阅读说明：本技术 激活宫颈癌HeLa细胞中LRIG1基因表达的双链saRNA分子 (Double-stranded saRNA molecule for activating LRIG1 gene expression in cervical cancer HeLa cells ) 是由 黄益玲 黄利鸣 严静 胡火军 鲁华 尤程程 向珺昱 夏玉桦 黄紫弦 王霖 汪聪 于 2021-06-03 设计创作，主要内容包括：本发明公开了一种激活宫颈癌HeLa细胞中LRIG1基因表达的双链saRNA(small activating RNA)分子。saRNA由正义链5’-UUC UUC CUG ACU GGG ACA U[dT][dT]-3′和反义链5’-AUG UCC CAG UCA GGA AGA A[dT][dT]-3′组成。本发明的saRNA分子可靶向结合LRIG启动子区,抑制人宫颈癌HeLa细胞增殖和侵袭迁移,诱导HeLa细胞发生凋亡,可用于制备宫颈癌治疗的药物。(The invention discloses a double-stranded sarRNA (small activating RNA) molecule for activating LRIG1 gene expression in a cervical cancer HeLa cell. The sarRNA consists of a sense strand 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3' and an antisense strand 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'. The saRNA molecule can be combined with the LRIG promoter region in a targeted manner, inhibits the proliferation and invasion migration of HeLa cells of human cervical cancer, induces the HeLa cells to generate apoptosis, and can be used for preparing medicaments for treating the cervical cancer.)

1. The double-stranded saRNA molecule of the cancer suppressor gene LRIG1 is characterized by consisting of a sense strand and an antisense strand with the following sequences:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

2. A composition for a cancer suppressor gene LRIG1 comprising a double-stranded saRNA molecule, wherein said double-stranded saRNA molecule consists of a sense strand and an antisense strand of the sequence:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

3. The composition of claim 2, further comprising at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable adjuvant.

4. The composition of claim 2, wherein the composition is a recombinant plasmid.

5. A method for increasing LRIG1 gene expression for non-therapeutic diagnostic purposes, wherein a double-stranded saRNA molecule is designed and introduced into a cell, said double-stranded saRNA molecule consisting of a sense strand and an antisense strand of the sequence:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

6. Use of the double-stranded saRNA molecule of any one of claims 1-4 in a medicament for activating expression of LRIG1 gene.

7. The use of claim 6, wherein the double-stranded saRNA molecule activates LRIG1 gene expression for the manufacture of a medicament for treating cervical cancer.

Technical Field

The invention belongs to the field of biological medicines, and provides a double-stranded small activating RNA molecule capable of activating expression of a cancer suppressor gene LRIG1 in a cervical cancer HeLa cell and application thereof in preparation of a cervical cancer treatment medicine.

Background

Cervical Cancer is a common malignancy in the female reproductive system, and recent statistical data from Cancer staticistics 2020 shows that about 60 million new cases of cervical Cancer and about 34 million deaths occur in 2020 worldwide. Although the incidence and mortality of cervical cancer gradually decline with the progress of cervical cancer screening and the application of HPV vaccines, the absolute incidence and mortality of cervical cancer still rise due to global population growth and population aging, and the health and life of women are seriously threatened. Therefore, the development of small molecular nucleic acid drugs for treating cervical cancer has important scientific significance and clinical application prospect.

The LRIG1 gene is located on chromosome 3p14, encodes a transmembrane protein rich in repetitive leucine sequences and immunoglobulin-like domains, also called LIG1 or LIG-1, and is widely expressed in normal tissues, and LRIG1mRNA is detected in all tissues of the human body. A large number of clinical data prove that LRIG1 is closely related to survival and prognosis of tumor patients, analysis studies such as Zhang are carried out on 2043 clinical patients, the positive expression rate of LRIG1 in cancer tissues is obviously lower than that of normal tissues, and high-expression LRIG1 is related to good prognosis of malignant tumors. Immunohistochemical results of clinical specimens from tumor patients showed that LRIG1 might be a good prognostic factor. The research report related to the cervical cancer shows that the high expression of LRIG1 is related to the good prognosis of cervical squamous cell carcinoma patients. So far, no report is found on the target point of applying the gene to the cervical cancer treatment at home and abroad.

