CN108866179A - LncRNA-SCARNA10 is in preparation liver fibrosis detection kit and the purposes for the treatment of hepatic fibrosis medicines - Google Patents

Abstract

The invention discloses the use of lncRNA-SCARNA10 in the preparation of a liver fibrosis detection kit and a drug for treating liver fibrosis. Application of the specific sequence against SCARNA10 in the preparation of a serum detection kit for diagnosing liver fibrosis and the application of the specific sequence capable of targeting interference with SCARNA10 in the preparation of products for the treatment of liver fibrosis. In the present invention, by studying the expression of SCARNA10 in human liver tissue and serum and its function and mechanism in the process of liver fibrosis, it is found that the expression of SCARNA10 in liver tissue and serum of patients with liver fibrosis is significantly increased, and it inhibits the expression of SCARNA10 in hepatic stellate cells. SCARNA10 can reduce the progression of liver fibrosis. The invention has important reference significance for the early non-invasive diagnosis and treatment strategy selection of liver fibrosis in the future.

Description

lncRNA-SCARNA10 in the preparation of liver fibrosis detection kit and treatment of liver fibrosis chemical drug use

technical field

The invention relates to the application of lncRNA-SCARNA10 in the preparation of a liver fibrosis detection kit and a drug for treating liver fibrosis.

Background technique

Hepatic fibrosis is a common liver disease, a common pathological process of chronic liver disease, and a reversible injury-healing response of the liver, mainly caused by alcohol abuse, viral infection, bile duct obstruction, and HBV, HCV, non-alcoholic Caused by chronic fatty liver and other etiologies, it is very common in Chinese and Western populations. It is characterized by the excessive production of extracellular matrix (ECM) and excessive deposition in the liver, which affects the blood flow of liver cells and the function of liver cells. The liver function is damaged, the basic biological metabolism function is lost, and eventually progresses to cirrhosis, Chronic liver failure. For patients with advanced liver fibrosis and liver cancer, the only effective treatment is liver transplantation. Currently, liver biopsy is the gold standard for diagnosis. Histopathological identification can grade the degree of liver fibrosis according to inflammation and necrosis. There are great limitations in application, so it is urgent to find an accurate non-invasive method for diagnosing liver fibrosis.

Numerous studies on the genome have shown that 98% of genes cannot encode proteins, while 80% of genes are considered to have biological functions, and their transcripts are called non-coding RNAs, which are mainly divided into small RNAs less than 200bp according to their length. Noncoding RNAs include miRNAs, small nuclear RNAs, etc., and long noncoding RNAs (lncRNAs) larger than 200bp. For a long time, lncRNAs were considered to be by-products of the transcription process without biological functions. In recent years, with the research progress of miRNAs, it has been revealed that non-coding RNAs play a very important role in human gene post-transcriptional regulation, cell growth, differentiation and proliferation. At the same time, it is also suggested that compared with miRNAs, lncRNAs with a higher proportion of transcription in cells may have more complex and important biological functions, and it is no longer considered "dark matter", and its function and mechanism of action are becoming more and more important. Therefore, many studies have focused on this in recent years.

The discovery of free RNA in plasma and serum, including miRNA, snoRNA, lncRNA and circRNA, is a major breakthrough in the history of scientific development. The traditional concept has always believed that free RNA cannot stably exist in plasma and serum, because RNA itself is extremely unstable and a large number of nucleases in blood circulation will also affect its stability. Until the late 1980s, several reports clearly elucidated the existence of amplifiable circulating RNA in blood circulation. At present, the source of circulating RNA in circulating blood and the mechanism of its stable existence have not been fully elucidated. Previous studies have speculated that the sources of circulating RNA include passive processes and active processes. The passive process means that free RNA enters the circulating blood after disease cell necrosis; the active process refers to the release of circulating RNA from cells in the form of apoptotic bodies or microsomes. Another study pointed out that the stable existence of plasma RNA is due to its ability to form hybrids with DNA or combine with lipoprotein complexes to avoid degradation by RNase. The convenience and relative non-invasiveness of collection of circulating biomarkers enables them to be used as screening tools for disease. Therefore, identifying circulating ncRNAs that are directly related to the process of liver fibrosis and can be used as diagnostic markers of liver fibrosis has far-reaching clinical significance.

