KR20040067251A - Lung cancer cell-growth inhibition drugs comprising RNAi sequences against MGC5627 gene, preparation method thereof and use thereof - Google Patents

Abstract

PURPOSE: Provided is a preparation for inhibiting the proliferation of lung cancer cells which expresses RNAi with respect to MGC5627 genes, and effectively inhibits the growth of the lung cancer tissues, and the colony formation and the anchorage independency thereof. CONSTITUTION: The lung cancer cell proliferation inhibitor comprises the RNAi sequence identified by SEQ ID NO 2 for inhibiting the gene expression of MGC5627 identified by SEQ ID NO 1 to treat lung cancers. The RNAi sequence is expressed by the human U6 promoter cassette. The RNAi expression vector with the concentration of 2 micro grams/1x100000 is introduced into the lung cancer cell together with cationic liposome of 1-20 micro grams/1x100000 several times for 4-15 days.

Description

Lung cancer cell proliferation inhibitor comprising RNC sequence for MMCC5627 gene, method for preparing same and method for inhibiting lung cancer proliferation using same {Lung cancer cell-growth inhibition drugs comprising RNAi sequences against MGC5627 gene, preparation method

The present invention relates to a base sequence of RNAi, an expression vector thereof, and a method of inhibiting the proliferation of lung cancer cells using the MGC5627 gene inhibiting the growth of cancer tissue by inhibiting the expression of MGC5627 gene in lung cancer cells.

Recently, as strategies for treating cancer are diversified, basic studies are being conducted to increase cancer survival by inhibiting cancer cell metastasis or inhibiting neovascularization, which is essential for cancer cell growth and metastasis.

For example, Australian Patent No. 6,699,301, US Patent Publication No. 2002-159989, US Patent Publication No. 2002-151457, and the like, particularly in connection with lung cancer, US Patent Publication No. 2002-160388, US Patent Publication No. 2002- 147143 and US Pat. No. 5,692,918.

However, these treatment methods also have a high risk of side effects due to systemic administration of angiogenesis and metastasis inhibitors (chemical and protein preparations) as conventional chemotherapy involves side effects from systemic administration of anticancer drugs. There is a need for a new treatment method for selectively introducing and expressing genes for cancer treatment in cancer cells and cells in cancer tissues.

Meanwhile, recent gene therapy studies have introduced foreign genes selectively into target cells to induce long-term expression and actually improve gene transfer efficiency when used in clinical treatment, and genes involved in cancer (cancer genes and Genes involved in tumor suppressor genes and immune responses against cancer cells (cytokine genes) have been identified, suggesting that cancer is a disease that can be treated using various methods of gene therapy.

Therefore, in the present invention, as a result of searching various genes to complete a method of inhibiting lung cancer growth by inhibiting the expression of genes involved in lung cancer cell growth, it was confirmed that the effect of inhibiting the proliferation of lung cancer cells through inhibition of the MGC5627 gene. It was possible to complete the present invention based on this.

Accordingly, an object of the present invention to provide a lung cancer cell proliferation inhibitor comprising an RNAi sequence for the MGC5627 gene that can inhibit lung cancer cell proliferation.

Another object of the present invention is to provide a vector into which the RNAi base sequence for the MGC5627 gene is introduced.

Another object of the present invention to provide a method for inhibiting the proliferation of lung cancer cells using the inhibitor.

In order to achieve the above object, the lung cancer cell proliferation inhibitor of the present invention comprises an RNAi nucleotide sequence represented by SEQ ID NO: 2 for the MGC5627 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.

In order to achieve the above another object, the recombinant vector of the present invention is composed of the RNAi sequence for the MGC5627 gene is introduced and expressed under the control of a human U6 promoter cassette.

In order to achieve the above another object, the lung cancer cell proliferation inhibition method of the present invention is introduced 1 μg and 10 μg of the cationic liposome per 1 × 10 ⁵ cells together, and then 1 to 4 days It consists of introducing several times.

1 is a map of the U6 + 27 / RNAi (SiRNA) plasmid structure, an RNAi expression vector.

