CN112294960B

CN112294960B - Use of micro-molecule RNA-30b-5p as target molecule

Info

Publication number: CN112294960B
Application number: CN202011189559.5A
Authority: CN
Inventors: 刁宏燕; 王凯航; 陈文标; 姜晶晶; 章旭君; 毕珂凡
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2022-02-08
Anticipated expiration: 2040-10-30
Also published as: CN112294960A

Abstract

The invention relates to application of micro-molecular RNA-30b-5p as a molecular target for resisting hepatocellular carcinoma related to hepatitis B virus. The invention discloses application of miR-30b-5p as a target molecule in preparation of a drug for resisting HBV-related hepatocellular carcinoma. By inhibiting the transcription of miR-30b-5p or leading the miR-30b-5p to lose the function of combining with a target gene, the growth and the invasion of HBV related liver cancer cells are inhibited.

Description

Use of micro-molecule RNA-30b-5p as target molecule

Technical Field

The invention relates to application of micro-molecular RNA-30b-5p as a molecular target for resisting hepatocellular carcinoma related to hepatitis B virus.

Background

Primary liver cancer is a disease seriously threatening human health, has high malignancy degree, easy recurrence and metastasis and poor disease prognosis, and is the fourth cause of death of malignant tumors around the world. The primary liver cancer mainly comprises three pathological tissue types of Hepatocellular carcinoma, intrahepatic cholangiocellular carcinoma and mixed cell carcinoma, wherein Hepatocellular carcinoma (HCC) is one of the most common cases of the primary liver cancer.

Liver cancer can be induced by a variety of causes including viral infection, alcohol consumption, obesity, exposure to chemical agents, long-term intake of moldy food, etc. In China, the condition that Hepatitis and cirrhosis caused by chronic infection of Hepatitis B Virus (HBV) finally develop into liver cancer is particularly serious. HBV is a causative agent of hepatitis B, belongs to the genus orthopathovirus of the family Hemophilic viridae, and its genome is a double-stranded circular DNA mainly encoding a surface antigen (HBsAg), a core antigen (HBcAg), a P protein and an X protein of the virus. There are a number of studies to demonstrate that after HBV infection, the signal pathway of host cells is affected and thus the normal function of hepatocytes is affected. Among them, the relationship between HBV and the mutual regulation of non-coding RNA in host cells and the proliferation, invasion and metastasis of hepatoma carcinoma cells is also a research hotspot in recent years.

Micro-molecular RNA (microRNA, miRNA) is a non-coding RNA with the length of about 18-25 nucleotides, and is combined with target mRNA through base complementary pairing to inhibit translation of the mRNA or promote degradation of the mRNA, so that the protein expression level is regulated and controlled. In published academic papers or patent inventions, it is reported that various mirnas play important roles in the generation, development, diagnosis and treatment of liver cancer, for example: xuguangxian et al disclose the use of miR-1247-5p as a molecular target in anti-hepatoma cell HepG 2; dengxin et al disclose a liver cancer diagnostic kit, wherein 13 miR including miR-30b-5p are used for auxiliary detection of suspected patients with liver cancer; yangchun et al discloses a drug prepared from artemisinin for resisting liver cancer cells HepG2 and Huh7, and finds that inhibition of Huh7 cell proliferation is mainly realized through MAPK signal pathway, and 9 miRNA levels including miR-30b-5p of host cells are regulated and controlled to be reduced in the process.

hsm-miR-30b-5p：UGUAAACAUCCUACACUCAGCU。

Disclosure of Invention

The technical problem to be solved by the invention is to provide a new target point for effectively inhibiting the disease progression of HBV-related hepatocellular carcinoma.

In order to solve the technical problems, the invention provides application of miR-30b-5p as a target molecule in preparation of a drug for resisting HBV-related hepatocellular carcinoma.