Contents of the invention

In order to solve the problems in the prior art, the present invention provides the use of lncRNA-SCARNA10 in the preparation of liver fibrosis detection kits and drugs for the treatment of liver fibrosis, which solves the problem that the detection accuracy of liver fibrosis in the prior art is not high, and there is no specific drug effective Stop or reverse liver fibrosis.

Technical scheme of the present invention is:

Use of the specific sequence for lncRNA-SCARNA10 in the preparation of a serum detection kit for liver fibrosis.

Use of the specific sequence of targeted knockdown lncRNA-SCARNA10 in the preparation of drugs for treating liver fibrosis.

The invention provides the application of serum detection of SCARNA10 in the preparation of a detection kit for diagnosing liver fibrosis and the use of substances capable of inhibiting SCARNA10 in the preparation of products capable of treating liver fibrosis. In the present invention, by studying the expression of SCARNA10 in human liver tissue and serum and its function and mechanism in the process of liver fibrosis, it is found that the expression of SCARNA10 in liver tissue and serum of patients with liver fibrosis is significantly increased, and it inhibits the expression of SCARNA10 in hepatic stellate cells. SCARNA10 can reduce the progression of liver fibrosis. The invention has important reference significance for the early non-invasive diagnosis and treatment strategy selection of liver fibrosis in the future.

Description of drawings

Figure 1: Determination of the full-length sequence of SCARNA10; taking the human hepatic stellate cell line LX-2 as the research object, the full-length sequence of human SCARNA10 was determined by rapid amplification of cDNA ends (RACE);

Figure 2: The expression of SCARNA10 in the liver tissue of patients with liver fibrosis/cirrhosis is increased; (A) HE, Masson and Sirius Scarlet were performed on 65 cases of human liver tissue (including normal people, patients with liver fibrosis and patients with liver cirrhosis) Staining and histological grading; (B) total RNA was extracted from 65 cases of human liver tissues, and the expression of SCARNA10 was detected by qRT-PCR technology; (C) SCARNA10 was detected in 65 cases of liver tissues (normal and fibrotic/cirrhotic liver tissues) and Col1α1 expression and correlation analysis, *p<0.05;

Figure 3: The expression of SCARNA10 is increased in the serum of patients with liver fibrosis/cirrhosis; total RNA was extracted from the serum of normal people, patients with liver fibrosis and patients with liver cirrhosis, and the expression of SCARNA10 was detected by qRT-PCR technology, *p<0.05;

Figure 4: SCARNA10 is overexpressed in mouse fibrotic liver tissue and activated HSCs; (A) Total RNA extracted from liver tissue at 0, 2, 4, 6, 8, and 10 weeks after intraperitoneal injection of CCl4, qRT-PCR Detection of expression changes of α-SMA (Acta2) and SCARNA10; (B) Total RNA was extracted from liver tissue on day 0, day 3, day 14 and day 21 after bile duct ligation (BDL), and qRT-PCR was used to detect α- The expression changes of SMA and SCARNA10; (C) The RNA of primary HSCs from normal mice and mice with liver fibrosis were extracted respectively, and the expression of SCARNA10 was detected by qRT-PCR; (D) The first primary HSCs cultured in vitro were extracted respectively 3, 7, 14 days RNA, qRT-PCR technology to detect the expression of α-SMA and SCARNA10; (E) qRT-PCR technology was used to detect the expression of α-SMA and SCARNA10 after the primary HSCs were treated with TGFβ stimulating factor for 24 hours on the 3rd day of in vitro culture - levels of SMA, col1α1 and SCARNA10, *p<0.05;

Figure 5: Knockdown of SCARNA10 can inhibit CCl ₄ -induced liver fibrosis in mice; the mice were divided into four groups: control group, CCl ₄ group, interference SCARNA10 group, interference SCARNA10+CCl ₄ group; high The titer of lentivirus (lenti-shSCARNA10) or control lentivirus (lenti-NC) interfering with SCARNA10, and at the same time construct the mouse liver fibrosis model induced by CCl ₄ ; (A) observe the liver appearance and HE staining of the four groups of mice , Sirius Red staining, and IHC of α-SMA, Col1α1, and PCNA proteins to determine the state of liver fibrosis; (B) Western blot detection of the expressions of α-SMA, Col1α1, MMP2, and PCNA in different mouse liver tissues, and GAPDH as a control; (C) Measuring the content of hydroxyproline in the liver tissues of different mice; (D, E) Extracting RNA from the liver tissues of the four groups of mice, and detecting the expression of genes related to liver fibrosis and inflammation by qRT-PCR. */ #p<0.05. *p<0.05 vs control group; #p<0.05 vs control+CCl ₄ group.