Small activating RNAs (sarnas) are newly discovered small double-stranded RNAs that target the promoter region of a gene and induce gene expression at the transcriptional level. Compared with RNA interference (RNAi), the RNA activation (RNAa) technology has more stable effect in cells and more durable effect on target genes. saRNA has cell specificity and sequence specificity: dsEcad-215 upregulated Ecad expression in PC-3 and DU-145 but had no effect on Ecad expression in Hela cells, dsVEGF-706 upregulated target gene expression in HeLa cells, and HEK293 did not respond to upregulation of dsVEGF-706. The first clinical study of the saRNA drug MTL-CEBPA worldwide started by the company MiNA in 2016 for the treatment of advanced liver cancer. The saRNA drug carries out gene activation from the level of transcription and after transcription, has obvious advantages of high specificity, high efficiency, long-acting property and the like compared with the traditional drug which plays a role in protein level, and has great potential in the field of tumor treatment.

Disclosure of Invention

The activation of saRNA is cell-specific and sequence-specific. In order to obtain the saRNA medicine capable of effectively treating cervical cancer, the inventor designs three pairs of saRNA molecules by taking LRIG1 gene as a target spot, and finds that dsLRIG1-393 has the strongest activation effect on LRIG1 in cervical cancer HeLa cells. The composition of dsLRIG1-393 is as follows:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGAA [ dT ] [ dT ] -3'

A double-stranded sarRNA molecule (dsLRIG1-393) of a cancer suppressor gene LRIG1 consists of a sense strand and an antisense strand of the following sequences:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

A composition for a cancer suppressor gene LRIG1 comprising a double-stranded saRNA molecule consisting of a sense strand and an antisense strand of the sequence:

sense strand: 5 '-UUC UUC CUG ACU GGG ACAU [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

The composition further comprises at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable adjuvant.

The composition is a recombinant plasmid.

A method for increasing LRIG1 gene expression for non-therapeutic diagnostic purposes, wherein a double-stranded sarRNA molecule is designed and introduced into a cell, and the double-stranded sarRNA molecule consists of a sense strand and an antisense strand with the following sequences:

sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'

Antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

The double-stranded saRNA molecule is used for activating the LRIG1 gene expression drug.

The double-stranded saRNA molecule activates LRIG1 gene expression and is used for preparing a medicine for treating cervical cancer.

The medicine adopts subcutaneous injection, intravenous injection or liquid oral preparation.

The dsLRIG1-393 has high and long-acting effect on the activation of LRIG1 in HeLa cells, the dsLRIG1-393 is transfected into HeLa at the concentration of 50nM, total RNA and total protein are respectively extracted after 72h, and the expression condition of LRIG1mRNA and protein is detected. After the dsLRIG1-393 transfection, the expression of LRIG1mRNA is up-regulated by 5.75 +/-1.97 times on average, and the expression of LRIG1 protein is up-regulated by 3.3 +/-0.02 times.

The cell-entering efficiency of dsLRIG1-393 reaches 90% after 72h of transfection, and the dsLRIG1-393 can enter a nucleus partially and activate the expression of the dsLRIG1 promoter region by combining with the promoter region.

The dsLRIG1-393 has obvious inhibition effect on proliferation and invasion migration of cervical cancer HeLa cells and can promote HeLa cell apoptosis.

