6 orifice plates; Overnight incubation carries out transfection when cell density reaches 40%.Change serum free medium after 37 DEG C of cultivation 24h to continue to cultivate 24h; Peptic cell prepares single cell suspension, and dilution is 5 × 10 ⁵individual cell/ml, often kind of cell kind 3 cells, add 100 μ l in each cell, add the substratum of 500 μ l containing 10%FBS as lower liquid, Transwell cell is put into hole in 24 orifice plates, continue to cultivate 12-24h and take out cell; Wipe the cell little chamber internal surface not being worn film with PBS, formaldehyde fixes 30min, violet staining 30min, with basis of microscopic observation.Cell counting under 20 × visual field, amounts to several four visuals field, averages.

Claims (5)

1.miR-146a, as liver cancer serum molecular marker, is characterized in that, described nucleotide sequence is as shown in SEQ ID No.1 in sequence table.

2. detect a primer for miR-146a expression amount, it is characterized in that, the nucleotide sequence of described primer as SEQ ID No.2 in sequence table, shown in SEQID No.3.

3. a liver cancer serum detection kit, is characterized in that, described test kit comprises primer according to claim 1, standard model, internal reference primer, ThermoScript II, dNTPs, buffer, RNA enzyme inhibitors and aqua sterilisa.

4. a kind of liver cancer serum detection kit according to claim 2, is characterized in that, described standard model is the cDNA that the RNA reverse transcription in untreated human serum sample becomes.

5. a liver cancer serum detection method for non-diagnostic object, is characterized in that, carries out in accordance with the following steps:

(1) total serum IgE of measuring samples is extracted;

(2) be cDNA by the total serum IgE reverse transcription that step (1) obtains;

(3) utilize a kind of liver cancer serum detection kit described in claim 3 or 4, the cDNA obtained with step (2), for template, does quantitative PCR, detects the expression amount of miR-146a in sample;

(4) if the expression amount of measuring samples is significantly higher than the expression amount of standard model, then this measuring samples takes from the biological sample of liver cancer patient.