Detailed ways

The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

Example 1

The full-length sequence of SCARNA10

Taking the human hepatic stellate cell line LX-2 as the research object, rapid amplification of cDNA ends (RACE) (purchased from Clontech Company RACE 5'/3' kit) to determine the full-length sequence of human SCARNA10 (SEQ ID NO.1). (As shown in Figure 1)

Method: 3'RACE: Gene-specific primer GSP1 as upstream primer (3' outer primer: GATTACGCCAAGCTTGGTCTGTAATCTTGGTGGGCGATACAGA (SEQ ID NO.2); 3' inner primer: GATTACGCCAAGCTTTGTGTTCACTGTAAGGGCAGACCAAC (SEQ ID NO.3)), and universal primer UPM as downstream Primers (containing part of the linker sequence) are used to amplify the DNA fragment at the 3' end of the target gene by PCR. The specific primer GSP2 is the downstream primer (5'outer primer: GATTACGCCAAGCTTTAGGACCCTTGGCCCTGATACCCTG (SEQ ID NO.4); 5'inner primer: GATTACGCCAAGCTTTGCCCTGTTCCTACTCT CTCACTCC (SEQ ID NO.5)), amplified by PCR on the cDNA fragment at the 5' end of the target gene The picture shows the schematic diagram of PCR, and then the gel was recovered and sent to the gene company for sequencing.

Example 2

In order to better study the role of SCARNA10 in liver fibrosis, we collected 65 cases of human liver tissue specimens. First, we performed pathological analysis on these 65 specimens. According to international general standards, through histological routine staining techniques ( HE, Masson and Sirius scarlet staining) these 65 cases of liver tissue specimens were divided into 5 grades.

HE staining steps:

(1) Dewaxing: Put the paraffin sections in xylene I for 20 minutes first, then put them in xylene II and let them stand at room temperature for 20 minutes (when the room temperature is too low, put the xylene in a 37°C incubator);

(2) Hydration: Take out paraffin sections from xylene II, put them into gradient absolute ethanol to rehydrate 100% Ⅰ, 100% Ⅱ, 95%, 90%, 80%, and 70% for 5 minutes each. The channel can be drained slightly; then wash with distilled water 3 times, 5min each time;

(3) Cell nucleus staining: blot the water around the tissue block dry, put the glass slide into 5 staining cups filled with hematoxylin, completely immerse the glass slide, and let it stand at room temperature for 8 minutes;

(4) Bleeding: Prepare ten pieces of staining vats filled with tap water, rinse the slides with hematoxylin, and place the slides for 20 minutes. The nuclei will gradually turn blue. Observe under the microscope and proceed to the next step after the blue color is obvious;

(5) Cytoplasmic staining: Blot up the water around the tissue block, put the glass slide into a staining cup filled with eosin, completely immerse the glass slide, let it stand at room temperature for 3 minutes, and observe the red color of the cytoplasm under a microscope. ;

(6) Transparency: Use tweezers to pick up the glass slides and wash them in 70%, 80%, 90%, and 95% ethanol in sequence, and then let them stand in 100% absolute ethanol Ⅰ and Ⅱ for 1 min and 3 min respectively. ; Take the slides out of 100% absolute ethanol II, drain slightly, and let stand in xylene I for 5 minutes and II for 8 minutes;

(7) Sealing: Take out the slide, dry the xylene, place it horizontally, drop an appropriate amount of neutral resin on one side of the slide, take the cover slip with tweezers and slowly lower it from the resin side until the tissue block is completely sealed ; Keep tissue blocks moist during all manipulation steps.

Sirius scarlet dyeing steps:

(1) Conventional dewaxing and hydration paraffin sections;

(2) Take out the slice from the PBS, and dry the residual PBS around the tissue with a paper towel;

(3) Sirius scarlet staining solution was drip-stained for 45 minutes;

(4) Rinse slightly with running water to remove the dye solution on the surface of the slice;

(5) Quickly wash the slides in 70%, 80%, 90%, 95% graded ethanol, let stand in 100% absolute ethanol I for 2 minutes, and let stand in 100% absolute ethanol II set for 3 minutes;

(6) Transparent: xylene Ⅰ 3-5 minutes, xylene Ⅱ 5-10 minutes

(7) Add neutral gum dropwise to the tissue block, and seal the slide with a cover glass. After natural drying, it can be observed under a microscope.