The invention discloses a double-stranded small activating RNA molecule (dsLRIG1-319) for activating LRIG1 gene expression in a cervical cancer HeLa cell and application thereof. The dsLRIG1-319 consists of a sense strand 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3' and an antisense strand 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'. The double-stranded small activating RNA molecule can be combined with a promoter region of an LRIG1 gene in a human cervical cancer HeLa cell in a targeted manner, can activate the expression of LRIG1 at the mRNA level and the protein level, can inhibit the proliferation and invasion migration of the human cervical cancer HeLa cell, can induce the HeLa cell to undergo apoptosis, and can be used for preparing a medicament for treating cervical cancer.

Drawings

FIG. 1 shows that dsLRIG1-393 activates mRNA and protein expression of LRIG1 gene in HeLa cells after transfection, A: detecting the expression condition of LRIG1mRNA by semiquantitative PCR; b: performing grey scale statistical analysis on LRIG1 mRNA; c: LRIG1 protein expression; d: LRIG1 protein gray scale statistical analysis, x: p <0.05, x: p < 0.01.

FIG. 2 shows that there is a time-long effect on the activation of LRIG1 protein expression after dsLRIG1-393 transfected HeLa cells, A: 24h, 48h, 72h and 7d after the dsLRIG1-393 transfects HeLa cells, the expression of LRIG1 protein; b: dsLRIG1-393 group time gradient LRIG1 protein expression; c: LRIG1 protein expression time gradient gray scale statistical analysis,: p <0.05, x: p < 0.01.

FIG. 3 shows that dsLRIG1-393 are targeted to bind to LRIG1 promoter region after transfection of HeLa cells, and LRIG1 gene promoter region is searched by NCBI database.

FIG. 4 shows the complete complementary pairing of LRIG1 gene promoter region with dsLRIG 1-393.

FIG. 5 shows the sequencing verification of plasmid pGL4 promoter in LRIG1 gene promoter region.

Fig. 6 is a dual luciferase reporter system demonstrating targeted binding of dsLRIG1-393 to LRIG1 gene promoter region: p <0.05, x: p < 0.01.

FIG. 7 shows the inhibition of HeLa cell proliferation after transfection of HeLa cells with dsLRIG1-393, A: MTT experiment detects the proliferation inhibition rate of dsLRIG1-393 on HeLa cells; b: p-ERK1 and ERK1/2 protein expression; c: statistical analysis of protein expression grayscales,: p <0.05, x: p < 0.01.

FIG. 8 shows the inhibition of HeLa cell migration after transfection of HeLa cells with dsLRIG1-393, A: cell scratch wound healing capacity (100 ×); b: statistical analysis of scratch healing capacity,: p <0.05, x: p < 0.01.

FIG. 9 shows the induction of HeLa cell apoptosis after dsLRIG1-393 transfection, A: detecting apoptosis by a flow cytometer; b: performing apoptosis cell statistical analysis; c: apoptosis signaling pathway-related protein expression; d: statistical analysis of protein expression grayscales,: p <0.05, x: p <0.01, x: p < 0.001.

Detailed Description

Example 1

dsRNA design dsLRIG1-393 is a dsRNA molecule which is designed to be complementary to a promoter region of LRIG1 gene and is matched with the promoter in a position close to a transcription starting point-393 and has the length of 21 nucleotide base pairs. The dsRNA (dsControl) of the control group is non-homologous with the known human genome sequence and is also a dsRNA sequence with the length of 21 nucleotide base pairs. The Genebank database is utilized to find the sequence of the LRIG1 gene promoter region, and the sequence aiming at the upstream-393 of the promoter is used as a template for designing dsRNA, and the specific sequence is as follows: sense strand: 5 '-UUC UUC CUG ACU GGG ACA U [ dT ] [ dT ] -3'; antisense strand: 5 '-AUG UCC CAG UCA GGA AGA A [ dT ] [ dT ] -3'.