As an improvement of the application of the invention: by inhibiting the transcription of miR-30b-5p or leading the miR-30b-5p to lose the function of combining with a target gene, the growth and the invasion of HBV related liver cancer cells are inhibited. The mode for inhibiting miR-30b-5p transcription or inhibiting miR-30b-5p from being combined with a target gene is as follows: by transfecting miR-30b-5p specific siRNA.

The invention firstly provides that miR-30b-5p has significant difference in the level of HBV-related hepatocellular carcinoma and non-HBV-related hepatocellular carcinoma. According to the embodiment of the invention, the content of miR-30b-5p in HBV-related hepatocellular carcinoma tissues is remarkably higher than that in non-HBV-related hepatocellular carcinoma tissues.

Further, the inventor finds that miR-30b-5p has a significant correlation with the growth of HBV-related liver cancer cells, but is not correlated with non-HBV-related liver cancer cells. According to the embodiment of the invention, the growth and invasion rates of the tumor cells are not obviously reduced when miR-30b-5p is inhibited in non-HBV related liver cancer cells, and the activity of the tumor cells can be effectively inhibited when the level of miR-30b-5p is reduced in HBV related liver cancer cells. In addition, the activity of the tumor cells can be further improved when the level of miR-30b-5p is up-regulated in HBV-related liver cancer cells.

Furthermore, the invention discovers that P protein coded by HBV is the main reason for mainly influencing the miR-30b-5P level in HBV-related liver cancer cells. According to the embodiment of the invention, the content of miR-30b-5P can be obviously improved when the P protein coded by HBV is over-expressed in non-HBV related liver cancer cells.

It should be noted that: liver cancer is caused by a variety of factors, and even if liver cancer cells belong to the same pathological tissue type but are induced by different risk factors, the change conditions of intracellular non-coding RNAs such as miRNA and lncRNA have differences. The invention discloses a miRNA, namely miR-30b-5p, which has obvious difference in transcriptional abundance in HBV-related liver cancer cells and non-HBV-related liver cancer cells. Different from the disclosed invention patents, the following steps are carried out: xuguangxian et al report that transcriptional level up-regulation of miR-30b-5p can be detected in blood of liver cancer patients, but do not mention the level difference of miR-30b-5p in HBV infection-related and non-HBV infection-related liver cancer patients; yang Gacisn et al reported that a drug prepared by artemisinin could reduce the transcription level of 9 miRNAs including miR-30b-5p through MAPK signaling pathway to achieve the purpose of inhibiting the proliferation of Huh7 cells, but did not mention whether the growth rate of Huh7 cells could be inhibited only by reducing the transcription level of miR-30b-5 p. However, it is worth noting that the inhibition of miR-30b-5p transcription level in Huh7 cells (non-HBV related hepatoma cell line) can not effectively inhibit the growth rate of the cells, and the inhibition of miR-30b-5p transcription in Hep3B cells (HBV related hepatoma cell line) can significantly reduce the growth rate of the cancer cells in cells and animal models. Namely, the present invention confirmed that: when miR-30b-5p transcription is inhibited in Huh7 cells (non-HBV related liver cancer cell lines), the proliferation and migration of liver cancer cells cannot be inhibited, and when miR-30b-5p transcription is inhibited in Huh7 or Hep3B (HBV related liver cancer cell lines) transfected with HBV, the growth rates of two liver cancer cell lines can be obviously inhibited. The fact that the inhibition of the growth of the liver cancer cells by inhibiting the transcription level of miR-30b-5p is not applicable to all types of liver cancer cells, but only has the application in hepatocellular carcinoma related to HBV is shown, so that the method is obviously different from the prior art.

The invention has the following technical advantages:

the invention proves that miR-30b-5P has obvious relevance with hepatocellular carcinoma related to HBV, namely P protein coded by HBV can up-regulate the level of host cell miR-30b-5P so as to promote the growth and invasion of liver cancer cells. When the miR-30b-5p is inhibited, the activity of HBV-related liver cancer cells can be effectively inhibited, and the growth and invasion capabilities of the HBV-related liver cancer cells can be reduced. The invention proves that miR-30b-5p can be used as an important target for effectively inhibiting HBV-related liver cancer cell proliferation.