2A is a DNA sequence listing showing the MGC5627 gene sequence,

2B is a sequence listing showing RNAi target positions for MGC5627.

Figure 3a is a photograph observed on day 4 of the culture cultured human lung cancer cell line transformed with the RNAi expression vector for the MGC5627 gene using a medium containing 600 µg / ml G418,

3b is a photograph showing cell clones that were fully screened and cultured 15 days after the start of the selective culture of human lung cancer cell lines transformed with RNAi expression vector for the MGC5627 gene using a medium containing 600 μg / ml G418.

Figure 3c is agarose gel electrophoresis confirmed by RT-PCR mRNA reduction of MGC5627 target gene in human lung cancer cell line transformed with RNAi expression vector for MGC5627 gene. Lane 1 is a size marker, lane 2 is a negative control indicating that there is no effect on MGC5627 gene expression in cells not transformed with a vector expressing RNAi for the MGC5627 gene, and lane 3 is RNAi for the MGC5627 gene showed reduced mRNA expression of the MGC5627 gene, lanes 4 and 5 are actin controls showing the same amount of RNA was used in lanes 2 and 3.

Figure 4 is a photograph showing the colony formation assay (colony formation assay) results of lung cancer cells transformed with RNAi expression vector for the MGC5627 gene. U6 is a negative control not transformed with RNAi expression vector for MGC5627 gene, U6 / HYK is a negative control transformed with vector with antibiotic resistance gene only, MGC / HYK is RNAi sequence and antibiotic resistance gene for MGC5627 gene Cells transformed with the vector.

Figure 5 is a graph showing the results of cell counting (cell counting assay) of lung cancer cells transformed with RNAi expression vector for MGC5627 gene.

FIG. 6 is a graph showing anchorage independency through soft agar assay of lung cancer cells transformed with RNAi expression vector for MGC5627 gene. U6 / HYK is a negative control transformed with a vector with antibiotic resistance genes only, MGC / HYK is a cell transformed with a vector with RNAi sequence and antibiotic resistance gene for MGC5627 gene.

Hereinafter, the present invention will be described in more detail.

As described above, the present invention provides a lung cancer cell proliferation inhibitor comprising an RNAi sequence for the MGC5627 gene that inhibits the expression of the MGC5627 gene, a method for preparing the same and a method for inhibiting the proliferation of lung cancer cells using the same.

In the present invention, the RNAi sequence for the MGC5627 gene was prepared, synthesized, and inserted into a multicloning site (MCS) of a pBluescript KS (+) vector to be introduced into cancer cells. The MGC5627 gene sequence is registered under GenBank Accession No. GI 13129100, whose function is unknown. However, in the present invention, it was confirmed on the protein that the expression of the MGC5627 gene is reduced in the course of cancer treatment after treatment with cancer cells.

The lung cancer cell proliferation inhibitor according to the present invention was obtained by inserting the RNAi nucleotide sequence for the MGC5627 gene into a vector which can be expressed under a human U6 promoter. In the present invention, pBluescript KS (+) was used, but various expression vectors are possible. According to the purpose of the other expression vector can be used easily.

The prepared vector is transduced into lung cancer cells in a conventional manner, it can be expressed with high efficiency to effectively inhibit lung cancer cell growth.

The concentration of the RNAi expression vector for the MGC5627 gene administered to the lung cancer cells is 0.1 to 2 µg / 1 × 10 ⁵ cells, preferably 1 µg / 1 × 10 ⁵ cells, and 1 to 20 µg / 1 × cationic liposomes. 10 ⁵ cells, preferably 10 μg / 1 × 10 ⁵ cells, may be administered once or in divided portions for 4 to 15 days. However, the actual dosage of the active ingredient may be determined differently depending on the type, characteristics, treatment conditions and administration methods of cancer cells.

