CN114832105B

CN114832105B - Application of inhibitor of C7orf50 gene or protein

Info

Publication number: CN114832105B
Application number: CN202110136133.1A
Authority: CN
Inventors: 黄晓武; 毛丽; 李佳
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2023-08-04
Anticipated expiration: 2041-02-01
Also published as: CN114832105A

Abstract

The invention discloses application of an inhibitor of a C7orf50 gene or protein, and provides the application that RNAi knockdown of the C7orf50 gene can effectively inhibit proliferation, invasion and migration of liver cancer cells and lung metastasis of liver cancer, and animal experiments (a mouse in-situ tumor model) prove that the liver tumor volume of a mouse with the RNAi knockdown of the C7orf50 gene is obviously smaller than that of a control group, and the RNAi knockdown of the C7orf50 gene can obviously inhibit lung metastasis of liver cancer of the mouse. The invention provides a molecular target for effectively treating liver cancer, and provides a new direction for research and development of new drugs for treating liver cancer and clinical diagnosis and treatment.

Description

Application of inhibitor of C7orf50 gene or protein

Technical Field

The invention relates to application of an inhibitor of a C7orf50 gene or protein, belonging to the technical field of molecular biology.

Background

Although liver cancer is the sixth most common tumor and the third cancer cause of death in the world currently, only 5-10% of liver cancer patients can undergo surgical excision. Since its symptoms are not apparent at an early stage, most patients are diagnosed already at a late stage of the disease, and the available treatments are extremely limited. The pathogenesis of liver cancer is complex, and its occurrence and development are a multifactorial process, driven by the accumulation of various genetic and non-genetic factors. Abnormal inactivation of an oncogene (e.g., p 53), abnormal activation of a proto-oncogene (e.g., K-ras), abnormal changes in a signaling pathway (e.g., PI3K, MAPK, JAK/STAT, NF- κB, wnt/β -catenin), abnormal regulation of epigenetic events, etc., may be involved in the development and progression of liver cancer. Liver cancer also develops from abnormal growth to life-threatening metastatic tumors with the accumulation of various factor changes. Therefore, research on molecular mechanisms for regulating and controlling the occurrence and development of liver cancer is urgently needed in the field, so that pathogenesis of liver cancer is better known, and medicines for effectively treating liver cancer or inhibiting metastasis of liver cancer are developed.

Disclosure of Invention

The invention solves the technical problems that: how to develop molecular targets for effectively treating liver cancer and medicines capable of effectively treating liver cancer or inhibiting liver cancer metastasis.

In order to solve the problems, the invention provides application of an inhibitor of a C7orf50 gene or protein, which is characterized in that the inhibitor is used for preparing medicines for treating liver cancer and/or medicines for inhibiting liver cancer metastasis.

Preferably, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient which is an inhibitor of the C7orf50 gene or protein.

Preferably, the inhibitor of the C7orf50 gene or protein comprises RNAi specific for the C7orf50 gene, microRNA specific for the C7orf50 gene, shRNA of the C7orf50 gene, siRNA of the C7orf50 gene, antibody of the C7orf50 protein, or inhibitor of activity of the C7orf50 protein.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses the function of C7orf50 gene or protein with unknown function, and based on the regulation function of C7orf50 gene or protein in liver cancer proliferation and metastasis, the proliferation, invasion and migration of liver cancer cells can be effectively inhibited and liver cancer lung metastasis can be inhibited by RNAi knockout of C7orf50 gene, a molecular target capable of effectively treating liver cancer is provided, and a new direction is provided for research and development of new drugs for liver cancer and clinical diagnosis and treatment.