In addition, the key to the clear distinction over published related studies is that: according to the embodiment of the invention, the inhibition level of miR-30b-5p in HBV-related liver cancer cells can effectively inhibit the growth and invasion of tumor cells, but the inhibition effect is not achieved in non-HBV-related liver cancer cells. In addition, no relevant research report exists at present on the specific mechanism of miR-30b-5p up-regulation caused by HBV infection.

Therefore, it is of great significance to research the function of miR-30b-5p in HBV-related hepatocellular carcinoma and whether the function can be used as a molecular target for resisting HBV-related hepatocellular carcinoma.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the sequencing results of transcriptome of HBV-associated hepatocellular carcinoma tissues and non-HBV-associated hepatocellular carcinoma tissues. Wherein, HBV + positive is HBV-related hepatocellular carcinoma, and HBV + negative is non-HBV-related hepatocellular carcinoma;

HBV + positive-T1-T7 respectively represent liver cancer tissue samples of 7 patients with HBV-related hepatocellular carcinoma, and HBV + negative-T1-T7 respectively represent liver cancer tissue samples of 7 patients with non-HBV-related hepatocellular carcinoma.

FIG. 2 shows that the influence of HBV-infected hepatoma cell line Huh7 on miR-30b-5p is determined by real-time fluorescent quantitative PCR. Among them, "Huh 7+ EV" is Huh7 cell transfected with blank control plasmid, and "Huh 7+ HBV" is Huh7 cell transfected with HBV.

FIG. 3 is a graph showing the effect of inhibition of miR-30b-5p on the growth viability and invasiveness of non-HBV related liver cancer cells by a scratch test;

in fig. 3:

a is the observation of the migration of Huh7 cells and Huh7 cells transfected with miR-30b-5p inhibitors at 0h, 24h and 48h after scratching; b is the mobility of the cells 48h after scratching in the A is calculated; c is the observation of the cell migration of the HBV transfected Huh7 cells without or with the addition of the miR-30b-5p inhibitor; d is the migration rate of the cells 48h after scratching in the step C;

wherein, A, B is a non-HBV related liver cancer cell line Huh7, C, D is a Huh7 cell line infected with HBV. "Huh 7+ control" is a control group, and "Huh 7+ Inhibitor miR-30b-5 p" is used for transfecting siRNA specific to the miR-30b-5p into cells for inhibiting the transcription of the siRNA.

FIG. 4 is a graph comparing the effect of decreasing or increasing miR-30b-5p levels on cell proliferation viability in HBV-associated liver cancer cells by a scratch experiment;

in fig. 4:

a is the migration condition of Hep3B cells at 0h, 24h and 48h after scratching when miR-30b-5p inhibitor is transfected or excessive miR-30b-5p is transfected; b is the mobility of the cells 48h after scratching in the A is calculated;

wherein, the 'Inhibitor miR-30b-5 p' is a Hep3B cell transfected with miR-30b-5p specific siRNA, and the 'mimics miR-30b-5 p' is a Hep3B cell transfected with excessive miR-30b-5p molecules.

FIG. 5 shows that the effect of miR-30b-5p on HBV-related liver cancer cell proliferation is verified by a nude mouse tumorigenesis experiment;

in fig. 5:

the left picture in A is a picture of a test group mouse (left and middle) and a control group mouse (right) when the tumor formation is observed for 26 days, and the right picture in A is a picture of a tumor body stripped from the test group mouse (lower dotted line) and the control group mouse (upper dotted line) when the tumor formation is observed for 26 days; b is the change condition of the tumor volume of the nude mice inoculated with the tumor cells subcutaneously along with time;

wherein, the 'Control' is a mouse subcutaneously inoculated with Hep3B cells, and the 'Inhibitor miR-30b-5 p' is a mouse subcutaneously inoculated with Hep3B cells transfected with miR-30b-5p specific siRNA.