Example 2

Cell culture and transfection

Human cervical cancer HeLa cells were purchased from the cell reservation center of Wuhan university and cultured and stored in the tumor microenvironment of the Sanxia university and key laboratories of the Hubei province of immunotherapy. The culture conditions are as follows: 5% CO₂Wet incubator at 37 deg.c, 1640 culture medium containing 10 vol% fetal calf serum, 1X 10⁵U/L penicillin and 100mg/L streptomycin. The experiments were divided into 3 groups: a control group (transfection dsLRIG1-393, dsLRIG1-365 and dsLRIG1-712 (specific sequences are shown in Table 1), wherein the concentration of dsRNA of each experimental group is 50nmol/L, the expression level of target gene LRIG1mRNA and protein after the dsLRIG1-393 transfection of HeLa cells is detected by a semi-quantitative PCR method and Western Blot, the cells are inoculated in a 6-well plate, gene transfection is carried out when the density reaches 60-70 percent, PBS washes each well plate cell twice, the growth state of the cells is determined to be good, 1.8mL of DMEM complete culture medium is added for standby, 1.5mL of EP tubes are placed on a tube frame, 200 muL of DMEM basic culture medium is respectively added in each tube, 5 muL of dsRNA which is slowly dissolved is respectively added in each EP tube, the tubes are gently mixed, 5 muL of Tubofect is respectively added in each tube after the tubes are placed for standby, the tubes are gently mixed, then placed on ice for 15min, short chain labeling is prepared, and fluorescent labels are paid attention to the complete nucleic acid labels which are respectively added in 6 wells kept away from light Short-chain nucleic acid complex of the body, preAnd shaking up in the left and right directions to ensure that the complex is uniformly distributed, placing the complex in a cell culture box for continuous culture, observing the state of the cells, and collecting the cells in each hole for subsequent experiments.

TABLE 1 SalRIG1 position and sequence

Example 3

The construction and target combination verification of the luciferase reporter gene plasmid selects a 2000bp region upstream of the transcription start point of the LRIG1 gene to construct into a luciferase reporter gene vector pGL4 basic, and the dual-luciferase reporter system is used for verifying the target relation between dsLRIG1-393 and a promoter region by using a pRL-TK sea cucumber luciferase plasmid as an internal reference for sequencing verification. Preparing a luciferase detection reagent according to the reagent instruction: adding 10mL of luciferase detection buffer solution into a bottle of freeze-dried luciferase detection substrate, shaking to fully mix the luciferase detection buffer solution and the freeze-dried luciferase detection substrate, subpackaging by using 1.5mL of EP tubes, and storing at-80 ℃ in a dark place. Collecting each group of cells in 1.5mL EP tube, adding 80 μ L of 1 × lysis solution into each tube, fully resuspending the cell precipitate, placing on ice for 15 minutes, flicking the EP tube once every 5 minutes to ensure full lysis, centrifuging at 4 deg.C and 12000rpm for 10min after completion, and taking the supernatant in a new EP tube. And (3) uniformly mixing the protein samples after each group of gene transfection with a luciferase detection reagent, and detecting and recording luciferase activity numerical values by using an enzyme-labeling instrument. The results show that the construction of the reporter plasmid in LRIG1 promoter region is successful, and compared with the control group, the fluorescence activity can be increased by 14.18 +/-4.00 times after the dsLRIG1-393 is transfected, and the results are shown in FIGS. 3-6. A2000 bp region (shown in figure 3) upstream of the transcription start point of the LRIG1 gene is constructed into a luciferase reporter gene vector pGL4 basic (shown in figure 4), sequencing verification is carried out (shown in figure 5), a pRL-TK sea cucumber luciferase plasmid is used as an internal reference, and a dual-luciferase reporter system is used for verifying the targeting relationship between dsLRIG1-393 and a promoter region. The results show that the construction of the reporter plasmid in LRIG1 promoter region is successful, and compared with the control group, the fluorescence activity can be increased by 14.18 +/-4.00 times after the dsLRIG1-393 is transfected, and the results are shown in FIG. 6.