Subsequently, we designed 2 pairs of specific primers for SCARNA10 (upstream primer 1: CTTGGTGGGCGATACAGAGT (SEQ ID NO.6); downstream primer 1: CTTTAGGACCCTTGGCCCTG (SEQ ID NO.7); upstream primer 2: AATCTTGGTGGGCGATACAG (SEQ ID NO.8) ; downstream primer 2: CAGCAACTGGAAGAATCACC (SEQ ID NO.9)), and using qPCR technology to detect the expression of SCARNA10, it was found that compared with normal liver tissue, the expression of SCARNA10 in fibrosis and cirrhosis liver tissue was significantly increased. At the same time, the expression of Col1α1 was detected and correlated with the expression of SCARNA10. The above results indicated that SCARNA10 was closely related to the degree of liver fibrosis.

The results are shown in Figure 2, the expression of SCARNA10 in the liver tissues of patients with liver fibrosis/cirrhosis increased (A) HE, Masson and Sirius scarlet staining and histological grading; (B) Total RNA was extracted from 65 cases of human liver tissue, and the expression of SCARNA10 was detected by qRT-PCR. The results confirmed that SCARNA10 was significantly expressed in the liver tissue of patients with liver fibrosis and cirrhosis (C) The expressions of SCARNA10 and Col1α1 were detected in 65 cases of liver tissues (normal and fibrotic/cirrhotic liver tissues) and the correlation analysis was carried out. It was found that SCARNA10 was significantly correlated with liver fibrosis marker Col1α1. *p<0.05.

Example 3

In order to better study the role of SCARNA10 in liver fibrosis and explore its potential as a serum marker for early diagnosis of liver fibrosis, we collected serum from 35 normal people, 38 patients with liver fibrosis and 45 patients with liver cirrhosis. RNA was extracted from the patient's serum, and the expression of SCARNA10 was detected by using the above-mentioned 3 pairs of specific primers. It was found that compared with normal human serum, the expression of SCARNA10 in the serum of patients with fibrosis was significantly increased, and in the serum of patients with liver cirrhosis The expression was higher than that of the serum of patients with liver fibrosis. The above results show that SCARNA10 is closely related to the degree of liver fibrosis, and gradually increases with the occurrence and development of liver fibrosis, revealing that SCARNA10 has the potential to be used as a serum marker for detecting fibrosis in clinical practice, so that it can be detected early by non-invasive means And diagnose liver fibrosis, take timely intervention measures to alleviate the progress of liver fibrosis, and achieve the purpose of reversing liver fibrosis.

As shown in Figure 3, the expression of SCARNA10 was increased in the serum of patients with liver fibrosis/cirrhosis. Serum total RNA was extracted from normal people, patients with liver fibrosis and patients with liver cirrhosis, and the expression of SCARNA10 was detected by qRT-PCR. *p<0.05.