FIG. 6 shows the effect of different HBV-encoded proteins on the level of miR-30b-5p in HBV-associated liver cancer cells, as determined by real-time fluorescent quantitative PCR; wherein, HBV-P is HBV P protein, HBV-X is HBV X protein, HBV-c is HBV core antigen, and HBV-s is HBV surface antigen.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

In the specific embodiment of the invention, clinical samples are provided by the first hospital affiliated to the Zhejiang university medical college, 7 cases of HBV-related hepatocellular carcinoma patients are used in the experimental group, and 7 cases of non-HBV-related hepatocellular carcinoma patients are confirmed by CT, histopathology and serology methods in combination with related medical history. The pathological tissues of the patients were obtained before their treatment. The experimental methods and procedures involved in the examples of the present invention were approved by the ethical committee of university of Zhejiang.

Description of the drawings: as used herein, the term "non-HBV-associated hepatocellular carcinoma" means that the liver cancer of a patient is caused by a non-HBV infection, and the term "non-HBV-associated hepatocellular carcinoma cell line" means that the liver cancer cell line does not contain any HBV-associated genetic information.

Example 1: analysis of the transcriptional level difference of miR-30b-5p in HBV-related or non-HBV-related hepatocellular carcinoma tissues

Tumor tissues of 7 experimental groups and 7 control groups of liver cancer patients obtained by surgical resection were added with Trizol reagent (1 mL per 100mg of tissue), ground with an electric homogenizer, and left to stand for 5min after completion. To the tissue homogenate was added chloroform (200. mu.L per 100mg of tissue homogenate), shaken vigorously, allowed to stand for 5min, and centrifuged at 4 ℃ for 15min (12000 rpm). And transferring the water phase of the layered liquid to another centrifuge tube after the centrifugation is finished, adding isopropanol with the same volume, uniformly mixing, and standing for 20-30min at room temperature. Centrifuging for 10min again, discarding supernatant to obtain white precipitate at the bottom of the tube, and naturally drying the tube in an ultraclean bench. Adding appropriate amount of DEPC water into the centrifuge tube to fully dissolve the extracted RNA. The prepared RNA samples were stored at ultra-low temperature and whole transcriptome sequencing was entrusted to Norhe Pogenic science and technology, Inc. And carrying out differential gene analysis on the sequencing data. The results are shown in FIG. 1, and it can be seen that the transcription level of miR-30b-5p in HBV-associated hepatocellular carcinoma tissue is higher than that in non-HBV-associated hepatocellular carcinoma tissue, which indicates that the transcription of miR-30b-5p in liver cancer cells is positively regulated under the influence of HBV infection, i.e. the content of miR-30b-5p in the cells is increased.

Further, the inventors transfected HBV in a non-HBV related liver cancer cell line Huh7 and used Huh7 cells as a control, and centrifuged 48h after transfection to collect cells and extract total RNA of the cells. RNA extracted from cells is first Reverse transcribed into cDNA using Reverse transcription PCR (RT-PCR). Add 2. mu.L of PCR buffer, 2. mu.L of cDNA, and 8. mu.L of DEPC water to the reaction tube in sequence and mix well. The reaction conditions are as follows: 15min at 37 ℃; at 85 ℃ for 3 min; storing at 4 ℃. Then, the relative transcription level of the gene is determined by using real-time fluorescent Quantitative PCR (Quantitative real-time PCR, qPCR). The reaction system is as follows: mu.L of each of 10. mu.M forward and reverse primers, 0.5. mu.L, 1. mu.L of cDNA, 5. mu.L of LPCR buffer, and 3. mu.L of DEPC water were added sequentially and mixed well. The reaction conditions are as follows: at 95 ℃ for 30 s; 40 PCR cycles (95 ℃, 30 s; 60 ℃, 30 s); 95 ℃ for 15 s; 60 ℃ for 60 s; 95 ℃ for 15 s.