On the other hand, the inhibitor may be prepared in the form of an isotonic aqueous solution or suspension, and may contain an adjuvant such as a salt or buffer for controlling osmotic pressure, a hydrating agent, a stabilizer, and other therapeutically useful substances.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1

Cell culture

Human lung cancer cell line (H460) was purchased from the American Cell Line Bank (ATCC) and contained RPMI culture medium (GIBCO / BRL, Grand Island, NY) containing 10% fetal calf serum, 100 units / ml penicillin, and 100 μg / ml streptomycin. Incubated at 37 ° C. and 5% CO ₂ .

Example 2

RNAi Vector Manufacturing

1 is a structure of U6 + 27 / RNAi (SiRNA) which is a vector of the present invention, referring to this may help understanding of the present embodiment.

By analyzing the mRNA sequencing of the MGC5627 gene, one target position was determined by a conventional RNAi target determination method and its oligonucleotides were customized. The produced oligonucleotide synthesizes a DNA fragment capable of expressing target RNAi using exTaq polymerase, and then ecoR I / downstream of the human U6 promoter in the human U6 promoter cassette. An expression vector system was prepared by subcloning into the Xba I site. Human U6 cassettes were prepared by amplifying the U6 gene (+27) from the human U6 promoter (-303) by PCR and subcloning into multicloning sites (Apa I / EcoR I) of pBluescript KS (+).

Figure 2a is the gene sequence (Genbank, GI 13129100) of MGC5627, Figure 2b is the RNAi target position.

Example 3

Transformation and RNAi Expression of Human Lung Cancer Cell Lines

Human lung cancer cell line (H460) (1 × 10 ⁵ ) was transferred to a 60 mm culture dish and incubated for 24 hours under the same conditions as in Example 1. After washing the cells twice with serum-free medium, 10 μg of cationic liposomes (GIBCO / BRL, Grand Island, NY) and the control vector expressing the lacZ gene, neomycin resistance gene Cells were incubated for 4 to 6 hours each in 200 µl of serum-free medium containing 2 µg of the pHYK expression vector mixture having 2 µg or a plasmid expressing RNAi targeting lacZ mRNA and 2 µg of the pHYK mixture having neomycin resistance genes. The cells were washed twice with serum free medium and cultured for 48 hours after the addition of the culture medium. After inducing the expression of the SiRNA vector for 48 hours, the selective culture of surviving cells was performed by treatment with the neomycin homolog, 600 µg / ml G418. The new medium and G418 were replaced every two days. After about one week, all human lung cancer cell lines that were not resistant to G418 died. About 15 days later, all human lung cancer cell lines that were resistant to G418 were completely selected. FIG. 3a shows the results of day 4 of starting the selection culture using a medium containing 600 µg / ml G418, and FIG. 3b is 15 days after starting the selection culture using the medium containing 600 µg / ml G418. Selected cultured cell clones are shown.

In addition, to recover the colonies formed by introducing the RNAi expression vector for the MGC5627 gene and pHYK, a G418 selective vector, the total RNA was isolated to confirm the mRNA quantitative change of the target gene by RNAi expression. , RT-PCR was used to quantify RNA. As a result, it was confirmed that the mRNA amount of the MGC5627 gene was reduced in the RNAi-expressing cells as shown in Figure 3c.

Example 4

Cancer Cell Growth Inhibitory Effect of RNAi Sequence of MGC5627 Gene by Colony Formation Analysis

Lung cancer cells (1 × 10 ⁵ ) were transferred to a 60 mm culture dish and incubated for 24 hours under the same conditions as in Example 1. After washing the cells twice with serum-free medium, 10 μg of cationic liposomes (GIBCO / BRL, Grand Island, NY), 1 μg of neomycin resistance gene expression vector (pHYK) and 1 μg of RNAi expression vector 200 μl of serum-free medium containing the mixture was used to transform human lung cancer cell line H460 together, followed by a screening process for about 15 days to compare the ability to produce colonies resistant to G418. As a result, as shown in Figure 4 it was observed that the production capacity of the RNAi-expressing colony for the MGC5627 gene is significantly lower than the control.