Drawings

FIG. 1A is a diagram showing the expression of the C7orf50 gene extracted from the TCGA database in liver cancer tissues;

FIG. 1B is a graphical representation of C7orf50 gene expression versus liver cancer prognosis extracted from the TCGA database;

FIG. 1C is a graph showing the expression level of the C7orf50 gene in 20 pairs of clinical liver cancer samples, wherein T is cancer tissue and P is paracancestor tissue;

FIG. 1D is a graph showing the result of PCR detection of the expression level of the C7orf50 gene in a liver cancer cell line;

FIG. 1E is a graph showing the result of detecting the expression level of the C7orf50 gene in a liver cancer cell line by Western blotting;

FIG. 2A is a diagram showing expression of C7orf50 mRNA in a stable virus strain for constructing a liver cancer cell line;

FIG. 2B is a diagram showing expression of C7orf50 protein in a stable virus strain for constructing a liver cancer cell line;

FIG. 2C is a graphical representation of the effect of a cell proliferation assay (CCK-8) to detect C7orf50 knockdown or overexpression on proliferation potency of liver cancer cell lines;

FIG. 2D is a graph showing the effect of C7orf50 knockdown or over-expression on proliferation potency of hepatoma cell lines;

FIG. 2E is a graph showing the effect of cell migration invasion assay to the ability of migration and invasion in liver cancer cell lines after C7orf50 knockdown or overexpression;

FIG. 2F is a graph showing the effect of cell scratch assay on migration ability in liver cancer cell lines after C7orf50 knockdown or over-expression;

FIG. 3A is a graph of in vivo imaging results of a mouse model of liver cancer (in situ tumor) after C7orf50 knockdown or over-expression;

FIG. 3B is a graph showing lung metastasis results of a mouse model of liver cancer (in situ tumor) after C7orf50 knockdown or overexpression (HE staining results observed with 100X and 200X microscopes);

FIG. 3C is a graph showing the results of fluorescence quantification of in situ liver tumors after C7orf50 knockdown or over-expression;

FIG. 3D is a graph showing the results of fluorescence quantification of lung metastases after C7orf50 knockdown or overexpression;

FIG. 3E is a graph of the weight quantification result of hepatic carcinoma in situ after C7orf50 knockdown or over-expression;

FIG. 3F is a graph showing the quantitative results of lung metastasis nodules after C7orf50 knockdown or over-expression.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

The experimental materials and general experimental methods in the following examples of the present invention are as follows:

1. patient and follow-up

Quick frozen tumor tissues of patients subjected to radical excision (Shanghai, china) at the liver cancer institute of the affiliated Zhongshan hospital of the double denier university were collected and used for Western Blotting (WB) analysis. All patients received histopathological diagnosis of liver cancer according to the world health organization standard. Each patient was informed consent, and was included in the study, and the ethical committee of the study affiliated with the middle mountain hospital at the complex university approved the use of human subject tissue. And (3) carrying out unified follow-up monitoring after radical excision on all patients.

2. Experimental cell strain

Human hepatoma cell lines MHCC97L, MHCC97H and HCCLM3, which show gradually increasing lung metastasis potential, respectively, were developed by the liver cancer institute of the affiliated Zhongshan hospital at the university of double denier. The normal liver cell line L02 and the non-metastatic human liver cancer cell lines HepG2 and PLC/PRF/5 were purchased from the American type culture Collection. All cell lines were cultured in DMEM (Invitrogen) with 10% fetal bovine serum (Gibco), 37℃at CO ₂ The concentration was 5%.

3. Immunoblotting

Protein lysates were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane, and after blocking with 5% skimmed milk, probed with primary and species-specific secondary antibodies.

RT-qPCR detection method

Total RNA samples were isolated from tissue specimens and cell lines of the present study using TRIzol reagent (Invitrogen, USA). cDNA synthesis was performed by PrimeScript RT kit (TaKaRa, dai of China). Real-time polymerase chain reaction (qPCR) was performed using SYBR Green Premix Ex Taq (TaKaRa, chinese da).

5. Stable strain construction

The overexpression and knock-down virus of C7orf50 are synthesized by Ji Kai gene company, and then liver cancer cell strain transfection and puromycin screening are carried out.

6. Cell proliferation, migration and invasion assays

(1) Cell proliferation assay: the cells were placed in 96-well plates using CCK-8 kit according to the protocol, and CCK-8 solution was added on day 0, day 1, day 2, day 3, day 4, and day 5, respectively, and OD values were observed and recorded to make a growth curve.

(2) Cell clone formation experiments: cells were seeded in six well plates, cultured in a cell incubator, fixed with paraformaldehyde and stained with crystal violet stain after 14 days, photographed and counted.