Example 4

Total RNA extraction and RT-PCR analysis of mRNA expression levels after transfection for 72h, cells were harvested, total RNA was extracted with Trizol reagent, 1. mu.l Oligo dT was added to the PCR tube in order of 1. mu.g RNA template, 12. mu.l Oligo dT was added with DEPC water, and the EP tube was placed at 70 ℃ for 5 minutes and immediately on ice for 1 minute. Mu.l of 5 XBuffer, 1. mu.l of 20U/. mu.l RNase inhibitor, 2. mu.l of 10mM dNTPmix and 1. mu.l of reverse transcriptase are added, mixed well and centrifuged instantaneously. The mixture was placed in a PCR instrument to complete the subsequent reaction: 5min at 37 ℃, 60min at 42 ℃ and 10min at 70 ℃. The target genes LRIG1 and GAPDH were amplified using the reverse transcribed cDNA as a template. The primer sequence is as follows: LRIG1 upstream 5'-ATCATCACCCAGCCAGAAAC-3', downstream 5'-CTACCGTGGTCCCATCCTT-3'; GAPDH upstream 5'-AACGGATTT GGTCGTATTGGG-3', downstream 5'-CCTGGAAGATGGTGATGGGAT-3', reaction parameters: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 45sec, and 35 cycles, and full extension at 72 ℃ for 10min, wherein the reaction system is shown in Table 2. The PCR product fragments were detected by gel electrophoresis, photographed, and the gray values were counted (as shown in FIGS. 1A and 1B).

TABLE 2 semi-quantitative PCR reaction System

Example 5

Western Blot detection of dsRNA of related protein expression level after transfecting cells for 72h, collecting the cells for analysis of protein expression, washing the cells with PBS, adding protein lysate (RIPA lysate comprises 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40 and 0.1% SDS), cracking on ice, centrifuging and collecting supernatant, determining OD value of protein solution with BCA kit, adjusting protein concentration of samples to make protein amount of each sample 20ug, using actin as reference protein, performing SDS-PAGE gel electrophoresis on the extracted protein, after protein transfer, sealing at room temperature for 2h, reacting with anti-target protein antibody (primary antibody) and horseradish peroxidase labeled secondary antibody, and tracing target protein by electrochemical luminescence method. The target proteins to be examined are: LRIG1, caspase3, caspase8, caspase9, Bax, Bcl-2, etc. The experiment uses beta-actin as an internal reference protein. The experimental result shows that the dsLRIG1-393 has the most obvious activation effect on the expression of LRIG1 protein in HeLa cells after transfection (so the dsLRIG1-393 is selected in subsequent experiments). dsLRIG1-393 upregulated LRIG1 protein expression on average 3.3. + -. 0.02 fold after 72h transfection (as shown in FIGS. 1C, 1D). A significant up-regulation of protein content was still detectable after 7d transfection with dsLRIG1-393, see FIG. 2.

dsLRIG1-393 increased the proportion of HeLa cells that were apoptotic (as shown in FIGS. 9A, 9B). After dsLRIG1-393 transfection, the apoptosis pathway execution protein casepase3 is significantly activated, and the exogenous apoptosis signal pathway protein casepase8 and the endogenous apoptosis signal pathway protein casepase9 can be simultaneously activated; the endogenous apoptosis signaling pathway upstream protein Bax is up-regulated and Bcl-2 is down-regulated (as shown in FIGS. 9C and 9D).