Example 5

Cancer Cell Growth Inhibitory Effect of RNAi Sequence of MGC5627 Gene by Cell Counting Analysis

MGC5627 was measured by recovering colonies formed by transforming RNAi expression vector and G418 selection vector (pHYK) together to reduce the growth rate of cancer cells and decrease the number of cells observed through inhibition of oncogene expression. The cancer cell growth inhibitory function of the RNAi sequence for the gene was investigated.

After selection, the number of cells is measured and each 10,000 cells are dispensed in 24 wells. The cell number was examined by cytometry for about one week from the next day. The average and standard deviation of three experiments per sample were calculated and shown in a graph (see FIG. 5). The results showed a significant decrease in the growth rate of cancer cells. This result is evaluated to be consistent with the results of the mRNA expression and colony forming ability shown above.

Example 6

Anchorage Independent Inhibitory Effect of RNAi Sequence on MGC5627 Gene

In order to determine whether expression of RNAi for MGC5627 gene leads to a decrease in anchorage independence for lung cancer cell line H460, the neomycin resistance gene expression vector and RNAi expression vector for MGC5627 are transformed together in human lung cancer cell line H460, followed by a screening process. After harvesting and incubating the formed G418-resistant colonies, a soft agar assay was performed to determine whether inhibition of MGC5627 gene expression reduced the oncogenicity of the cells. As a result, as shown in the above study results, a significant decrease in the number of colonies in cells in which mRNA expression of the MGC5627 gene was suppressed was observed (see FIG. 6).

As described above, the cancer cell proliferation inhibitor expressing RNAi for the gene MGC5627 according to the present invention was effectively introduced into the lung cancer cell line and expressed with high efficiency to obtain an effective effect of inhibiting the growth and anchorage independence of lung cancer tissues and lung cancer cells of the RNAi In addition to the overall understanding of lung cancer cell proliferation mechanism through the ability to inhibit the proliferation, it is expected to have a synergistic effect in the treatment of lung cancer in combination with gene therapy using conventional tumor suppressor genes.

<110> SK corporation <120> Lung cancer cell-growth inhibition drugs comprising RNAi          sequences against MGC5627 gene, preparation method according to          use <130> Lee02-422 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 513 <212> DNA <213> human <400> 1 atgtctgtgg ccggtgggga gattcgtggg gacacggggg gagaggacac tgctgctccc 60 ggccggttca gcttcagccc ggagcccacg ctcgaggaca tccgccgcct ccatgctgag 120 tttgctgcgg aacgagactg ggaacagttc catcagcctc ggaatctcct cctggccttg 180 gttggggaag tgggggagct ggcagaactc tttcagtgga aaaccgatgg ggaacctggc 240 ccccaaggct ggtcccccag ggaacgggca gcccttcaag aggagcttag tgacgtcctc 300 atctacctgg tggcattagc agcccgctgc cgtgtggatc tgccgctagc agtgctctcc 360 aaaatggaca tcaaccggcg acgctaccca gcccatctgg cccgcagctc ttcccgcaag 420 tatacagaat tgccccatgg ggccatctct gaagaccagg ctgtggggcc tgcggacatt 480 ccctgtgact ccacaggcca gacctcaacc tag 513 <210> 2 <211> 21 <212> DNA <213> human <400> 2 caaggctggt cccccaggga a 21

Claims (3)

A cancer cell proliferation inhibitor comprising an RNAi sequence represented by SEQ ID NO: 2 that inhibits MGC5627 gene expression represented by SEQ ID NO: 1 for the treatment of lung cancer. A recombinant vector designed to express the RNAi sequence for the MGC5627 gene represented by SEQ ID NO: 2 according to claim 1 under the control of a human U6 promoter cassette. The introduction into the RNAi expression vector of the MGC5627 according to 2, wherein from 0.1 to 2㎍ / 1 × 10 ⁵ cell concentration of 1 to 20 ㎍ / 1 × 10 ⁵ lung cancer cells with a cationic liposome to the cells of, and from 4 to 15 Lung cancer proliferation inhibition method using a lung cancer cell proliferation inhibitor comprising RNAi sequence for the MGC5627 gene, characterized in that the introduction divided into 1 to several times during the day.