(3) Transwell cell migration challenge experiment: according to the procedure of the Transwell filter chamber (BD Biosciences, USA) instructions, matrigel gel was placed or not in the upper chamber, then 600 μl of complete medium was placed in twenty-four well plates (i.e. the lower chamber), 200 μl of serum-free cells were placed in the upper chamber, placed in a conventional incubator for culturing, after 48 hours, the cells were taken out, rinsed with clear water, fixed in paraformaldehyde and stained in crystal violet stain, the cells attached to the upper chamber were carefully wiped with a cotton swab and rinsed with clear water, and the cells passed through the lower chamber were observed under a microscope, and cell counts and photographs were taken.

(4) Scratch experiment: cells were inoculated into six well plates, cultured in a cell incubator, scored after confluence, photographed at 0h,24h,48h, respectively, and cell growth recorded. .

7. Animal model

Male nude mice (BALB/c nu/nu) four to six weeks old were purchased from Shanghai institute of matter medicine, national academy of sciences, and all animals were kept under specific pathogen free animal (SPF) conditions. Mice were subcutaneously injected 3X 10 ⁶ Liver cancer cells. Tumor volumes were measured at specified time intervals. Mice bearing tumors were sacrificed 30 days post inoculation and tumors were removed for further study. All experiments were approved by the institutional animal care and use committee of Shanghai cancer research.

Example 1

Verifying that the C7orf50 gene or protein is associated with liver tumor metastasis and prognosis:

1.1 extracting the expression of the C7orf50 gene in liver cancer tissues and paracancerous tissues through a TCGA database, wherein the expression of the C7orf50 gene in the cancerous tissues is higher than that in the paracancerous tissues as shown in figure 1A; and extracting the relation between the C7orf50 gene expression quantity and the prognosis of the patient, wherein the surface C7orf50 gene expression quantity is closely related to the prognosis of the patient as shown in FIG. 1B. The expression level of C7orf50 protein was examined by randomly extracting 20 cases from the liver cancer samples collected in the past, and as shown in FIG. 1C, the C7orf50 protein was highly expressed in most samples (16/20).

1.2 detecting the expression level of C7orf50 mRNA in liver cancer cell lines with different metastatic potential by adopting an RT-qPCR detection method, wherein the result is shown in a figure 1D, the C7orf50 mRNA is expressed relatively high in high metastatic potential cell lines (HCCLM 3, MHCC97H and MHCC 97L), expressed relatively low in low metastatic potential cell lines (HepG 2 and PLC/PRF/5), and expressed lowest in normal liver cell lines L0-2; the immunoblotting method is used for further detecting the expression level of the C7orf50 protein in liver cancer cell lines with different metastatic potential, as shown in FIG. 1E, the C7orf50 protein is expressed relatively high in the high metastatic potential cell lines (HCCLM 3, MHCC97H and MHCC 97L), expressed relatively low in the low metastatic potential cell lines (HepG 2 and PLC/PRF/5), and expressed lowest in the normal liver cell line L0-2. The C7orf50 gene or protein is described as being associated with liver tumor metastasis.

Example 2

RNAi knockdown of the C7orf50 gene can inhibit proliferation, invasion and migration capacity of liver cancer cells, and can inhibit lung metastasis of liver cancer cells:

2.1 constructing a C7orf50 gene knockdown and knockdown back stable transgenic cell strain in HCCLM3 by using a slow virus, constructing a C7orf50 gene over-expression stable transgenic cell strain in PLC/PRF/5 by using a slow virus, detecting the expression level of C7orf50 mRNA in the stable transgenic cell strain, as shown in FIG. 2A, and detecting the expression level of C7orf50 protein in the stable transgenic cell strain, as shown in FIG. 2B.

2.2 measurement of in vitro proliferation potency of HCCLM3 and PLC/PRF/5 cell stable transformants using cell counting kit 8 (CCK-8) (Dojindo, japan), operating according to the instructions of CCK-8, experimental results showed that knockdown of C7orf50 gene in HCCLM3 cells reduced proliferation potency of liver cancer cells, while proliferation potency of HCCLM3 cells that restored the C7orf50 gene after knockdown was significantly restored, and proliferation potency of PLC/PRF/5 cells that overexpressed C7orf50 gene in PLC/PRF/5 cells was also significantly enhanced, as shown in FIG. 2C.