The RT-PCR primers used above were synthesized by Rebo Biotechnology Ltd, Suzhou, with sequence 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGagctgagt-3'.

The qPCR primers used above were synthesized by scomitrella biotechnology, inc, with the forward primer sequence 5'-GCCGAGtgtaaacatccta-3' and the reverse primer sequence 5'-CTCAACTGGTGTCGTGGA-3'.

The qPCR experiment result is shown in FIG. 2, and it can be seen that compared with control Huh7, the expression level of miR-30b-5p in Huh7 cells infected with HBV is significantly improved, which also indicates that the transcription of miR-30b-5p in liver cancer cells is promoted under the influence of HBV infection.

The above results show that the transcription level of miR-30b-5p in liver cancer tissue has significant correlation with whether HBV is infected, and the gene activates the transcription of the gene after HBV infection and increases the content of the gene in the liver cancer tissue.

Example 2: inhibiting the level of miR-30b-5p in HBV-related liver cancer cells can inhibit the growth and invasion of tumor cells

In this example, the inventor firstly selected a non-HBV related hepatoma cell line Huh7, and compared the effect of miR-30b-5p on cell growth and invasion ability when infected or not infected with HBV by scratch test. miR-30b-5 p-specific siRNA was transfected into Huh7 cells or HBV-infected Huh7 cells and compared with Huh7 cells, respectively. After 24h of transfection, the cell culture dish was scratched with a pipette tip and washed twice with PBS to remove the scraped cells. The cells were observed and photographed at 0h, 24h and 48h after the scratch, and the migration area and migration rate of the cells on both sides of the scratch were analyzed and calculated using Image J software, and the results are shown in fig. 3. It can be seen that there is no significant difference in the cell distance between the experimental group and the control group on both sides of the scratch at 24h and 48h after the above siRNA was transfected into Huh7 cells (FIGS. 3A and 3B). However, when the above siRNA was transfected into HBV-infected Huh7 cells, it was observed that the growth of cells on both sides of the scratch was significantly reduced, and the confluency of cells was significantly reduced at 24h and 48h after transfection compared to the control group (fig. 3C and 3D). The miR-30b-5p specific siRNA used in the method is synthesized by Ribo Biotechnology Co., Suzhou with the sequence of 5'-CTCAACTGGTGTCGTGGA-3'.

Further, another HBV-related hepatoma carcinoma cell Hep3B was selected in the invention to analyze the influence of miR-30b-5p on the growth and invasion of hepatoma carcinoma cells in the inhibition and activation states, respectively. miR-30b-5 p-specific siRNA (for) or synthesized miR-30b-5p is transfected into Hep3B cells respectively and takes Hep3B cells as a control, a cell scratching experiment is carried out, the cells are observed and photographed at 0h, 24h and 48h after scratching, and the migration area and the migration rate of the cells on both sides of the scratching are analyzed and calculated by using Image J software. Description of the drawings: the miR-30b-5p specific siRNA is used for inhibiting miR-30b-5p transcription, and miR-30b-5p enables the intracellular miR-30b-5p level to be increased. The results are shown in fig. 4A and fig. 4B, and it can be seen that Hep3B cells in the control group had high growth activity, and the cells had significantly migrated toward the median line at 24h after scratching, and the cells at both sides of the scratch had substantially confluent at 48 h. Compared with the cells of the control group, the cells are further promoted to grow and migrate when the synthesized miR-30b-5p is transfected in the Hep3B cells, and it can be seen that the migration area of the cells on two sides of the scratch at the 24h after transfection is obviously higher than that of the control group, and the cells are completely confluent by the 48 h. However, a reduction in the growth and migration rate of the cells was clearly observed in Hep3B cells transfected with miR-30b-5 p-specific siRNA, and the two cells remained unconjugated at 48h after scratching compared to the other two groups of cells.