Example 4

Since the activation of HSCs is the core link of liver fibrosis, the occurrence and development of liver fibrosis can be promoted by activating HSCs. Therefore, we explored whether SCARNA10 has a function in the activation of HSCs. First, we extracted hepatic stellate cells from the livers of healthy mice and diseased mice, and extracted RNA on the first day of culture to detect the expression levels of α-SMA and SCARNA10. Before extraction, a large number of lipid droplets were observed in primary HSCs of normal mice under the microscope, indicating that they were in a resting state, while lipid droplets disappeared in primary HSCs of fibrotic mice, indicating that they were in the process of liver fibrosis already activated. The results of qRT-PCR showed that compared with primary HSCs of normal mice, the expression of α-SMA was significantly increased in primary HSCs of fibrotic mice, and the expression of SCARNA10 was also significantly increased in HSCs of fibrotic mice, indicating that SCARNA10 is highly expressed in activated HSCs in vivo. Studies have shown that resting HSCs will gradually activate during in vitro culture. In order to verify the above experimental results, we extracted primary HSCs from normal mice for in vitro culture, and extracted them on the 3rd, 7th, and 14th days respectively. RNA. Through these three time points, the resting state, partially activated state and fully activated state of primary HSCs were reflected. The expression levels of α-SMA and SCARNA10 were detected by qRT-PCR. It was found that the expression level of α-SMA was significantly higher on the 14th day On the 7th day, and the 7th day was significantly higher than the 3rd day, indicating that the expression of α-SMA increased with the activation of HSCs (Fig. The 3rd day is the resting state, the 7th day is the partial activation state, and the 14th day is the full activation state. Our results showed that the expression of SCARNA10 was consistent with the expression trend of α-SMA, which was significantly higher on the 14th day than on the 7th day, and the 7th day was significantly higher than that on the 3rd day, indicating that the level of SCARNA10 also gradually increased with the primary HSCs cultured in vitro. Activation process increases. In addition, since TGFβ is an important cytokine for the activation of HSCs, we further examined whether TGFβ could regulate the expression of SCARNA10. The normal mouse primary HSCs we isolated were treated with 10ng/ml TGFβ for 24 hours on the 3rd day of in vitro culture, and RNA was extracted to detect the expression levels of α-SMA, col1α1, and SCARNA10. The results showed that TGFβ treatment stimulated the activation of HSCs, and the expression of α-SMA, col1α1 and other genes related to HSCs activation increased, and the expression level of SCARNA10 increased significantly, indicating that SCARNA10 may be regulated by TGFβ and participate in the promotion of HSCs activation.

As shown in Figure 4, (A) Total RNA was extracted from liver tissue at 0, 2, 4, 6, 8, and 10 weeks after intraperitoneal injection of CCl ₄ , and the expression changes of α-SMA (Acta2) and SCARNA10 were detected by qRT-PCR ; The results confirmed that the expressions of α-SMA and SCARNA10 gradually increased in fibrotic liver tissue; (B) Total RNA was extracted from liver tissue on day 0, day 3, day 14 and day 21 after bile duct ligation (BDL) , qRT-PCR detected the expression changes of α-SMA and SCARNA10; the results confirmed that the expression of α-SMA and SCARNA10 gradually increased in fibrotic liver tissue. (C) RNA was extracted from primary HSCs of normal mice and mice with liver fibrosis, and the expression of SCARNA10 was detected by qRT-PCR; the results confirmed that the expression of SCARNA10 was significantly increased in activated HSCs. (D) Extract the RNA of primary HSCs on the 3rd, 7th, and 14th day of in vitro culture, and detect the expression of α-SMA and SCARNA10 by qRT-PCR; the results confirm that the expression of α-SMA and SCARNA10 increases with Activation increases gradually. (E) qRT-PCR was used to detect the levels of α-SMA, col1α1 and SCARNA10 after primary HSCs were treated with TGFβ-stimulating factors on the 3rd day of in vitro culture; the results confirmed that the expression of SCARNA10 was significantly increased in TGFβ-stimulated HSCs . *p<0.05.

Example 5

In order to explore the relationship between SCARNA10 and the development of liver fibrosis in vivo. First, we divided the experimental animals into four groups, namely the negative control group, the CCl ₄ group, the interference SCARNA10 group, and the interference SCARNA10+CCl ₄ group. The negative control group was injected with olive oil intraperitoneally, and the negative control lentivirus (lenti-NC) was injected into the tail vein of the mice; the mice in the CCl ₄ group were injected with 5% CCl ₄ intraperitoneally, and the control lentivirus (lenti-NC) was injected into the tail vein of the mice; the interference SCARNA10 group Mice were intraperitoneally injected with olive oil to produce high-titer lenti-shSCARNA10 lentivirus (lenti-shSCARNA10: siRNA sequence 1: GGGACCUUUGGCCUGUUAA (SEQ ID NO.10); siRNA sequence 2: CACAGAUCUUUGGUAAUCU (SEQ ID NO.11); siRNA Sequence 3: CCAAGGGUCCUAAAGGACU (SEQ ID NO.12)) was injected into the tail vein of the mouse; the interfering SCARNA10+CCl ₄ group was intraperitoneally injected with 5% CCl ₄ , and the mice were injected with a high-titer lnc-SCARNA10 interfering lentivirus into the tail vein. The CCl ₄ group and the interfering lnc-SCARNA10+CCl ₄ group were intraperitoneally injected with carbon tetrachloride for 14 days, and then injected the rat tail vein with lentivirus once, and then continued to inject CCl ₄ for 6 weeks, twice a week, after the end of time They were sacrificed under anesthesia and the liver tissue was frozen and embedded in paraffin.