2.3 cell clone formation experiments can detect cell population dependence and proliferation capacity, the same number of logarithmic growth phase cells in each group shown in figure 2D are taken in a culture dish, the cells are fixed after 2 weeks, and clone numbers are recorded by crystal violet staining, so that the result shows that the knockdown C7orf50 gene can effectively inhibit proliferation of liver cancer cells, as shown in figure 2D.

2.4 cell migration invasion assay (transwell filter, BD Biosciences, USA) and scratch assay can detect invasion and migration ability of liver cancer cells, and the result shows that knocking down the C7orf50 gene can inhibit invasion and migration ability of liver cancer cells, while over-expressing the C7orf50 can enhance invasion and migration ability of liver cancer cells, and the result of cell migration invasion assay is shown in fig. 2E, and the result of scratch assay is shown in fig. 2F.

Example 3

RNAi knockdown of the C7orf50 gene can effectively inhibit the growth of liver tumors and liver cancer lung metastasis:

3.1 constructing a C7orf50 gene knockdown and knockdown back-complement stable transgenic cell strain in HCCLM3 by using a slow virus, constructing a C7orf50 gene over-expression stable transgenic cell strain in PLC/PRF/5 by using the slow virus, injecting the constructed C7orf50 gene knockdown and knockdown back-complement stable transgenic cell strain into the subcutaneous of a mouse, taking the injected normal HCCLM3 cell strain as a control group, injecting the constructed C7orf50 gene over-expression stable transgenic cell strain into the subcutaneous of the mouse, taking the injected normal PLC/PRF/5 cell strain as the control group, and transplanting small subcutaneous tumors into the liver of a nude mouse in situ after the tumor formation (a mouse in-situ tumor model). The in-vivo imaging result of the mouse in-situ tumor model is shown in fig. 3A, the tumor volume of the HCCLM3 cell strain knocked out of the C7orf50 gene is obviously smaller than that of the control HCCLM3 cell strain, the tumor volume of the knocked-out back C7orf50 genome is increased compared with that of the C7orf50 knocked-out group, and the tumor volume of the PLC/PRF/5 cell strain over-expressing the C7orf50 gene is obviously larger than that of the control PLC/PRF/5 cell strain, so that the RNAi knocked out of the C7orf50 can inhibit the growth of liver tumors. The fluorescent quantitative result of the C7orf50 gene knockdown or over-expressed hepatoma is shown in FIG. 3C, the quantitative result of the weight of the C7orf50 gene knockdown or over-expressed hepatoma is shown in FIG. 3E, and the weight of the C7orf50 gene knockdown group hepatoma is obviously lower than that of the control group and the anaplerotic group, so that the RNAi knockdown of the C7orf50 can inhibit the proliferation of liver cancer and the growth of liver tumor.

3.2 taking lung tissue of liver cancer mice to perform HE staining, as shown in FIG. 3B, the lung metastasis capability of in-situ tumor of C7orf50 gene knockdown group is obviously smaller than that of control group, and the lung metastasis capability of in-situ tumor of reintegration C7orf50 gene after knockdown is increased compared with that of C7orf50 knockdown group, and in-situ tumor over-expressing C7orf50 gene has obvious lung metastasis compared with control group. The fluorescent quantitative result of the C7orf50 gene knockdown or over-expressed lung metastasis is shown in fig. 3D, the quantitative result of the C7orf50 gene knockdown or over-expressed lung metastasis nodules is shown in fig. 3F, and the number of lung metastasis nodules in the C7orf50 gene knockdown group is obviously smaller than that in the control group and the reinjection group, so that the RNAi knockdown of C7orf50 can effectively inhibit liver cancer lung metastasis.

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. Use of an inhibitor of a C7orf50 gene or protein for the preparation of a medicament for treating liver cancer, wherein the inhibitor of a C7orf50 gene or protein comprises RNAi specific for a C7orf50 gene, microRNA specific for a C7orf50 gene, shRNA of a C7orf50 gene, siRNA of a C7orf50 gene, an antibody to a C7orf50 protein, or an inhibitor of activity of a C7orf50 protein.

2. Use of an inhibitor of the C7orf50 gene or protein as in claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient that is an inhibitor of the C7orf50 gene or protein.