The results show that miR-30b-5p only plays a role in promoting cell growth and invasion in HBV-related liver cancer cells, and the growth activity of the HBV-related liver cancer cells can be reduced when miR-30b-5p is inhibited from transcribing or loses the function of combining with a target gene. However, miR-30b-5p does not have this function in non-HBV related hepatoma cells.

Example 3: inhibition of miR-30b-5p in animal models can inhibit tumor growth

In this example, miR-30b-5 p-specific siRNA was transfected into Hep3B cells, and Hep3B cells were used as a control group. Description of the drawings: hep3B is a human liver cancer cell strain integrated with complete HBV genome, and HBV virions can be detected in supernatant during the growth and proliferation process of the human liver cancer cell strain.

After transfection, the cells were collected and resuspended in serum-free DMEM medium to a cell concentration of 2X 10⁷cell/mL. Equal amounts of Hep3B cells and Hep3B cells transfected with the above siRNA were inoculated subcutaneously into nude mice with a syringe, three cells per group, and tumor growth was observed and tumor size was recorded after inoculation. The mice were sacrificed 26 weeks after inoculation, and the size was measured and recorded by photographing after the tumor body was detached. As shown in FIG. 5A, it can be seen that the tumor formation was clearly observed in the subcutaneous tissues of nude mice inoculated with Hep3B cells at day 9, and the growth rate of tumor was significantly increased with the lapse of time, and the tumor volume was over 200mm by day 26³(FIG. 5B). However, when the siRNA-transfected Hep3B cells were inoculated subcutaneously into nude mice, although tumors were also formed, it was observed that the tumor growth rate was significantly inhibited, and the tumor size was about 30mm at 26 days after inoculation³。

The results show that after the transcription of miR-30b-5p is inhibited in HBV-related liver cancer cells or the miR-30b-5p loses the capacity of combining with a target gene, the growth of tumor cells at the inoculation part of an experimental animal can be effectively inhibited, and the miR-30b-5p can be used as a potential target spot for inhibiting HBV-related hepatocellular carcinoma.

Example 4: p protein coded by HBV can up-regulate transcription of miR-30b-5P in liver cancer cells

In this example, the encoding genes of P protein, X protein, core antigen (C protein) and surface antigen (S protein) of HBV in NCBI database (NC — 003977) were cloned and constructed into overexpression vectors, and transfected into HBV-associated hepatoma cell line Hep3B, respectively, with the original Hep3B cells as controls. Cells were harvested by centrifugation 48h after transfection and cellular RNA was extracted and the relative transcript levels of miR-30b-5p in each group were determined using RT-PCR and qPCR. As shown in FIG. 6, it can be seen that the relative transcription level of miR-30b-5P in the Hep3B cell is obviously improved after the plasmid capable of expressing HBV P protein is transfected into the cell, and the other three HBV coding proteins have no such function.

The above results indicate that HBV-encoded P protein is the main reason for its major cause of miR-30b-5P upregulation in hepatoma cells.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

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Claims

1. The application of specific siRNA for inhibiting miR-30b-5p transcription or inhibiting miR-30b-5p transcription and target gene combination in preparation of anti-HBV related hepatocellular carcinoma drugs.

2. The application of the specific siRNA for inhibiting miR-30b-5p transcription or inhibiting miR-30b-5p transcription and target gene combination in preparation of the medicine for resisting HBV-related hepatocellular carcinoma is characterized in that: by inhibiting the transcription of miR-30b-5p or leading the miR-30b-5p to lose the function of combining with a target gene, the growth and the invasion of HBV related liver cancer cells are inhibited.

3. The application of the specific siRNA for inhibiting miR-30b-5p transcription or binding with a target gene in preparation of a drug for resisting HBV-related hepatocellular carcinoma is characterized in that: p protein coded by HBV can up-regulate the level of host cell miR-30b-5P, thereby promoting the growth and invasion of liver cancer cells.