By observing the general appearance of the liver tissue, it was found that compared with the control group, the liver surface of the mice in the CCl ₄ group was rough and dark, with more nodules, while the liver surface of the interference SCARNA10+CCl4 group was smoother and the color was darker than that of the CCl ₄ group. Yes, the nodules are reduced. Then the liver tissue sections were stained with HE, it can be seen that the liver structure of the mice in the CCl ₄ group was destroyed, the liver cells were necrotic, and the arrangement of the liver cords was disordered. Compared with the CCl ₄ group, the interfering SCARNA10+CCl ₄ group significantly restored the liver structure, decreased hepatic cell necrosis, and the hepatic cords arranged radially around the central vein. The liver tissue sections were stained with Sirius scarlet and immunohistochemical staining of α-SMA, Col1α1 and PCNA. Observation of the results showed that the extracellular matrix and collagen fibers in the liver of the mice in the CCl ₄ group were significantly increased, and the proliferation markers of HSCs were obvious. The degree of liver fibrosis was obvious, and the degree of fibrosis in the interference SCARNA10+CCl ₄ group was significantly alleviated. Compared with the CCl ₄ group, the extracellular matrix and collagen fibers in the mouse liver were significantly reduced, and HSCs proliferation markers were reduced. In addition, we judged the amount of collagen and indirectly reflected the state of liver fibrosis by measuring the amount of hydroxyproline in the liver. The results showed that the hydroxyproline content of the mice in the CCl ₄ group was significantly increased, while the interfering SCARNA10+CCl ₄ group Compared with the CCl ₄ group, the content of hydroxyproline was significantly decreased. The above morphological results can show that the degree of fibrosis in the interference SCARNA10+CCl ₄ group was significantly reduced.

Experimental steps for the determination of hydroxyproline content:

(1) Weighing the wet weight: take out the tissue from -80°C, cut the tissue block with autoclaved scissors and tweezers, then accurately weigh the wet weight of the tissue, and transfer it to a test tube;

(2) Alkaline hydrolysis: Use a 1ml pipette gun to accurately add 1ml of alkaline hydrolyzate, mix well, seal with plastic wrap, put it in a water bath at 95°C, hydrolyze for 20 minutes, and mix well during the period;

(3) Adjust the pH to 6.0 to 6.8: add a drop of indicator, and the solution turns red at this time; add 1ml of pH solution A, and the solution is still red at this time; slowly add pH solution B, each drop needs to Mix well until the solution turns yellow-green, that is, when the red just disappears; add 8ml of double distilled water to the solution and mix well;

(4) Activated carbon adsorption: take 4ml of the above-mentioned hydrolyzate, and add about 30mg of activated carbon;

(5) Centrifuge at room temperature for 10 minutes at 3500 rpm;

(6) Preparation of testing samples

Blank tube: 1ml double distilled water + 0.5ml reagent one + 0.5ml reagent two + 0.5ml reagent three

Standard tube: 1ml standard product + 0.5ml reagent one + 0.5ml reagent two + 0.5ml reagent three

Measurement tube: 1ml supernatant + 0.5ml reagent one + 0.5ml reagent two + 0.5ml reagent three

After mixing, it is used for the detection of hydroxyproline;

(7) Detection: measure the absorbance of each tube at a wavelength of 550nm, and make a record;

(8) Calculate.

In addition, we also carried out the detection of genes related to molecular biology. The RNA of the liver tissues of _four groups of mice was extracted, and the genes related to liver fibrosis and inflammation were detected by qRT-PCR technology. The results showed that α-SMA, Col1α1, Mmp2, Timp1, Ctgf, Tnfα , IL-1β, Mcp-1 expression was significantly increased, and after knocking down the expression of SCARNA10 and injecting CCl ₄ , compared with the CCl ₄ group, the expression of these genes was significantly decreased. Then, the proteins of different mouse liver tissues were separated and detected by protein electrophoresis technology. The results showed that interference with SCARNA10 inhibited the high expression of fibrosis-related genes α-SMA, Col1α1, MMP2, and PCNA caused by CCl ₄ .