Example 6

MTT experiment for detecting cell proliferation capacity after transfection of hLa cells with sarRNA at 50nM concentration for 2d, pancreatin digestion to collect cells, preparation of cell suspension and counting, and further culture after spreading 96-well plates with 3000 cells/well. Plates were removed at different time periods (3d, 5d, 7d), 20. mu.L MTT (5mg/mL) was added to each well, plates were gently shaken to mix well and incubated for an additional 4h in the incubator. And (3) taking out the culture plate, removing the supernatant, adding 150 mu L DMSO into each well, keeping out of the sun, shaking gently for 15min at room temperature by using a shaking table, measuring the OD value of the microplate reader at 490nm, detecting the proliferation inhibition rate of dsLRIG1-393 on HeLa cells, and recording and analyzing the result. MTT detection results show that dsLRIG1-393 can obviously inhibit HeLa cell proliferation compared with NC groups. After 72h, extracting total protein, and detecting the protein content change of ERK/MAPK signal channels ERK1/2 and p-ERK1 by using a Western Blot technology. As a result, dsLRIG1-393 was found to reduce the protein content of p-ERK1, as shown in FIG. 7, compared with the NC group, dsLRIG1-393 was found to significantly inhibit HeLa cell proliferation (as shown in FIG. 7A). After 72h, total protein is extracted, and changes of protein contents of ERK/MAPK signal channels ERK1/2 and p-ERK1 are detected by using a Western Blot technology (as shown in FIG. 7B), and dsLRIG1-393 is found to reduce the protein content of p-ERK1 (as shown in FIG. 7C).

Example 7

Cell scratch test for detecting cell migration ability HeLa cells in logarithmic growth phase are laid on a 6-hole plate, and each hole is about 2 multiplied by 10⁵And slightly scratching each cell 12 hours after the cells adhere to the wall by using a ruler as reference and a 10ul gun head, wherein at least 2 lines are scratched in each hole, and the interval between every two lines is 0.5 cm. Old medium was removed, cells were washed 3 times with PBS, scratched cells were removed, and dsLRIG1-393 sequences were subsequently transfected into HeLa at a concentration of 50nM, cultured using incomplete medium containing 2% FBS, photographed at 0h, 24h, 48h, 72h, and continuously observed under an optical microscope for scratch wound healing. The experimental results show that the dsLRIG 1-39372 h transfected in HeLa cells can reduce the healing capacity of cell scratch wounds by 46 percent and obviously inhibit the migration of HeLa cells, as shown in figure 8, and the dsLRIG 1-39372 h transfected in HeLa cells can reduce the healing capacity of cell scratch wounds by 46 percent and obviously inhibit the migration of HeLa cells (as shown in figures 8A and 8B).

Example 8

Detecting apoptosis by flow cytometry, dsLRIG1-393 is transfected into HeLa at the concentration of 50nM, each group of cells is digested by pancreatin without EDTA after 72h, the cells are centrifuged at 800rpm for 3min to collect suspended and adherent cells, the cells are washed twice by PBS, and the cells are resuspended by a proper amount of Annexin V binding solution to ensure that the cell density is 1 x 10⁶And L. And (3) adding a staining solution (the ratio of Annexin V-FITC binding solution to the staining solution is 100: 1) into the cell suspension, gently mixing uniformly, incubating in an ice box in a dark place for 15min, adding 2 mu L of PI staining solution, incubating in an ice box in a dark place for 5min, and detecting by an up-flow cytometer. The results are shown in FIG. 9, which is the induction of HeLa cell apoptosis after dsLRIG1-393 transfection, A: detecting apoptosis by a flow cytometer; b: performing apoptosis cell statistical analysis; c: apoptosis signaling pathway-related protein expression; d: statistical analysis of protein expression grayscales,: p<0.05，**：P<0.01，***：P<0.001. The experimental results show that dsLRIG1-393 can increase the proportion of HeLa cell apoptosis (as shown in FIGS. 9A and 9B). After dsLRIG1-393 transfection, the apoptosis pathway execution protein casepase3 is significantly activated, and the exogenous apoptosis signal pathway protein casepase8 and the endogenous apoptosis signal pathway protein casepase9 can be simultaneously activated; endogenous source of endogenousThe protein upstream of the apoptosis signaling pathway, Bax, is up-regulated and Bcl-2 is down-regulated (as shown in FIGS. 9C and 9D).