As shown in Figure 5, knockdown of SCARNA10 can inhibit _CCl4 -induced liver fibrosis in mice

The mice were divided into four groups: control group, CCl ₄ group, interference SCARNA10 group, interference SCARNA10+CCl ₄ group. A high-titer SCARNA10-interfering lentivirus (lenti-shSCARNA10) or a control lentivirus (lenti-NC) was injected through the tail vein of mice, and a CCl ₄ -induced mouse liver fibrosis model was constructed at the same time. (A) Observation of liver appearance, HE staining, Sirius Red staining, and IHC of α-SMA, Col1α1, and PCNA proteins in the four groups of mice to determine the state of liver fibrosis; the results confirmed that the severity of fibrosis in the control + CCl ₄ group was greater than that of interfering SCARNA10 +CCl ₄ groups. (B) Western blot test for the expression of α-SMA, Col1α1, MMP2 and PCNA in the liver tissues of different mice, GAPDH was used as the control; the results confirmed that the protein levels of α-SMA, Col1α1, MMP2 and PCNA were control + CCl ₄ group > interference SCARNA10+CCl ₄ group. (C) Measuring the content of hydroxyproline in the liver tissues of different mice; the results confirmed that the content of hydroxyproline in the mouse liver tissues of the control+CCl ₄ group>the content of the interference SCARNA10+CCl ₄ group. (D, E) RNA was extracted from the liver tissues of the four groups of mice, and the expression of genes related to liver fibrosis and inflammation were detected by qRT-PCR; the results confirmed that the expression levels of pro-fibrosis and pro-inflammatory related genes were both in the control + CCl ₄ Group > interference SCARNA10+CCl ₄ group. */ #p<0.05. *p<0.05 vs control group. #p<0.05 vs control+ _CCl4 group.

sequence listing

<110> Tianjin Medical University

<120> Use of lncRNA-SCARNA10 in the preparation of liver fibrosis detection kits and drugs for the treatment of liver fibrosis

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 330

<212> RNA

<213> gene sequence()

<400> 1

gccacatgat gatatcaagg ctgttgtgat tcagttggtt tggctaagcc cagggacctt 60

tggcctgtta aaggtctgta atcttggtgg gcgatacaga gttatgtgtg ttcactgtaa 120

gggcagacca acaagaactt tttccctactt ttgagctacc tctttttaat aggggtgatt 180

cttccagttg ctggagagaa attgtggtaa ctggagtgag agagtaggaa cagggcatgt 240

tcagggtatc agggccaagg gtcctaaagg acttagcttg tgttatggcc actgagagat 300

gaaacacaga tctttggtaa tctgatggct 330

<210> 2

<211> 43

<212>DNA

<213> artificial sequence ()

<400> 2

gattacgcca agcttggtct gtaatcttgg tgggcgatac aga 43

<210> 3

<211> 41

<212>DNA

<213> artificial sequence ()

<400> 3

gattacgcca agctttgtgt tcactgtaag ggcagaccaa c 41

<210> 4

<211> 40

<212>DNA

<213> artificial sequence ()

<400> 4

gattacgcca agctttagga cccttggccc tgataccctg 40

<210> 5

<211> 40

<212>DNA

<213> artificial sequence ()

<400> 5

gattacgcca agctttgccc tgttcctact ctctcactcc 40

<210> 6

<211> 20

<212>DNA

<213> artificial sequence ()

<400> 6

cttggtgggc gatacagagt 20

<210> 7

<211> 20

<212>DNA

<213> artificial sequence ()

<400> 7

ctttaggacc cttggccctg 20

<210> 8

<211> 20

<212>DNA

<213> artificial sequence ()

<400> 8

aatcttggtg ggcgatacag 20

<210> 9

<211> 20

<212>DNA

<213> artificial sequence ()

<400> 9

cagcaactgg aagaatcacc 20

<210> 10

<211> 19

<212> RNA

<213> artificial sequence ()

<400> 10

gggaccuuug gccuguuaa 19

<210> 11

<211> 19

<212> RNA

<213> artificial sequence ()

<400> 11

cacagaaucuu uggaaucu 19

<210> 12

<211> 19

<212> RNA

<213> artificial sequence ()

<400> 12

ccaagggucc uaaaggacu 19

Claims (2)

1. being directed to the specific sequence of lncRNA-SCARNA10 in the purposes for preparing hepatic fibrosis serum detection kit.

2. purposes of the specific sequence in preparation treatment hepatic fibrosis medicines that low lncRNA-SCARNA10 is struck in targeting.